Comparison of Rhizosphere Microbial Diversity in Soybean and Red Kidney Bean Under Continuous Monoculture and Intercropping Systems

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript reports the results of a 3-year field-based study to better understand the consequences of intercropping (red kidney bean & soybean) on soil microbial communities through the use of amplicon sequencing.

The topic area is of scientific merit and is current given the global focus to improve the environmental consequences of food production. This study adds to the increasing literature on the topic.

The experimental design is appropriate but it is unfortunate that notwithstanding the level of replication, the study is based only on a single field site which consequently limits the wider applicability of the results to a more local interpretation. 

Missing from the methods is an indication of the resistant/tolerant/susceptible status of the two cultivars towards Alternaria. Simply put if one cultivar has known susceptibility and the other cultivar is known to be either resistant or tolerant, the results regarding Alternaria burden are challenged.

The manuscript is well-written, with clarity regarding the objectives.

I congratulate the authors for not using Bray-Curtis as the dissimilarity index (line 157) which is frequently used by default in the R package vegan without the user having knowledge of the statistical limitations of Bray-Curtis.

I would ask the authors to reflect upon the following:

keywords: several of the keywords are also in the title and therefore useless as keywords, please replace

Lines 45-47: and also increase yield (see Brooker et al, Plant and Soil, 2024)

Line 69: ",soybean" should read ", soybean"

Lines 156-159: which packages were used to determine beta diversity

Line 169: should "36" read "63"?

Lines 177-182: English grammar requires improving - seems to be a broken sentence

Line 186: the text deviates from the cited Figure. While the text states statistical significance, Figure 1b shows non-significant, Shannon p=0.16 and Pielou p=0.14

Lines 187-189: but there is no statistical significance therefore this can not be stated or inferred, please delete

Line 203: I suggest replacing "separated" with "distinct"

Line 204: please delete "indicating unique bacterial composition under R" as the text adds nothing to the paper

Lines 206-207: please delete this sentence as it is not appropriate to combine the variance of 2 different plots

Figures 2a and 2b please ensure that the x-axis scale are harmonised in terms of decimal places - please select one decimal place

Lines 282-283: unclear how a conclusion that an increased number of unique ASVs leads to an increased pathogen burden, appears to be a cause and effect assumption

Lines 331-334: as written, the sentence is incomplete

Lines 343-347: the text on soil remediation of smelter/mine areas is not relevant to this study - please delete

Lines 353-355: this is an incorrect statement. Figure 5b clearly shows that there is no statistically support for a decrease in Alternaria relative abundance. It is therefore wrong to state "significant reduction in the relative abundance of pathogenic fungi, such as Alternaria and Mortierella". The statement is correct for Mortierella.

References: it is perhaps the journal style to have a free form reference list but please not that a mixture of reference formatting is used. A single format should be used throughout. Also, some page numbers are missing (e.g., see line 419). There are some formatting/spelling issues with some journal names.

 

Author Response

Thank you for your thoughtful evaluation of this manuscript and for acknowledging the scientific relevance of our work on intercropping’s impact on soil microbial communities.  

 

We sincerely thank you for highlighting the experimental limitations. In future experimental designs, we will implement multi-year and multi-location cultivation trials and observations. This manuscript represents the initial phase of this ongoing research endeavor. 

 

Regarding Alternaria resistance, the soybean cultivar exhibited strong field resistance while the red kidney bean showed marked susceptibility—a divergence we intentionally leveraged to demonstrate crop-specific pathogen profiles (Fig. S1). Continuous intercropping of these cleistogamous crops significantly mitigated disease vulnerability, evidenced by recurrent Alternaria outbreaks in monocropped kidney bean plots versus negligible incidence in soybean monocultures. Mechanistically, soybean acted as a physical barrier aboveground, impeding pathogen dispersal to kidney bean, while simultaneously restructuring the soil microbiome belowground to suppress pathogen proliferation, as confirmed in our study.

 

We appreciate your insightful comments and your expertise greatly improved the manuscript’s rigor. All revisions are highlighted in yellow in the resubmitted text. Should further clarifications be needed, we are happy to provide them. In the following responses, I will address each of your queries point-by-point, with all replies in red font.

 

I would ask the authors to reflect upon the following:

 

Comments 1: keywords: several of the keywords are also in the title and therefore useless as keywords, please replace

 

Response 1:

Lines 37-38: Keywords: Legume crops; amplicon sequencing; rhizosphere microbiome assembly; agroecosystem sustainability

 

Thank you for pointing this out. We agree with this comment. The above modifications have been implemented and highlighted in yellow at the revised manuscript.

 

Comments 2:

Lines 45-47: and also increase yield (see Brooker et al, Plant and Soil, 2024)

 

Response 2:

Lines 50-52: In contrast, intercropping systems, where two or more crops are grown simultaneously, have been shown to improve resource use efficiency, suppress diseases, enhance microbial diversity , and also increase yield.

 

We appreciate your identification of this limitation and have implemented corresponding revisions in the manuscript.

 

Comments 3:

Line 69: ",soybean" should read ", soybean"

 

Response 3:

Lines 78-80: For example, intercropping soybeans with maize or wheat enhances the abundance of Bradyrhizobium and Pseudomonas, which are known for nitrogen fixation and biocontrol, respectively

 

Thank you for pointing this out. We agree with this comment, and have revised the manuscript accordingly.

 

Comments 4:

Lines 156-159: which packages were used to determine beta diversity

 

Response 4:

Lines 180-181: Using the ape package in R to perform PCoA analysis, output the PCoA coordinates of the sample points, and plot them as a two-dimensional scatter plot.

Thank you for pointing this out. We have revised the manuscript accordingly.

 

Comments 5:

Line 169: should "36" read "63"?

 

Response 5:

Thank you for your attention to this detail. As shown in Fig. S1, we collected a total of 36 samples throughout the entire growth period.

 

Comments 6:

Lines 177-182: English grammar requires improving - seems to be a broken sentence

 

Response 6:

Lines 189-197: The number of reads per sample group before and after quality control is shown in Table S1. In total, 2,067,744 high-quality bacterial sequences and 4,337,126 high-quality fungal sequences were recovered after quality control. There are three cultivation groups and four growth stages, with a total of 36 samples. The mean sequencing depth reached 57,437 reads per sample for bacteria and 120,476 reads for fungi. As summarized in Table S2, the average sequence read length for both domains was 418 bp. At a 100% identity threshold, we detected 92,444 distinct bacterial amplicon sequence variants (ASVs) and 4,273 fungal ASVs. Notably, bacterial ASV richness (Table S3) substantially exceeded fungal ASV counts (Table S4).

Thank you. We have revised the manuscript accordingly.

 

Comments 7:

Line 186: the text deviates from the cited Figure. While the text states statistical significance, Figure 1b shows non-significant, Shannon p=0.16 and Pielou p=0.14

 

Response 7:

Lines 207-215: Fungal communities (Figure 1b) exhibited distinct patterns. Intercropping (SR) moderately influenced fungal richness (Chao1: p= 0.057; observed species: p= 0.054), while it slightly enhanced community evenness (Shannon: p=0.16; Pielou-e: p=0.14). Notably, the SR system achieved the highest fungal evenness among all the treatments, suggesting that intercropping may optimize community structure by suppressing pathogen accumulation in continuous cropping systems. These results indicate that intercropping exerts limited influence on bacterial diversity but potentially improves fungal community diversity and stability, likely through ecological niche complementarity and pathogen suppression mechanisms.

 

I sincerely apologize for this oversight. I have carefully reviewed the labeling in Figure 1 and revised the corresponding text accordingly. Thank you again for pointing out this error.

 

Comments 8:

Lines 187-189: but there is no statistical significance therefore this can not be stated or inferred, please delete

 

Response 8:

Thank you for pointing out this issue. We have made the corresponding revisions as shown in the previous response, and these changes have also been incorporated into the manuscript.

 

Comments 9:

Line 203: I suggest replacing "separated" with "distinct"

 

Response 9:

Thank you for your valuable suggestion. We have incorporated the modification as indicated in Line 227 of the revised manuscript.

 

Comments 10:

Line 204: please delete "indicating unique bacterial composition under R" as the text adds nothing to the paper

 

Response 10:

Thank you for your valuable suggestion. We have incorporated the modification as indicated in Line 228 of the revised manuscript.

 

Comments 11:

Lines 206-207: please delete this sentence as it is not appropriate to combine the variance of 2 different plots

 

Response 11:

Thank you for your valuable suggestion. We have incorporated the modification as indicated in Lines 230-231 of the revised manuscript.

 

Comments 12:

Figures 2a and 2b please ensure that the x-axis scale are harmonised in terms of decimal places - please select one decimal place

 

Response 12:

We are grateful for your observation. The suggested modifications have been incorporated into Fig. 2.

 

Comments 13:

Lines 282-283: unclear how a conclusion that an increased number of unique ASVs leads to an increased pathogen burden, appears to be a cause and effect assumption

 

Response 13:

Lines 310-316: Crucially, the R system exhibited substantially higher unique bacterial and fungal ASVs compared to SR and S systems. This indicates that SR and S systems share closer microbial diversity profiles, whereas the unique ASVs exclusively present in R may potentially be related to its increasingly severe field disease incidence over successive years. As observed in Figure S1, the R system demonstrated markedly higher field disease prevalence compared to both SR and S systems.

 

Thank you for your inquiry. We have incorporated the suggested changes and marked the corresponding modifications within the manuscript

 

Comments 14:

Lines 331-334: as written, the sentence is incomplete

 

Response 14:

Lines 354-359: The findings of this study demonstrate that the intercropping of soybeans and red kidney beans reshapes microbial community structure compared to continuous monoculture. These results align with previous research highlighting intercropping’s positive effects on soil microbial communities and their functional roles in agroecosystems [34]. Below, we discuss the implications of our findings regarding microbial diversity, functional microbial enrichment, and pathogen suppression.

 

Thank you for your inquiry. We have incorporated the suggested changes and marked the corresponding modifications within the manuscript

 

Comments 15:

Lines 343-347: the text on soil remediation of smelter/mine areas is not relevant to this study - please delete

 

Response 15:

Lines 377-379:The application of Streptomyces and Bacillus consortium in smelter/mines-contaminated soil affected the physicochemical characteristics of the soil. The extractable Zn decreased, while Cd increased at each harvest [40].

 

We appreciate your suggestion and have deleted the corresponding content in the manuscript.

 

Comments 16:

Lines 353-355: this is an incorrect statement. Figure 5b clearly shows that there is no statistically support for a decrease in Alternaria relative abundance. It is therefore wrong to state "significant reduction in the relative abundance of pathogenic fungi, such as Alternaria and Mortierella". The statement is correct for Mortierella.

 

Response 16:

We sincerely appreciate you identifying this error. The phrase 'and Mortierella' has been deleted at Line 388 in the revised manuscript.

 

Comments 17:

References: it is perhaps the journal style to have a free form reference list but please not that a mixture of reference formatting is used. A single format should be used throughout. Also, some page numbers are missing (e.g., see line 419). There are some formatting/spelling issues with some journal names.

 

Response 17:

We appreciate your meticulous review. We have verified and corrected reference formatting, page numbers, and journal name spellings throughout the revised manuscript.

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Prof. Dr. Editor;

You find enclosed my comments about the revision of the Manuscript,  agronomy-3725957 entitled " Comparison of Rhizosphere Microbial Diversity in Soybean and Red Kidney Bean Under Continuous Monoculture and Intercropping Systems”.

• This study assesses the diversity of rhizosphere bacterial and fungal microbiota in in two leguminous plants, soybean and red kidney bean, in continuous monoculture and intercropping system, across different cultivation stages. The methodology adopted is well-developed, both in terms of design and molecular analyses performed to elucidate the diversity and dynamic of each studied microbial taxonomic group. Thus, the results are convincingly interpreted using indices recognized for biodiversity assessment.
• The conclusions of the initial results on the importance of intercropping as a good strategy for soil health management and high yield were also convincing and well supported by the comparative interpretations of diversity indices.

Overall, the work is well written. Despite this effort, the redaction of the paper still require amelioration in order valorize this work.  

• My comments are listed as follows:
• Q1: Why did you choose to focus the study on the third and final year of cultivation?
• Q2 : What do you think about possible changes in diversity and dynamic of microbial communities from one year to the next?
• Q3: Is there any information on crop yield in the cropping systems used to further demonstrate the positive effect of intercropping?
• Overall, the manuscript can be accepted minor revision.

Author Response

Thank you sincerely for your thorough review of our manuscript and for providing such insightful comments. We greatly appreciate the time and expertise you dedicated to evaluating our work, particularly your constructive feedback on both the strengths of the methodology and the interpretation of results regarding rhizosphere microbial diversity under different cropping systems. Your recognition of the significance of our findings on intercropping is highly encouraging.

 

We have carefully considered all your suggestions for improving the manuscript. In the following sections, we will address each of your points specifically and detail the revisions made to enhance the clarity, precision, and overall presentation of the paper. Our goal is to fully incorporate your valuable insights to strengthen the manuscript’s impact and academic rigor.

 

All revisions are highlighted in yellow in the resubmitted text. Should further clarifications be needed, we are happy to provide them. In the following responses, I will address each of your queries point-by-point, with all replies in red font.

 

My comments are listed as follows:

 

Q1: Why did you choose to focus the study on the third and final year of cultivation?

 

Response 1: 

Thank you for raising this point. We acknowledge that red kidney bean is a crop susceptible to continuous cropping obstacles, and that its pathogens differ from those affecting soybean. Consequently, we designed this experiment to explore whether intercropping red kidney bean with soybean could mitigate its continuous cropping obstacles.

 

In our experiment, we observed an intensification of disease in red kidney beans over two consecutive years of monocropping. As shown in Fig. S1, the disease severity became particularly pronounced in the third year of continuous cropping, presenting a stark contrast to both the intercropped red kidney beans and the continuously cropped soybeans. Therefore, in the third year, we conducted amplicon diversity analysis on rhizosphere microorganisms throughout the entire growth period under different cultivation modes.

 

It is important to note that the experiment did not conclude in the third year. This ongoing study has now progressed to its sixth year. We have collected rhizosphere soil samples annually and will continue to analyze these in future publications. This will further elucidate the shifts in rhizosphere microbial communities under continuous red kidney bean monocropping, red kidney bean-soybean intercropping, and soybean monocropping. Additionally, we aim to investigate the mechanisms underlying crop-rhizosphere microorganism interactions across these cultivation systems.

 

Q2 : What do you think about possible changes in diversity and dynamic of microbial communities from one year to the next?

 

Response 2: 

Thank you very much for your question. During the experimental design phase, we collected rhizosphere soil samples annually. Initially, we hypothesized that as the disease severity in red kidney beans progressively increased over consecutive years, there would be a clear accumulation of pathogenic microorganisms within the soil microbial community. However, plants facing disease stress can recruit beneficial bacteria as a self-rescue mechanism. This response may lead to increased complexity in the rhizosphere or phyllosphere microbial communities, rather than necessarily exhibiting a straightforward, year-by-year accumulation of pathogens.

 

In subsequent research, we successfully isolated pathogenic fungi from diseased red kidney bean leaves. While simultaneously, we also identified and screened antagonistic bacteria from the leaf surfaces that exhibited significant inhibitory effects against these pathogens. Therefore, based on years of experimental observations, we propose that although field disease symptoms under continuous red kidney bean monocropping show increasingly evident severity, the annual changes in the rhizosphere microbial community are likely more complex. It may not solely manifest as a progressive accumulation of pathogens but rather present a more intricate dynamic.

 

This hypothesis will be validated through amplicon sequencing or metagenomic sequencing conducted on the rhizosphere microbial communities collected each year.

 

Q3: Is there any information on crop yield in the cropping systems used to further demonstrate the positive effect of intercropping?

Overall, the manuscript can be accepted minor revision.

 

Response 3: 

Thank you very much for your question. During the experiment, we measured the plot yields.

 

We observed that continuous monocropping of red kidney beans resulted in a near-total crop failure by the third year. In contrast, intercropping maintained a certain yield level for the red kidney beans, although it did affect soybean yield to some extent. Notably, soybean yields remained relatively stable under continuous monocropping.

 

As this particular article focuses specifically on investigating the dynamics of rhizosphere microbial communities throughout the entire growth cycle, we did not include the yield data here. These yield results will instead be presented in a subsequent publication analyzing microbial diversity across multiple years. In that work, the yield data will be correlated with the microbial data to demonstrate the complexity of the observed microbial shifts.

 

We appreciate your understanding regarding this presentation of data across separate manuscripts.

 

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

As part of plant microbiome rhizosphere microorganisms have the potential to help enhance plant growth and the plant's defense against plant pathogens. Major advances in genome sequencing have led to a better understanding of the microbiome structure and function. A shift in the microbiome balance is linked to a broad range of plant diseases. This understanding may lead to potential opportunities to develop next-generation microbiome-based disease management options and diagnostic biomarkers. However, our understanding is limited given the nature of the plant microbiome and its complex and multidirectional interactions. The manuscript titled “Comparison of Rhizosphere Microbial Diversity in Soybean and Red Kidney Bean Under Continuous Monoculture and Intercropping Systems” by Huibin Qin et al. describes analysis of diversity of rhizosphere microbial communities of red kidney bean and soybean under monocropping and intercropping cultivation using high-throughput sequencing technology and bioinformatics analysis.

The manuscript is well-written and presents an interesting and careful bioinformatic analysis and visualization of changes in rhizosphere microbiome of red kidney bean and soybean under different cropping systems.

Page 3 Lines 90 -108

What is the cropping history of the site?

Red kidney bean and soybean cultivars used – what is their disease resistance or susceptibility characteristics?

Field management included standardized fertilization protocols – more specific details needed, and it is relevant to the study (soil microbiome).

Page 3 Lines 109 -130

This is a field study, and the focus is on soil microbiomes (rhizosphere). Basic information about the soil physicochemical characteristics needs to be provided.

Page 7 Lines 258 – 260

Cropping systems exerted systematic effects, with intercropping enriching nutrient cycling taxa (e.g., Fusarium, Botryotrichum CITATIONS? ) and suppressing pathogens, while monocultures favored stress-tolerant groups (e.g., Streptomyces in R, Ascomycota in S CITATIONS? ).

Page 8 Lines 282 – 284

This finding aligns with field observations of progressively severe kidney bean disease incidence under monocropping (Figure S1).

Why was the causal agent of severe kidney bean disease under monocropping not diagnosed? This is very pertinent to the study. The life cycle of the pathogen(s) both in soil and above ground needs to be discussed. Both red kidney beans as well as soybeans are susceptible to Alternaria alternata infection.

Page 9 Lines 332 – 334

which is strongly driven by the complementarity of below-ground structures (???) and their interactions with the soil microbiomeenhanced (???) soil microbial [36].

Page 10 Lines 343 – 346

Application of Streptomyces and Bacillus consortium in smelter/mines- contaminated soil affected the soil physico-chemical characteristics. The extractable Zn decreased, while Cd increased at each harvest [40].

In this context the citation 40 does not fit, use a more suitable in-text citation.

Page 10 Lines 353 – 358

One of the most striking findings of this study is the significant reduction in the relative abundance of pathogenic fungi, such as Alternaria and Mortierella, in intercropping systems. Alternaria section Alternaria is comprised of many species that infect a broad diversity of important crop plants and cause post-harvest spoilage [44]. Mortierella species are abundant and frequently isolated from soil and plant roots, particularly in soils with pathogenic fungi, and associated with declining Araucaria araucana trees [45].

In this context (red kidney bean or soybean being the host plant), it is not clear why stressing Mortierella species being pathogenic fungi is relevant. Yes, it is reported to be pathogenic to Araucaria araucana trees, but in general Mortierella species are considered to play a beneficial role.

Author Response

We sincerely thank the reviewer for their insightful evaluation of our manuscript and their recognition of our bioinformatic analysis exploring rhizosphere microbiome dynamics in legumes under different cropping systems. We appreciate the reviewer's constructive feedback regarding both the strengths of our work and the opportunities for enhancement. We have carefully considered each point raised and will systematically address all comments in our revisions. Where necessary, we will refine the text to improve clarity, strengthen data interpretation, and enhance the scientific rigor of the manuscript to better reflect the significance of microbiome shifts in plant health and disease management potential.

 

All revisions are highlighted in yellow in the resubmitted text. Should further clarifications be needed, we are happy to provide them. In the following responses, I will address each of your queries point-by-point, with all replies in red font.

 

 

Comments 1:

Page 3 Lines 90 -108

 

What is the cropping history of the site?

 

Red kidney bean and soybean cultivars used – what is their disease resistance or susceptibility characteristics?

 

Field management included standardized fertilization protocols – more specific details needed, and it is relevant to the study (soil microbiome).

 

Response 1:

Lines 102 -104: The soybean cultivar "Pindou 24" and red kidney bean cultivar "Pinjinyun 4" exhibited moderate disease resistance and were provided by the Center for Agricultural Genetic Resources Research, Shanxi Agricultural University.

 

Lines 108 -109: In 2018, the entire experimental field was uniformly monocropped with buckwheat (Fagopyrum esculentum Moench).

 

Lines 117 -119: At seeding, a basal fertilization of 15 kg·ha⁻¹ NPK compound fertilizer (N-P₂O₅-K₂O) was uniformly applied across all experimental plots, with identical fertilization regimes maintained for the three cultivation systems.

 

We sincerely appreciate your question and have supplemented the relevant content in the Methods section accordingly.

 

Comments 2:

Page 3 Lines 109 -130

 

This is a field study, and the focus is on soil microbiomes (rhizosphere). Basic information about the soil physicochemical characteristics needs to be provided.

 

Response 2:

Lines 120 -123: The experimental soil, collected from a homogeneous plot block, exhibited the following pre-experimental physicochemical characteristics: total nitrogen (TN 0.08%), available phosphorus (AP 19.52 mg/kg), available potassium (AK 107.4 mg/kg), organic matter (OM 14.49 g/kg) and pH 8.70.

 

We appreciate your identification of this omission. Soil nutrient properties had been determined prior to manuscript submission and have now been incorporated into the text as noted.

 

Comments 3:

Page 7 Lines 258 – 260

 

Cropping systems exerted systematic effects, with intercropping enriching nutrient cycling taxa (e.g., Fusarium, Botryotrichum CITATIONS? ) and suppressing pathogens, while monocultures favored stress-tolerant groups (e.g., Streptomyces in R, Ascomycota in S CITATIONS? ).

 

Response 3:

Lines 287 – 289, We appreciate your feedback and have now supplemented the references in the indicated sections as suggested.

 

Comments 4:

Page 8 Lines 282 – 284

 

This finding aligns with field observations of progressively severe kidney bean disease incidence under monocropping (Figure S1).

 

Why was the causal agent of severe kidney bean disease under monocropping not diagnosed? This is very pertinent to the study. The life cycle of the pathogen(s) both in soil and above ground needs to be discussed. Both red kidney beans as well as soybeans are susceptible to Alternaria alternata infection.

 

Respons4:

We appreciate your insight regarding the severe foliar disease observed in red kidney beans. While the isolation and identification of Alternaria and Colletotrichum pathogens from diseased leaves will be detailed in a companion paper (currently in preparation), this article does not address the pathogen isolation process in depth. Our experimental data confirmed pronounced disease pressure on red kidney beans within the same field plot. Notably, neither monocropped nor intercropped soybeans showed signs of Alternaria infection. We postulate that the underlying mechanisms warrant further investigation.

 

Comments 5:

Page 9 Lines 332 – 334

 

which is strongly driven by the complementarity of below-ground structures (???) and their interactions with the soil microbiomeenhanced (???) soil microbial [36].

 

Response 5:

Lines 364 – 367: In an intercropping system, the interplay between cereals and legumes, which is strongly driven by the complementarity of root architecture systems and their interactions with the soil microbiome, enhanced the soil microbial community.

 

We appreciate your insights and have implemented corresponding revisions in the revised manuscript.

 

Comments 6:

Page 10 Lines 343 – 346

 

Application of Streptomyces and Bacillus consortium in smelter/mines- contaminated soil affected the soil physico-chemical characteristics. The extractable Zn decreased, while Cd increased at each harvest [40].

 

In this context the citation 40 does not fit, use a more suitable in-text citation.

 

Response 6:

We appreciate your comment. As advised by another reviewer, the statement in question has been removed from the revised manuscript as it falls outside the primary scope of this study.

 

 

Comments 7:

Page 10 Lines 353 – 358

 

One of the most striking findings of this study is the significant reduction in the relative abundance of pathogenic fungi, such as Alternaria and Mortierella, in intercropping systems. Alternaria section Alternaria is comprised of many species that infect a broad diversity of important crop plants and cause post-harvest spoilage [44]. Mortierella species are abundant and frequently isolated from soil and plant roots, particularly in soils with pathogenic fungi, and associated with declining Araucaria araucana trees [45].

 

In this context (red kidney bean or soybean being the host plant), it is not clear why stressing Mortierella species being pathogenic fungi is relevant. Yes, it is reported to be pathogenic to Araucaria araucana trees, but in general Mortierella species are considered to play a beneficial role.

 

Response 7:

We appreciate your inquiry. This article primarily focuses on analyzing rhizosphere microbial shifts under different cropping systems, and as such, the pathological role of Mortierella has not been extensively addressed. Meanwhile, a companion paper currently under preparation details the isolation and identification of multiple Alternaria and Colletotrichum strains from diseased red kidney bean leaves, with laboratory-based pathogenicity tests confirming their infectious capacity.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Dear Authors,

Suggestions for corrections are described in the attached file.

Best regards,

Comments for author File: Comments.pdf

Author Response

We sincerely appreciate your insightful evaluation highlighting the significance of this work for sustainable legume production. Your constructive feedback, particularly regarding methodological refinement, in-depth discussion, and enhanced data clarity, is invaluable for strengthening the manuscript.

 

All revisions are highlighted in yellow in the resubmitted text. Should further clarifications be needed, we are happy to provide them. In the following responses, I will address each of your queries point-by-point, with all replies in red font.

 

Comments 1:

 

- Include quantitative data highlighting the main differences in microbial diversity between monoculture and intercropping systems.

- Specify the methodological approaches used for microbial characterization (e.g., 16S rRNA or ITS sequencing), even briefly.

- Clearly indicate which microbial genera were most affected by each cropping system.

- Conclude with a statement emphasizing the relevance of the findings for sustainable agricultural practices and soil management.

 

Response 1:

Lines 22-25: To elucidate the composition and diversity of rhizosphere microbial communities, we conducted amplicon sequencing targeting the V3-V4 hypervariable regions of the bacterial 16S rRNA gene and the ITS1 region of fungal ribosomal DNA across distinct growth stages. 

 

Lines 31-32: The monocropping of red kidney beans increased the richness of rhizosphere bacteria and fungi and promoted the accumulation of pathogenic microorganisms. In contrast, intercropping cultivation and soybean monocropping favored the accumulation of beneficial bacteria such as Bacillus and Streptomyce, reduced pathogenic fungi including Alternaria and Mortierell, and exhibited less microbial variation across different growth stages.

 

Lines 35-36: The findings of this study will inform sustainable agricultural practices and soil management strategies.

 

We appreciate your feedback and have implemented the corresponding revisions as noted.

 

Comments 2:

- Reinforce the importance of the rhizosphere microbiome for soil health, plant growth, and legume productivity.

- Provide theoretical background on the impacts of continuous monoculture and intercropping on microbial composition and function.

 

Response 2:

Lines 59-61: Colonizing the rhizosphere—the soil zone immediately surrounding plant roots—diverse microbial assemblages profoundly influence plant growth, nutrition, and health, as well as the structure of plant communities.

 

We appreciate your feedback and have implemented the corresponding revisions as noted.

 

Comments 3:

- Highlight gaps in the literature regarding direct comparisons between soybean and red kidney bean under different cropping systems.

- Clearly state the study’s objectives and, if possible, formulate a hypothesis about the expected effects of intercropping on microbial diversity.

- Justify the choice of the crops based on their agronomic significance and potential for symbiotic interaction with soil microbes.

 

Response 3:

Lines 52-56: While the pathogen-blocking efficacy of soybean–red kidney bean intercropping systems lacks prior documentation, the biological compatibility of these two legumes—characterized by analogous growth habits yet distinct pathogen profile-provides a compelling rationale for implementing this cropping pattern.

 

We appreciate your feedback and have implemented the corresponding revisions as noted.

 

Comments 4:

- Provide detailed information on DNA extraction techniques and primers used for microbial gene amplification.

- Report the quality control criteria applied during sequencing, such as quality filters and chimera removal.

- Specify the diversity indices used (e.g., Shannon, Simpson, Chao1) and how they were interpreted.

- Describe the full experimental design, including number of replicates, sampling units, and blocking (if applicable).

- Include environmental parameters (e.g., soil type, moisture, temperature, pH) that could have influenced microbial data.

 

Response 4:

Lines 158-161: Bacterial 16S rRNA gene V3-V4 regions were amplified using the primers 338F (5'-ACTCCTACGGGAGGCAGCA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') [17]. Fungal ITS1 regions were amplified with the primers ITS5F (5'-GGAAGTA AAAGTCGTAACAAGG-3') and ITS2R (5'-GCTGCGTTCTTCATCGATGC -3') [18].

 

Lines 170-173: Quality filtering, denoising, read merging, and chimera removal were performed using the DADA2 plugin [20]. Non-singleton amplicon sequence variants (ASVs) were aligned with MAFFT [21], and phylogenetic trees were constructed using FastTree2

 

Lines 173-176: Alpha diversity indices (Chao1 richness [23]), observed species, Shannon diversity [24, 25], and Pielou’ s evenness [26] were calculated using the ASV tables in QIIME2, and they were visualized as box plots

 

Lines 128-133: During the 2021 growing season, rhizosphere soil samples were collected from two leguminous species (Glycine max and Phaseolus vulgaris) using three cultivation modes at four growth stages: seedling, flowering, maturation, and post-maturation. We employed a five-point sampling method, positioning the central point at the intersection of field diagonals with four additional points equidistant along the diagonals (Figure S1: cultivation modes and sampling timeline).

 

Lines 117-123: At seeding, a basal fertilization of 15 kg·ha⁻¹ NPK compound fertilizer (N- P₂O₅-K₂O) was uniformly applied across all experimental plots, with identical fertilization regimes maintained for the three cultivation systems. The experimental soil, collected from a homogeneous plot block, exhibited _the following pre-experimental physicochemical characteristics: total nitrogen (TN 0.08%), available phosphorus (AP 19.52 mg/kg), available potassium (AK 107.4 mg/kg), organic matter (OM 14.49 g/kg) and pH 8.70.

 

We appreciate your feedback and have implemented the corresponding revisions as noted.

 

 

Comments 5:

 

- Present diversity data with clear numerical values (e.g., alpha diversity indices) and visible statistical analyses.

- Highlight differences in dominant microbial genera across cropping systems, and discuss their relationship to plant physiology.

- Analyze how intercropping affected functional microbial groups (e.g., nitrogen fixers, organic matter decomposers).

- Compare findings with previous studies to support the originality or consistency of the results.

- Discuss how root exudates may shape microbial communities differently under monoculture versus intercropping.

- Point out the agronomic implications of maintaining or losing microbial diversity under continuous monoculture.

 

Response 5:

Lines 354-359: The findings of this study demonstrate that the intercropping of soybeans and red kidney beans reshapes microbial community structure compared to continuous monoculture. These results align with previous research highlighting intercropping’s positive effects on soil microbial communities and their functional roles in agroecosystems [38]. Below, we discuss the implications of our findings regarding microbial diversity, functional microbial enrichment, and pathogen suppression.

 

Lines 392-394:Root exudation under maize/soybean intercropping system mediates the arbuscular mycorrhizal fungi diversity and improves the plant growth.

 

I sincerely appreciate your insightful critique and have implemented the revisions above based on comprehensive literature review.

 

Comments 6:

 

- Acknowledge the study’s limitations, such as the lack of deeper functional analyses (e.g., metagenomics or metabolomics).

- Recommend long-term studies to validate the effects on productivity and soil health.

- Reinforce the applicability of the findings to real-world legume cropping systems.

 

Response 6:

Lines 409-419: In conclusion, soybean–red kidney bean intercropping significantly restructures the rhizosphere microbiota compared to monoculture, enriching beneficial microbes, suppressing pathogens, and enhancing soil health—highlighting its potential as a sustainable solution for continuous cropping challenges. While these findings demonstrate how optimized plant-microbe interactions can foster resilient agroecosystems supporting global food security, we acknowledge limitations including the lack of functional analyses (e.g., metagenomics/metabolomics). Thus, long-term trials validating productivity metrics (yield stability) and soil parameters (organic carbon accrual) are recommended. Crucially, the field-relevant framework ensures direct applicability to legume production systems, informing sustainable rotations leveraging plant-microbe synergies.

 

We sincerely appreciate your reminder. Our long-term trial is ongoing, currently in its sixth year of cropping. We will employ metagenomic approaches to analyze annual dynamics of rhizosphere and endophytic microbiomes. Additionally, pathogen-antagonist isolation and identification for red kidney beans were conducted last year, with ongoing efforts to implement biological control strategies against its diseases. We reiterate our gratitude for your systematic and insightful perspectives.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

NA