Microbial Assemblages Associated with the Soil-Root Continuum of an Endangered Plant, Helianthemum songaricum Schrenk

ABSTRACT The microbial network of the soil-root continuum plays a key role in plant growth. To date, limited information is available about the microbial assemblages in the rhizosphere and endosphere of endangered plants. We suspect that unknown microorganisms in roots and soil play an important role in the survival strategies of endangered plants. To address this research gap, we investigated the diversity and composition of the microbial communities of the soil-root continuum of the endangered shrub Helianthemum songaricum and observed that the microbial communities and structures of the rhizosphere and endosphere samples were distinguishable. The dominant rhizosphere bacteria were Actinobacteria (36.98%) and Acidobacteria (18.15%), whereas most endophytes were Alphaproteobacteria (23.17%) as well as Actinobacteria (29.94%). The relative abundance of rhizosphere bacteria was higher than that in endosphere samples. Fungal rhizosphere and endophyte samples had approximately equal abundances of the Sordariomycetes (23%), while the Pezizomycetes were more abundant in the soil (31.95%) than in the roots (5.70%). The phylogenetic relationships of the abundances of microbes in root and soil samples also showed that the most abundant bacterial and fungal reads tended to be dominant in either the soil or root samples but not both. Additionally, Pearson correlation heatmap analysis showed that the diversity and composition of soil bacteria and fungi were closely related to pH, total nitrogen, total phosphorus, and organic matter, of which pH and organic matter were the main drivers. These results clarify the different patterns of microbial communities of the soil-root continuum, in support of the better conservation and utilization of endangered desert plants in Inner Mongolia. IMPORTANCE Microbial assemblages play significant roles in plant survival, health, and ecological services. The symbiosis between soil microorganisms and these plants and their interactions with soil factors are important features of the adaptation of desert plants to an arid and barren environment. Therefore, the profound study of the microbial diversity of rare desert plants can provide important data to support the protection and utilization of rare desert plants. Accordingly, in this study, high-throughput sequencing technology was applied to study the microbial diversity in plant roots and rhizosphere soils. We expect that research on the relationship between soil and root microbial diversity and the environment will improve the survival of endangered plants in this environment. In summary, this study is the first to study the microbial diversity and community structure of Helianthemum songaricum Schrenk and compare the diversity and composition of the root and soil microbiomes.

We appreciate the editor insightful comments, and sorry for our negligence. We have listed the amplicon sequencing.
Line140-144: Raw data has been uploaded and amplicon sequencing data has been deposited. ( 16Sr DNA:

Poor writing
We are thankful to the editor's insightful comments.This manuscript have already revised the grammatical errors by a native English speaker.
Thank you again for your comments. We hope we could learn more from you.

Reviewer's insightful comments
Thank you again for your comments. We hope we could learn more from you.
We have highlighted the changes in red according to reviewer #1 comments in the annotated version of the revised manuscript (Revision, changes marked).
1. The study is very primary, the description is unclear, and the conclusion regarding "sensitivities of community" is hard to be supported and even understand.
We appreciate the reviewer's insightful comments. We are very sorry for not clearly describing this content in the previous manuscript. We have added and changed some informative and key sentences in the texts.
Line19-21:We have revised the sentence"We suspect that unknown microorganisms in roots and soil play an important role in the survival strategies of endangered plants".
Line21: "we investigated" instead of "we used Illumina MiSeq high-throughput sequencing to investigate.
Line23-24: We have revised the sentence "and observed that the microbial communities and structures between the rhizosphere and endosphere samples were distinguished".
Rhizosphere bacteria relative abundance was higher than endosphere samples. Fungal rhizosphere and endophyte samples had approximately equal amounts of the Sordariomycetes (23%), while the Pezizomycetes were more abundant in the soil (31.95%) than root (5.70%). The abundance between root and soil samples of phylogenetic relationships also showed that the most abundant bacterial and fungal reads tended to be dominant in either the soil or root samples but not both.
Additionally, the Pearson correlation heatmap ananlysis showed that soil bacteria and fungal diversity and composition were closely related to pH, total nitrogen, total phosphorus and organic matter, of which pH, and organic matter were the main drivers. These results clarify the difference patterns of soil-root continuum of microbial communities in support of better conservation and utilization of desert endangered plants in Inner Mongolia".
Line 312-313: Changed "although root and soil microbiomes association have widely been researched in other related endangered plants as well" to "although microbiomes association have widely been researched in other related endangered plants as well".
Line 320-323:Changed "Additionally, the interactions between the fungal and bacterial communities; and soil parameters in the root and soil facilitated the development of intricate networks of species interactions" to "Compared with the rarefaction curves of fungi, the sequencing depth of bacteria was more OTUs, and the variability between rhizosphere and root was less.This may be due to the high variability of ITS sequence, which can not accurately identify all species (23,24)".
Line 327-330: Added "The number of OTU in the bacterial soil samples (2118) was much higher than in the root (314) . It may be due to the interaction between plants and soil microorganisms that plants will provide some nutrients to soil microorganisms, which is conducive to the growth of microorganisms.".
Line 359:Changed"Helianthemum songaricum soil-root continuum of bacterial and fungal communities differences"to"Comparison of the soil-root continuum of bacterial and fungal communities".
Line 377-380: Added "Compared with bacteria, the structure stability of fungal network in rhizosphere and root system was lower, which indicated that the structure of fungal community was prone to change in arid environment, resulting in the loss of fungal diversity.".
Line 395-396:Changed"in the root and soil the dominant bacterial class were Actinobacteria, Acidobacteria, Alphaproteobacteria, Deltaproteobacteria,Betaproteobacteria, Sphingobacteriia, Cytophagia and Gemmatimonadetes (Fig. 2)."to"three bacterial and two fugal class were considered as the host-specific microbes (Fig. S3)" Line 412-413:Added "This may be caused by scattered distribution and uneven colonization of bacteria in rhizosphere soil, which may lead to diversity differences.".
Line 428-434:Added "Rhizosphere and endosphere microorganisms play an important role in plant growth and evolution (53,54). Interestingly, the abundance of Pezizomycetes in rhizosphere soil (31.95%) is significantly higher than that in root system (5.7%), which may be due to the fact that Pezizomycetes is conducive to nutrient absorption by plants in arid and barren soil environment (55,56). Therefore, in order to adapt to environmental changes, plants have attracted some microbial groups, providing them with a habitat conducive to plant growth.". Similarly, the sensitivity of the communities of roots endosphere was far more than rhizosphere soil communities. The bacterial community, while having a lower diversity of species, was more stable than the fungal community because it had a higher number of more integrated and complicated networks and biomarkers. We We have highlighted the changes in yellow according to reviewer #2 comments in the annotated version of the revised manuscript (Revision, changes marked).

I would like to recommend the authors resubmit the manuscript after thorough and appropriate revisions in the texts.
We appreciate the reviewer's insightful comments. According to the comments given by the reviewer, we have appropriate revisions in the texts.
Line43-49:We have revised the sentence "The symbiosis between soil microorganisms and these plants and their interaction with soil factors is an important feature of desert plant adaptation to arid and barren environment. Therefore, make a profound study on the microbial diversity of desert rare plants can provide important data support for the protection and utilization of desert rare plants. Accordingly, in this study, high-throughput sequencing technology was applied to study microbial diversity in the plant root and rhizosphere soils".
Line64：Add a phrase "the propagations of".
Line65-66："can increase the adaptation of plants to extreme environments"instead of "may increase the capacity of applying these relationships to increase the adaptation of plants to extreme environments". Line70-71:"composition in the root and soil" instead of "dominant microbial".
Line71-72:"and the microbial species in the soil were more various than those in the endosphere" instead of "and the main microorganisms in the soil are much more various than in the endosphere".
Line73-75 ： We have revised "the soil-root continuum microbiome of a typical endangered plant, Helianthemum songaricum Schrenk, has not been studied currently, which, little is known about whether or not soil microbial variation is correlated with succession in these endangered plant".
Line79："It has been known that" instead of "Many of the previous findings reported that".
Line82-83："including the improvement of disease and stress resistance capacity, and the acceleration of plant growth rate" instead of "including promoting disease resistance, plant growth, and stress resistance".
Line83-86:"previously" instead of "in the previous literature","is" instead of "are","the external environments, thus, exhibits complex diversity" instead of "the external environment and exhibits complex diversity","structural feature" instead of "structure of the microbial community"."The soil-root continuum" instead of "in the previous literature" Line 89: Added"changes".
Line 94:Changed "among soil-root continuum microbial community groups and different in situ remains unclear" to "different in situ remains unclear".
Line 98-104:Changed "To address these issues, the high-throughput sequencing (Illumina MiSeq) analyzed the fungal and bacterial communities of Helianthemum songaricum Schrenk (in Ordos City of Qipanjing). Also, we identified the core microbiomes and the enriched microbial. The soil-root microbial communities in dry environments are very complicated, and the present study findings will provide light on this complexity for endangered plants" to "To address these issues, we analyzed the fungal and bacterial communities from Helianthemum songaricum Schrenk growing in Ordos City of Qipanjing via high-throughput sequencing technology, enabling direct comparisons between these microbial communities. The main purpose was to reveal the variation of soil-root microbial communities,elucidate the environmental factors that influence the root and soil microbial communities, and provide theoretical reference for the better conservation and utilization of desert endangered plants in Inner Mongolia".
Line 140-144: Added "Raw data has been uploaded and amplicon sequencing data has been deposited. ( 16Sr DNA: Line 149 Thank you again for your comments. We hope we could learn more from you.

Finally, we are very grateful to the reviewer's and editor's understanding and affirmation again. We wish the article can be published in Spectrum.
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Revision Notes
Dear Editor: Thank you for your kind letter of "Spectrum03389-22R1 -Editor decision -revise" on March 6, 2023.
Generally, we appreciate the editor and reviewer's insightful comments, which are helpful for improving the manuscript. We are thankful to reviewer #1, #2 and #3 comments, which are all valuable and very helpful for revising and improving our paper. Based on editor and reviewers' comments and requests, we have made major modification in the revised manuscript. Below is a summary of our responds to the reviewers' comments.
In revision notes, the line numbers refer to the PDF vision of the revised manuscript.
Thank you again for your comments. We hope we could learn more from you.
Editor insightful comments 1. Dada submission. Amplicon sequencing data need to be deposited.
We appreciate the editor insightful comments, and sorry for our negligence. We have listed the amplicon sequencing.
Line140-144: Raw data has been uploaded and amplicon sequencing data has been

Poor writing
We are thankful to the editor's insightful comments.This manuscript have already revised the grammatical errors by a native English speaker.
Thank you again for your comments. We hope we could learn more from you.

Reviewer's insightful comments
Thank you again for your comments. We hope we could learn more from you.
We have highlighted the changes in red according to reviewer #1 comments in the annotated version of the revised manuscript (Revision, changes marked).
1. The study is very primary, the description is unclear, and the conclusion regarding "sensitivities of community" is hard to be supported and even understand.
We appreciate the reviewer's insightful comments. We are very sorry for not clearly describing this content in the previous manuscript. We have added and changed some informative and key sentences in the texts.
Line19-21:We have revised the sentence"We suspect that unknown microorganisms in roots and soil play an important role in the survival strategies of endangered plants".
Line21: "we investigated" instead of "we used Illumina MiSeq high-throughput sequencing to investigate.
Line23-24: We have revised the sentence "and observed that the microbial communities and structures between the rhizosphere and endosphere samples were distinguished".
Line 320-323:Changed "Additionally, the interactions between the fungal and bacterial communities; and soil parameters in the root and soil facilitated the development of intricate networks of species interactions" to "Compared with the rarefaction curves of fungi, the sequencing depth of bacteria was more OTUs, and the variability between rhizosphere and root was less.This may be due to the high variability of ITS sequence, which can not accurately identify all species (23,24)".
Line 327-330: Added "The number of OTU in the bacterial soil samples (2118) was much higher than in the root (314) . It may be due to the interaction between plants and soil microorganisms that plants will provide some nutrients to soil microorganisms, which is conducive to the growth of microorganisms.".
Line 359:Changed"Helianthemum songaricum soil-root continuum of bacterial and fungal communities differences"to"Comparison of the soil-root continuum of bacterial and fungal communities".
Line 377-380: Added "Compared with bacteria, the structure stability of fungal network in rhizosphere and root system was lower, which indicated that the structure of fungal community was prone to change in arid environment, resulting in the loss of fungal diversity.".
Line 395-396:Changed"in the root and soil the dominant bacterial class were Actinobacteria, Acidobacteria, Alphaproteobacteria, Deltaproteobacteria,Betaproteobacteria, Sphingobacteriia, Cytophagia and Gemmatimonadetes (Fig. 2)."to"three bacterial and two fugal class were considered as the host-specific microbes (Fig. S3)" Line 412-413:Added "This may be caused by scattered distribution and uneven colonization of bacteria in rhizosphere soil, which may lead to diversity differences.".
Line 428 Similarly, the sensitivity of the communities of roots endosphere was far more than rhizosphere soil communities. The bacterial community, while having a lower diversity of species, was more stable than the fungal community because it had a higher number of more integrated and complicated networks and biomarkers. We identified the core microbes and host-specific microbes, which increased the understanding of the soil-root continuum microbial communities of Helianthemum songaricum. Also, soil-root continuum microbiomes of Helianthemum songaricum were significantly affected by the key potential drivers of AP, AN, OM and pH. The findings in the present study will spawn new theoretical advancements and developments on the microbial community implications regarding the diversity in the adaptation of endangered plants to arid conditions." to "This study was the first to report the soil-root continuum environment and its variation driving factors for the diversity of fungal and bacterial communities in the natural environment of Helianthemum songaricum. Bacterial dominant class were Actinobacteria 、 Acidobacteria and Alphaproteobacteria, and fungal dominant phyla were class were Sordariomycetes and Pezizomycetes. At the taxonomic level and individual OTU level, there are differences in the abundance of bacteria and fungi in the root and rhizosphere of Helianthum songaricum, indicating that the microbial community has a unique niche in plants and soil. Also, soil-root continuum microbiomes of Helianthemum songaricum were significantly affected by the key potential drivers of pH, organic matter and ammonium nitrogen. Helianthemum songaricum is an ancient and endangered species. Therefore, this study provides a theoretical basis for the future research and protection of the endangered plant biodiversity by studying the microbial community diversity of desert endangered plants. In the future research, the enriched in the corresponding group and have significant influence on the difference between groups. The light yellow nodes represent microbial groups that have no significant difference between groups, or have no significant effect on differences between groups. Circles indicate phylogenetic levels from Phylum to species. The diameter of each circle is proportional to the abundance of the group.

Fig6: Suggest providing the p values and indicates correlation with p<0.05.
We appreciate the reviewer's insightful comments. According to the comments given by the reviewer, we have appropriate revisions in the texts.
Note: The phylogenetic evolutionary tree is on the left. Each branch in the evolutionary tree represents a species. The branches are colored according to the advanced taxonomic level to which the species belongs. The bar chart on the right shows the proportion of Reads of species in different groups.
Thank you again for your comments. We hope we could learn more from you.
Finally, we are very grateful to the reviewer's and editor's understanding and affirmation again. We wish the article can be published in Spectrum.