Analysis of Microbial Community Structure Around Roots of Stipa Grandis

The root zone microbial structure is particularly complex for plants with rhizosheaths, which may play an important role in the future agricultural sustainable development. However, one of the important reasons for restricting our study of rhizosheath microbial structure is that there is no denite method for rhizosheath separation. The aim of this study was to explore the isolation methods of rhizosheath and the diversity and functional characteristics of microorganisms around the rhizosphere. In this study, we isolated the rhizosheath of Stipa grandis, a dominant species in desert steppe, and the microorganisms in the roots, root epidermis, rhizosheath, rhizosphere soil were extracted and sequenced by 16s RNA and ITS. The bacterial alpha diversity index was in the order rhizosphere soil > rhizosheath > root epidermis > endophytic, and the fungal alpha diversity index was rhizosphere soil and rhizosheath > root epidermis and endophytic. There were signicant differences in bacterial community structure between the root epidermis and endophytic, rhizosheath, rhizosphere soil, and the sum of relative abundance of the dominant bacterial populations Actinobacteria and Proteobacteria was 73.9% in the root epidermis. Different from bacterial community structure, the community structure of root epidermis fungi was similar to endophytic, but signicantly different from rhizosheath and rhizosphere soil. We suggest that the root epidermis can act as the interface between the host plant root and the external soil environment. This study will provide theoretical and technical guidance for the isolation of plant rhizosheath and the study of microorganisms in it. plant growth. This study will provide a new method and theoretical guidance for further exploring the function and ecological signicance of rhizosheath. This study provides a feasible method to separate rhizosheath and root epidermis. This will provide the possibility for further study on the microorganism of rhizosheath and root surface. There are signicant differences in bacterial diversity between root epidermis and roots, rhizosheath and rhizosphere soils, suggesting that some functions of root epidermis and rhizosheath may play an important but previously ignored role. We suggest that the root epidermis can be used as a compartment to separate the root system from the soil and that it may act as the interface between the host and the external environment, with microorganisms in this interface potentially having some important functions.


Introduction
The close interaction between plants and rhizosphere microorganisms has prompted people to regard plants as a superorganism (Cornéet al. 2014). Research on plant root -rhizosphere soil microorganisms aims to identify correlations between plant -soil -microorganisms and reveal their important roles in the ecosystem. However, the relationship between the root and the soil around the root of some plants is more complex, and these plants are plants with rhizosheaths. For example, barley (George et al. 2014), wheat (Delhaize et al. 2015), corn (Duell and Peacock 1985), rushes (Shane et al. 2009(Shane et al. , 2011, etc. even some scholars have proposed that there is a rhizosheath on the ne roots of some leguminous plants (Sprent 1975;Unno 2005). However, McCully (1999) suggest that this needs further research and con rmation. It has been more than 100 years (Volkens 1887) since the rhizosheath was rst described, initiating study of its structure (Bailey 1997), formation, function and genetic characteristics.
However, little is known about it even to this day.
Rhizosheath is particularly obvious in the root structure of Gramineae in arid areas. Price (1911) and young (1995) believe that the rhizosheath plays an important role in increasing the drought resistance of plants, and existing research supports this view (Pate and Dixon, 1996;Shane et al. 2010; Benard et al. 2016). This understanding is of great signi cance for improving agricultural sustainability in the context of future climate change, limited resources and a growing global population. Some scholars believe that the rhizosheath plants may play an important role in the second green revolution and future agricultural sustainable development (Lynch 2007;Brown et al. 2017).
The research on the structure and function of plant rhizosphere microorganisms has always been a hot topic, although most microorganisms in the environment have not been cultured, with the rapid development of highthroughput sequencing technology, the structure of microorganisms in the environment is gradually becoming known. Previous studies have shown that there are some differences in the composition of plant root microorganisms and rhizosphere and non-rhizosphere soil microorganisms, which directly affect the normal growth of host plants. For example, some high concentrations of molecules released by rhizosphere microorganisms inhibit the elongation of primary roots and promote the formation of lateral roots and root hairs (Zhang et al. 2017). Some rhizosphere bacteria or fungi produce auxin, which directly interferes with auxin signal transduction (Spaepen et al. 2014 Vries (2020) found little evidence for a coupling relationship between the drought tolerance mechanism of microorganisms and the functional characteristics of plant drought resistance, highlighting the need for further research. There are few reports on the structure and function of microorganisms in plant rhizosheath. Therefore, it will be a challenge to study the microorganisms around the rhizosheath plants York et al. (2016) summarized and de ned the generation process and semantics of "rhizosphere", and considered that rhizosheath is a mixture of soil particles adhered by mucus (the secretion of plant roots or microorganisms) (Volkens 1887;George et al. 2014). The epidermal cell layer attached to the rhizosheath was not a part of the rhizosheath, and they called the combination of the epidermal cell layer and the rhizosheath as "rhizoplane" (York et al. 2016). The di culty of rhizosheath separation is the separation of rhizosheath and root epidermis. And there is no standard method to isolate microorganisms from rhizosheath. In this study, using Stipa grandis as experimental material, we isolated the roots, root epidermis, rhizosheath and rhizosphere soil of Stipa grandis, extracted microbial DNA from roots, root epidermis, rhizosheath and rhizosphere soil, and sequenced 16S RNA and ITS. The purpose of our research is to explore the isolation methods of rhizosheath and root epidermis, and analyze the similarities and differences of microbial communities among the root, root epidermis, rhizosheath and rhizosphere soil of Stipa brevi ora, so as to provide new methods and suggestions for the future research of rhizosheath plants.

Materials And Methods
Overview of the research site Wear sterile gloves and sterilize the workspace with 70% EtOH. The forceps and scissors used in the experiment were wiped and disinfected with 70% EtOH. 3 Stipa plants were selected from each sampling site, and remove loose soil by kneading and shaking by hand and by patting the roots on the back of a gloved hand. The shaken soil is the rhizosphere soil sample. The soil that is not shaken off and adheres to the root surface is de ned as rhizosheath soil (Fig. 1). (2) Mixing and puri cation of PCR products The PCR product was detected by electrophoresis with 2% agarose gel. According to the concentration of PCR product, the samples were mixed equally, and then the PCR products were detected by agarose gel electrophoresis with 2% agarose gel. The gel recovery kit provided by Qiagen company was used to recover the target band.
(3) Library construction and sequencing Library construction used TruSeq® DNA PCR-Free Sample Preparation Kit (Building Database Kit). The library was quanti ed by qubit and Q-PCR, and after the library was quali ed, it was sequenced by novaseq6000.
Data analysis based on the Analysis Platform of Novogene company (1) Sequencing data processing According to the barcode sequence and PCR ampli cation primer sequence, each sample data was separated from the o ine data. After the barcode and primer sequences were cut off, the reads of each sample were spliced with  (Fig. 2, c, d). Table 1 shows that the number of Observed_species, Chao1 index and ACE number index were highest in rhizosphere soil, followed by rhizosheath soil, and were smallest in the root system, and there were signi cant differences between them. There was no signi cant difference in the Shannon index between rhizosphere soil and rhizosheath soil, which were both signi cantly higher than the Shannon index in root epidermis and roots, but the Shannon index of bacteria in root epidermis was signi cantly higher than in roots. The Simpson index of bacteria in rhizosphere soil, rhizosheath soil and root epidermis had no signi cant differences, but were signi cantly higher than those in the root system. The goods_coverage index of the samples was higher than 98%.
The observed_species, Shannon, Simpon, Chao1, ACE, PD_whole_tree index of fungi in rhizosphere soil were the highest, followed by rhizosheath soil, but there was no signi cant difference between the two groups. The observed_species, Shannon, Simpon, Chao1, ACE, index of rhizosphere soil and rhizosheath soil fungi were signi cantly larger than the root epidermis and root. There was no signi cant difference in observed_species, Shannon, Simpon, Chao1, ACE index between roots and root epidermis. The good_coverage indexes of the samples were all higher than 99%. The top 10 phylum level classi cation of bacterial communities is shown in Fig. 3 (a). The relative abundance of Cyanobacteria in the root system was 46.4%, while the relative abundance of Cyanobacteria in rhizosphere, rhizosphere and non-rhizosphere soil was only 5.0%, 2.1% and 1.1%, respectively. Actinobacteria was the dominant population in root epidermis, and its relative abundance was 43.0%, which was signi cantly higher than that in the root system, rhizosheath soil and rhizosphere soil. The relative abundance of Proteobacteria in root epidermis, rhizosheath soil and rhizosphere soil was 30.9%, 31.3% and 30.2%, respectively, while that in roots was only 11.7%.
The relative abundances of Acidobacteria, Gemmatimonadetes, Bacteroidetes and Verrucomicrobia in rhizosheath soil and rhizosphere soil were similar and signi cantly higher than those in root systems and root epidermis. The relative abundances in rhizosheath soil were 22.1%, 9.2%, 8.2% and 3.2%, respectively, and those in rhizosphere soil were 21.8%, 9.6%, 7.6% and 3.7%, respectively.
The top 10 phylum level classi cation of the sample fungal communities is shown in Fig. 3 (b). Different from bacterial communities, the analysis shows that Basidiomycota and Ascomycota have higher relative abundance in roots, root epidermis, rhizosheath and rhizosphere soil. The relative abundance of Basidiomycota in roots and root epidermis are 72.3% and 70.3%, respectively, which is signi cantly higher than in rhizosheath soil and rhizosphere soil. The relative abundance of Ascomycota in roots, rhizosheath and rhizosphere soil was 20.4%, 22.4% and 27.6%, respectively, while that in root epidermis was only 8.7%. The relative abundance of Others was in the order rhizosphere soil > rhizosheath soil > root epidermis > roots.
Results of non-metric multidimensional scaling (NMDS) analysis of bacteria in roots, root epidermis, rhizosheath and rhizosphere soil of Stipa grandis are shown in Fig. 3 (c). The microbial community structure of root epidermis, rhizosheath soil and rhizosphere soil are distinct and distant in the plot, while the distance between rhizosheath soil and rhizosphere soil is relatively close. The UPGMA cluster analysis of all samples also showed that the similarity of bacterial composition and relative abundance between rhizosheath soil and rhizosphere soil was high, and that there was a big difference in bacterial composition between soil, roots and root epidermis.
The results of NMDS analysis of fungi are shown in Fig. 3 (d). The fungal community structure of roots and root epidermis, and rhizosheath and rhizosphere soil were relatively close, while that of roots and root epidermis were relatively distinct from those of rhizosheath and rhizosphere soil. Through UPGMA cluster analysis of all the samples, in contrast to bacterial community clustering, rhizosheath soil and rhizosphere soil fungal communities can be clustered into one group, while root and root epidermis can be clustered into another group.
As shown in Table 2, the bacterial community composition in rhizosheath soil and rhizosphere soil of Stipa grandis was not signi cantly different (P > 0.05). The bacterial community composition of roots and root epidermis of Stipa grandis was signi cantly different at P < 0.05, and the differences in bacterial community among other groups was extremely signi cantly different (P < 0.01).
There was no signi cant difference in fungal communities between the roots and root epidermis of Stipa grandis or between rhizosheath soil and rhizosphere soil (P > 0.05). The difference of fungal community among other groups reached an extremely signi cant level (P < 0.01).

Discussion
Rhizoplane refers to the outer surface of plant roots and any closely attached soil or debris particles, which was proposed by Clark (1949 incorrect to call root epidermis as rhizoplane, which would greatly reduce the spatial range of rhizosphere and he agreed with Clark's (1949) de nition of rhizosplane. The results showed that there were signi cant differences in the composition of bacterial and fungal communities in root epidermis and rhizosheath, which could be inferred that they played different but important roles in plant growth. We agree with Clark (1949)  Our results showed that the bacterial and fungal community structure of root was signi cantly different from that of rhizosheath soil and rhizosphere soil, and although there was no signi cant difference in fungal community between root and root epidermis, there were differences in bacterial community. In addition, the total relative abundance of Actinobacteria and Proteobacteria in root epidermis was 73.9%, which was signi cantly different from that in root and soil. This indicates that there are different microbial community systems in root, root epidermis, rhizosheath and rhizosphere soil of rhizosheath plants, and our method of rhizosheath separation is feasible. Both sides of root epidermis are root system and rhizosheath, which can be used as a compartment to separate root system from soil. In terms of rhizosphere microbial ecology, Philippot (2013) mentioned that rhizosphere is the interface between plant roots and soil, and the rhizosphere environment is complex and dynamic. The interaction between various microorganisms affects plant growth and tolerance to biotic and abiotic stresses. However, we suggest that for plants with rhizosheath, the outer root epidermis is the interface between the root system and the external soil.
In In this study, phosphate buffer was used as a protective substance to separate roots, root epidermis, rhizosheath, and rhizosphere soil, which seems to be able to clarify the relationship between rhizosheath, root epidermis and microbial differences in root. Bergmann (Wullstein et al. 1979;Wullstein 1980Buckley 1982. There are few studies on other functions of microorganisms in root epidermis, which may be limited by experimental techniques. The differences in composition and function of microorganisms inside and outside the rhizosheath may prompt us to reexamine its potentially important role in plant growth. This study will provide a new method and theoretical guidance for further exploring the function and ecological signi cance of rhizosheath.

Conclusion
This study provides a feasible method to separate rhizosheath and root epidermis. This will provide the possibility for further study on the microorganism of rhizosheath and root surface. There are signi cant differences in bacterial diversity between root epidermis and roots, rhizosheath and rhizosphere soils, suggesting that some functions of root epidermis and rhizosheath may play an important but previously ignored role. We suggest that the root epidermis can be used as a compartment to separate the root system from the soil and that it may act as the interface between the host and the external environment, with microorganisms in this interface potentially having some important functions. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
All authors declare that they have no competing interests.  Root system of Stipa grandis and schematic diagram of root cross section and separation of root-root epidermisrhizosheath-rhizosphere soil. In gure 1, the left picture is the picture of the root system of Stipa grandis, the top right is the schematic diagram of the cross section of the root system, and the four gures in the lower right corner are the pictures of the root system, root epidermis, rhizosheath and rhizosphere soil separated from the root system.

Figure 2
Venn diagrams of samples and rarefaction curves for samples. In gure 2, a and b are Venn diagrams of bacteria and fungi, and c and d are OTU dilution curves of bacteria and fungi respectively. In Fig. 2(a, b), each circle in the Venn diagram represents a group of samples. The number of overlapped parts represents the number of OTUs shared between groups, and the number without overlap represents the number of OTUs unique to the sample group. In Fig. 2 (c, d), the abscissa is the number of sequencing pieces randomly selected from a sample, and the ordinate is the number of OTUs that can be constructed based on the number of sequencing pieces to re ect the sequencing depth. SG.EC, SG.RS, SG.RH, SG.SO refer to roots, root epidermis, rhizosheath, and rhizosphere soil of Stipa grandis, respectively.