Variation of soil microbiome in licorice rhizosphere driven with inoculating dark septate endophytes under drought stress

20-μL reaction system containing 4 μL of 5× FastPfu Buffer (for 16s v3-v4) / 2 μL of 10× Buffer (for ITS), 2 μL dNTPs, 0.8 μL of the aforementioned forward and reverse primers, 0.4 μL FastPfu Polymerase (16S v3-v4) / 0.2 μL rTaq polymerase (ITS), 0.2 μL bovine serum albumin (BSA), and 10 ng template DNA. PCR ampli�cation was conducted using the following thermal program: initial denaturation at 95 °C for 3 min followed by 28 denaturation cycles at 95 °C for 30 s, annealing at 55 °C for 30 s, elongation at 72 °C for 45 s, and a �nal extension at 72 °C for 10 min. The PCR products were detected by gel electrophoresis (2% (w/


Abstract
Background Dark septate endophytes (DSE) are facultative biotrophic ascomycetes that colonize plant roots either alone or with arbuscular mycorrhizal (AM) fungi.DSE may provide nutrients to their plant hosts and help them adapt to various abiotic and biotic stresses.DSE inoculation under drought stress increased the biomass, root exudates, and AM fungi in the licorice (Glycyrrhiza uralensis Fisch.)rhizosphere.We conducted a pot experiment to establish whether the responses of licorice to DSE inoculation under drought stress are caused by changes in the rhizosphere microbiome.Each pot was inoculated with either Acrocalymma vagum or Paraboeremia putaminum.One set of pots was inoculated with a sterile culture medium.All three DSE-treated and uninoculated pots were subjected either to a well-watered (70% eld water capacity, FWC) or drought stress (30% FWC) water regime.Rhizosphere microbiome compositions were measured by Illumina MiSeq sequencing of the 16S and ITS2 rRNA genes.

Results
In total, 1,278 fungal and 1,583 bacterial operational taxonomic units (OUTs) were obtained at a 97% sequence similarity level.Ascomycota were the predominant fungi and Proteobacteria, Actinobacteria, Chloro exi and Firmicutes were the predominant bacteria.DSE inoculation and water regime signi cantly in uenced the rhizosphere microbiome composition.However, the effects of DSE on the fungal community were greater than those on the bacterial community.Paraboeremia putaminum exerted a stronger impact on the licorice rhizosphere microbiome than Acrocalymma vagum under drought stress.The observed changes in edaphic factors (water condition, soil organic matter, available N, available P, and available K) caused by DSE inoculation could be explained by the variations in rhizosphere microbiome composition.A network analysis indicated that DSE inoculation augmented the relative abundance of bene cial symbiotrophic fungi and growth-promoting bacteria but diminished the relative abundance of pathogens in the licorice rhizosphere.

Conclusions
The present study showed that the licorice rhizosphere microbial community differed between the DSEinoculated and uninoculated plants.DSE had a stronger in uence on the fungal than on the bacterial rhizosphere community under drought stress.These give us the guidance to develop biofertilizers with DSE consortia to enhance the cultivation of medicinal plants by shaping soil microbial community structure in dryland agriculture.

Background
Drought is a major abiotic stressor that limits plant growth and agricultural production in arid and semiarid regions worldwide [1,2].Water de cit inhibits root growth and development and decreases plant nutrient and water uptake from the soil [3].Under drought stress, plants modify their root exudate abundance and composition [4].This response alters the rhizosphere microbiome, which, in turn, strongly in uences plant survival and growth and soil ecology [5].Several studies have shown that plant adaptation to abiotic stress is closely associated with rhizosphere microorganisms [6,7].Bene cial endophytic fungal colonization can alter root growth, increase plant biomass, reduce water loss, and help host plants adapt to arid environments [8,9].Moreover, dark septate endophytes (DSE) enhance viral resistance and heavy metal tolerance in plants [10,11].DSE are ubiquitous root-colonizing fungi characterized by dark, septate hyphae and melanized microsclerotia.They are integral and functional parts of plant roots [12].DSE inoculation facilitates plant growth, nutrient uptake, and biotic and abiotic stress tolerance [8,11].Previous studies demonstrated that rhizosphere-associated microbes indirectly affect plant growth by changing the rhizosphere microbial community composition and activity.These communities play key roles in soil nutrient cycling and structural formation [13,14].Fiorentino et al. [15] found that Trichoderma inoculation more strongly affected the eukaryotic community composition of low-N than N-fertilized soils.Changey et al. [16] found that arbuscular mycorrhizal (AM) fungal inoculation dramatically in uenced the rhizosphere microbial community composition.Tian et al. [17] reported that Glomus intraradices inoculation increased certain bene cial bacterial species and decreased certain pathogenic fungi in the Panax ginseng rhizosphere.Han et al. [18] proposed that Bacillus amyloliquefaciens promotes cucumber growth by modulating its rhizosphere microbial community composition.The direct effects of bene cial rhizosphere microbes on plant growth have been extensively examined [19,20].Nevertheless, there is relatively little information on the impact of DSE inoculation on the rhizosphere microbial community and its network structure.
Licorice (Glycyrrhiza uralensis Fisch.)belongs to Fabaceae.It is widely distributed in arid and semi-arid regions worldwide.It is regarded as an "essential herbal medicine" in traditional Chinese medicine and has been prescribed and administered for > 1,000 y.Its active principles include glycyrrhizin and glycyrrhizic acid [21].Licorice adapts very well to low-fertility soils and drought.In fact, it has been used in the ecological restoration of arid regions [22].In its natural habitats, the licorice rhizosphere harbors actinobacteria, rhizobia, other soil bacteria, AM, and DSE with plant growth-promoting activity [23,24].An ester-linked phospholipid fatty acid (PLFA) analysis in our previous study showed that DSE (Acrocalymma vagum and Paraboeremia putaminum) inoculation increased AM and gram-negative bacterial abundance in the licorice rhizosphere under drought stress [8].However, its effects on the composition of the microbial communities in the licorice rhizosphere remain unclear.Rhizosphere microbes play vital roles in plant growth, health, and abiotic stress tolerance.Here, we used Illumina MiSeq sequencing to reveal variations in the composition of the licorice rhizosphere microbial communities inoculated with DSE under drought stress.We hypothesized that DSE inoculation considerably alters the composition and network structure of the fungal and bacterial communities in the licorice plant rhizosphere.Furthermore, these changes are more pronounced under drought stress than well-watered conditions.Our objective was to test whether DSE inoculation alters the soil microbiome and increases drought tolerance in licorice.

Characterization of Illumina sequencing data
We obtained 2,304,715 fungal and 2,098,398 bacterial sequences.Rarefaction curve analysis displayed high 16S rRNA gene sequencing depth and strong potential for observing community diversity in each licorice rhizosphere (Fig. 1).Rank abundance curves showed that all six treatments had high species evenness and homogeneity (Fig. 2).The sequencing results covered the biological information of most of the fungi and bacteria in the soil samples.

Rhizosphere microbial diversity and richness
Under well-watered, inoculation with either P. putaminum or A. vagum increased the soil Simpson fungus index compared to the uninoculated condition.The A. vagum inoculation decreased the soil fungus Ace and Chao1 indices.Compared with well-watered, drought stress decreased the diversity and richness of the fungal community colonizing the licorice rhizosphere.Compared to the uninoculated condition, P. putaminum inoculation increased the soil fungus Shannon, Ace, and Chao1 indices but decreased the soil fungus Simpson index.However, A. vagum inoculation increased the soil fungus Ace and Chao1 indices (Table 1).DSE inoculation signi cant affected soil bacterial diversity and richness under all water regimes (Table 2).Under well-watered, DSE inoculation increased the soil bacteria Chao1 index but decreased the soil bacteria Simpson index compared with the uninoculated condition.Under drought stress, P. putaminum inoculation increased the soil bacteria Ace and Chao1 indices whereas A. vagum inoculation had no signi cant effect on soil bacterial community diversity and richness.Compared with the uninoculated condition, drought stress decreased the soil bacteria Simpson index.
Nonmetric multidimensional scaling (NMDS) ordination revealed that the rhizosphere fungal community composition signi cantly differed between the DSE inoculation and uninoculated treatments under wellwatered (Fig. 6).Compared to the uninoculated condition, P. putaminum inoculation substantially affected fungal community composition under drought stress.No dramatic effect was observed in response to A. vagum inoculation (Fig. 6).DSE inoculation signi cantly affected the composition of the bacterial community under well-watered.The A. vagum and P. putaminum inoculation had different effects (Fig. 6).Under drought stress, P. putaminum inoculation signi cantly affected the bacterial community composition relative to the uninoculated condition.In contrast, A. vagum inoculation had no signi cant impact on the bacterial community composition (Fig. 6).Permutational multivariate analysis of variance (PerMANOVA) indicated that the fungal (F = 7.435, R 2 = 0.264, P = 0.001; F = 6.224,R 2 = 0.331, P = 0.001) and bacterial (F = 6.125,R 2 = 0.392, P = 0.001; F = 5.648, R 2 = 0.440, P = 0.001) community compositions were signi cantly different between the well-watered and the drought stress treatments.Furthermore, drought stress and DSE inoculation more strongly affected the fungal than the bacterial community composition.

Structure of various treatment-rhizosphere microbe networks
The various treatment-rhizosphere fungal networks are shown in Fig. 8.Under well-watered, P. putaminum inoculation increased the relative symbiotroph abundance but decreased the relative abundance of saprotroph and other fungi.In contrast, A. vagum inoculation increased the relative abundance of symbiotrophs and other fungi and decreased the relative abundance of pathotrophs and saprotrophs compared with the uninoculated condition.Drought stress decreased the relative abundance of symbiotrophs, saprotrophs, and other fungi but increased the relative abundance of pathotrophs compared with the well-watered condition.However, P. putaminum inoculation increased the relative abundance of symbiotrophs and other fungi and decreased the relative abundance of pathotrophs.Moreover, A. vagum inoculation increased the relative abundance of symbiotrophs, saprotrophs, and other fungi and decreased the relative abundance of pathotrophs compared with the uninoculated condition.
The network of different treatments-rhizosphere bacteria is shown in Fig. 8. Bacillus,Pseudarthrobacter OTU3533, and other bacteria had variable relative abundance under different treatments.Oscillatoria was only distributed under NCK.Microbacterium OUT1007 was distributed under NCK, NAV, DCK, and DAV.Inoculation with A. vagum decreased the relative abundance of Oscillatoria compared with the uninoculated condition.Nocardioides was only distributed under DCK and DAV.Sporocytophaga was only detected under DAV.Arcnimonas only occurred under NAV and DAV.Ensifer was found under NPP, NAV, and DPP.Pedomicrobium was only seen under NPP.
The network of various treatment-rhizosphere microbe functional group is shown in Fig. 8. Compared to the uninoculated treatment, under well-watered, DSE inoculation increased the relative abundance of bene cial and neutral fungi and bacteria and decreased the relative abundance of pathogenic fungi and bacteria.Relative to the uninoculated condition, under drought stress, P. putaminum inoculation decreased the relative abundance of pathogenic fungi and bene cial bacteria but increased the relative abundance of neutral fungi and neutral and pathogenic bacteria.The A. vagum inoculation increased the relative abundance of bene cial fungi and neutral bacteria but decreased the relative abundance of pathogenic fungi and bacteria.

Discussion
The plant rhizosphere houses a complex microbiome that includes bacteria, archaea, and fungi.These microorganisms affect plant survival, growth, and adaptability [25,26].Here, we used Illumina MiSeq sequencing to reveal the changes in microbial community diversity and network structure in the rhizosphere of licorice inoculated with DSE under drought stress.The relative abundance of microbes colonizing licorice rhizosphere differed among treatments.However, Ascomycota, Proteobacteria, Actinobacteria, Chloro exi, and Firmicutes were the dominant phyla under both well-watered and drought conditions.The predominance of Ascomycota in arid and semi-arid regions was previously reported [27,28].Dai et al. [29] found that Actinobacteria, Proteobacteria, Saccharibacteria, Chloro exi, Acidobacteria, and Cyanobacteria were the predominant phyla in drought-treated and untreated peanut rhizosphere.Lundberg et al. [30] observed that Proteobacteria, Bacteroidetes, Actinobacteria, Acidobacteria, Firmicutes, Gemmatimonadetes, and Cyanobacteria dominated in Arabidopsis rhizosphere.Barraza et al. [31] also reported that the bacterial community structure of the common bean roots, mainly composed by Proteobacteria, Actinobacteria, Bacteroidetes, Acidobacteria, and Firmicutes.The major rhizosphere microbes vary widely among plant species.Nevertheless, Actinobacteria and Proteobacteria may be the most common bacterial phyla in plant rhizospheres.
Biotic and abiotic stressors alter rhizosphere microbe community structure and may augment or diminish certain microbial populations [32,33].He et al. [8] found that interactions between DSE inoculation and water regime markedly in uenced the soil organic matter content.Compared to the uninoculated condition, A. vagum inoculation under drought conditions increased soil available N and P. In contrast, P. putaminum inoculation decreased soil organic matter and available N relative to the uninoculated condition.The microbes colonizing the licorice rhizosphere exhibited distinct preferences for various soil factors.For instance, Phialophora OTU1296, Gibberella OTU1913, and Pseudarthrobacter OTU3533 showed a positive moisture preference whereas Vermispora OTU575, Xanthomonadales OTU7347, Actinobacteria OTU1174, and Streptomyces OTU5992 displayed a negative moisture preference.The norank-f-Anaerolineaceae OTU1186, norank-f-Sandaracinaceae OUT476, Mycoarthris OUT1539, and norank-c-Cynobacteria OTU5893 showed a positive soil organic matter preference while Lysinibacillus OTU9205 presented with a negative soil organic matter preference.These ndings were consistent with those previously reported that microbial inoculation broadly in uences plant rhizosphere microbial communities by altering soil chemical properties and indirectly affecting host plant growth [34,35].
Here, NMDS ordination disclosed that P. putaminum inoculation had a signi cant effect on fungal community composition under drought stress.However, no signi cant difference was found between A. vagum inoculation and the uninoculated condition even though A. vagum inoculation increased the fungal community Ace and Chao1 indices in the licorice rhizosphere.The P. putaminum inoculation increased the relative abundance of Basidiomycota and Zygomycota but decreased the relative abundance of Ascomycota in the licorice rhizosphere under drought stress.Previous studies showed that the three aforementioned fungal phyla predominated in different ecological environments [36].Certain Ascomycota including DSE form mycorrhizae in plant roots and enhance plant nutrient uptake and growth [37,38].Lin et al. [39] found that Ascomycota, Basidiomycota, and Zygomycota strongly tolerated heavy metal contamination.Moreover, Proteobacteria, Actinobacteria, Chloro exi, Firmicutes, and Acidobacteria predominated in the licorice rhizosphere under drought stress regardless of DSE inoculation.Compared to the uninoculated condition, inoculation with P. putaminum increased the relative abundance of Acidobacteria, Chloro exi, and Cyanobacteria but decreased the relative abundance of Actinobacteria.The A. vagum inoculation increased the relative abundance of Cyanobacteria and Bacteroidetes.Khan et al. [40] reported that Acidobacteria, Actinobacteria, Bacteroidetes, and Proteobacteria were highly abundant in medicinal plant rhizosphere microbiomes in arid soil.Proteobacteria, Acidobacteria, and Bacteroidetes had high heavy metal tolerance [41].Sanguin et al. [42] found that Proteobacteria enrichment increased the disease suppression capacity of the rhizosphere.Singh [43] reported that Cyanobacteria improved the soil environment and survived in arid soil by accumulating soil carbon and nitrogen.The P. putaminum inoculation increased Chloro exi abundance which was consistent with a previous study on micro ora in a biofertilizer soil [44].Wu et al. [45] found that Chloro exi does not produce oxygen during photosynthesis and inhibits nitrogen xation.In contrast, Acidobacteria decompose cellulose and modulate soil pH.Hence, the enrichment of speci c rhizosphere fungi and bacteria might enable plants to maintain active microbiomes that improve survival under drought stress [46].
It was unknown whether DSE-mediated changes in the rhizosphere microbial communities augment drought stress tolerance in licorice.Our previous study showed that DSE inoculation (A.vagum and P. putaminum) improve licorice growth and survival under drought conditions [8].In the present study, a network structure analysis disclosed that both soil fungal and bacterial networks were characterized by high specialization and modularity.Drought stress markedly affected the relative abundance of various microbial functional groups.Compared to the uninoculated condition under drought stress, P. putaminum and A. vagum inoculation increased symbiotrophic fungi by 78.2% and 34.6% and saprotrophic fungi by 0.4% and 12.1% and decreased pathotrophic fungi by 125.6% and 44.5%, respectively.Microascaceae, Trichoderma, and Aspergillus were the dominant fungi in DCK, Chaetomium and Pseudalescheria predominated in DPP, and Trichoderma and Aspergillus prevailed in DAV.The Microascaceae include both saprobes and plant pathogens.Certain species are intrinsically resistant to antifungal agents [47,48].Trichoderma is an effective biofertilizer, soil amendment, and biocontrol agent [49].Certain Aspergillus species such as A. avus are facultative plant pathogens under drought stress and can produce considerable amounts of a atoxin [50].In contrast, A. niger and A. fumigatus are metallotolerant [41].Chaetomiaceae degrade cellulose in the soil and increase soil organic matter [51].Pseudallescheria (Scedosporium) spp.are global pathogens that resistant most antifungal agents [52].
Compared to the fungal community, DSE inoculation and the water regime had less in uence on the composition of the bacterial community colonizing the licorice rhizosphere.Bacillus, Microbacterium, Nocardioides, and Pseudarthrobacter predominated and constituted 78.6% of the total abundance under the DCK treatment.Bacillus, Pseudarthrobacter, and Ensifer occupied 54.6% of the total abundance under the DPP treatment.Bacillus, Microbacterium, Nocardioides, Pseudarthrobacter, Sporocytophaga, and Arenimonas comprised 81.3% of the total abundance under the DAV treatment.These bacteria have high drought stress tolerance and most of them promote plant growth [53][54][55].Bacillus species are also effective biocontrol bacteria [56].Pseudarthrobacter and Sporocytophaga e ciently degrade cellulose, crude oil, and multibenzene compounds [57,58].Compared to the uninoculated condition, P. putaminum and A. vagum inoculation increased the relative abundance of bene cial (2.5% and 56.3%) and neutral (86.4% and 4.1%) fungi but decreased the relative abundance of pathogenic fungi (178.5% and 50.7%) under drought stress.Compared to the uninoculated condition, P. putaminum inoculation increased the relative abundance (107.4% and 55.4%) of neutral and pathogenic bacteria but decreased the relative abundance (92.8%) of bene cial bacteria under drought stress.The A. vagum inoculation increased the relative abundance (45.8%) of neutral bacteria but decreased the relative abundance (128.6%) of pathogenic bacteria.Our results indicated that DSE inoculation alters the licorice rhizosphere microbiome community composition.It enriched bene cial and neutral fungi and reduced harmful fungi under drought stress.Hence, DSE inoculation may be an important modality for the improvement of plant growth and drought resistance [8, 59,60].

Conclusion
DSE inoculation and water regime markedly affected the composition and diversity of the microbial communities colonizing licorice rhizospheres, and such impact on fungal community was greater than bacterial community.Of the two DSE species, P. putaminum exerted a stronger in uence on the licorice rhizosphere microbiome than A. vagum under drought stress.The edaphic factor changes caused by DSE inoculation and water regime partially account for the observed variations in licorice rhizosphere microbiome.DSE inoculation under drought stress enriched bene cial symbiotrophic fungi and growthpromoting bacteria but decreased the relative abundance of licorice rhizosphere pathogens.In this manner, it promoted licorice growth, strengthened pathogen resistance and drought tolerance, and facilitate licorice survival under drought stress.These guide us to develop e cient and ecofriendly biofertilizers with symbiotic fungal consortia for the cultivation of medicinal plants based on the soil characteristics and the microbial community that it harbors in dryland agriculture.The substrate used for the pot experiment was a 1:2 (w/w) mixture of sand (< 2 mm) and soil collected from the arid arable land of Northern China whereupon the licorice plants were naturally distributed.The physicochemical properties of the substrate were organic matter content, 21.57mg g -1 ; available nitrogen (N), 85.19 mg kg -1 ; and available phosphorus (P), 7.90 mg kg -1 .
Two licorice seedlings were transplanted to a sterile plastic pot (13 cm diameter × 12 cm height) lled with 800 g of nonsterile growth substrate.For the DSE inoculation treatments, 600 g of the growth substrate was poured into a pot, and 5-mm plugs were excised from the edges of actively growing DSE colonies on the culture media.The plugs were inoculated at 1-cm intervals near the licorice seedling roots.Then, 200 g of the growth substrate was added.For the uninoculated control, two plugs were excised from the fungus-free sterile medium and inoculated near the licorice seedling roots in each pot.The entire inoculation process was performed on a clean bench.All pots were maintained in a growth chamber under a 14 h/10 h photoperiod, 27 °C/22 °C (day/night), and 60% mean RH.
After 1 mo, half the DSE-inoculated and uninoculated pots were subjected to the WW treatment (70% eld water capacity), while the balance were subjected to the DS treatment (30% eld water capacity).Drought stress was applied according to the median soil moisture content recorded for the natural licorice habitat in Northern China.The soil moisture content in each pot was measured using a soil humidity recorder (L99-TWS-2; Hangzhou Loggertech Co. Ltd., Hangzhou, China).Lost water was replenished with sterile distilled water to maintain the desired eld capacity determined by regular weighing.Seedlings were grown for 3 mo.

Rhizosphere soil sampling and physicochemical properties
The rhizosphere soil strongly adhering to the root surfaces was collected from each pot.Each soil sample was passed through a 2-mm sieve and divided into two subsamples.One was naturally dried at about 25 °C and its physicochemical properties were measured.The other was frozen at -80 °C until the subsequent microbial community composition analysis.A 0.2-g dried soil sample was digested in 10 mL of a 10:1:2 mixture of perchloric acid (12.7 M), sulfuric acid (18 M), and water in a Mars 6 microwave reaction system (CEM Corporation, Matthews, NC, USA) until a clear liquid was obtained.The soil organic matter content, available nitrogen (N), available phosphorus (P), and available potassium (K) were quantified by dichromate oxidization in the presence of sulfuric acid [61], alkaline hydrolysis diffusion, chlorostannous-reduced molybdophosphoric blue [62], and ame photometry [63], respectively.

Bioinformatics analysis
Raw fastq les were demultiplexed, quality-ltered, and merged by Trimmomatic and fast length adjustment of short reads (FLASH; Johns Hopkins University, Baltimore, MD, USA) [66].Sequences that were < 50 bp long and had average quality score < 20 or ambiguous bases were removed.The ltered high-quality sequences were merged according to the overlap sequences between read pairs.Sequences with mismatches along the primer region were removed before the downward process.Non-chimeric sequences were dereplicated and singletons were discarded.The ltered non-chimeric sequences were clustered into operational taxonomic units (OTUs) at a 97% sequence level based on the UPARSE pipeline using USEARCH v. 8.0.The RDP Bayesian classi er algorithm was used to classify OTU representative sequences via the fungal (ITS) UNITE database v. 18.11.2018and the Silva (SSU123) 16S rRNA reference database at con dence threshold = 0.7.The RDP then collated the functional gene database from GeneBank (Release 7.3; http://fungene.cme.msu.edu/) and obtained species annotation data.To eliminate potential bias caused by divergent sequence depth across samples, all samples were subsampled to the minimum sequencing depth.The dilution curve, the Venn map, and the community composition analysis were conducted in R (R Core Team, Vienna, Austria; version 3.

Statistical methods
Two-way analysis of variance (ANOVA) was used to disclose the effects of DSE inoculation, water regime, and their interactions on fungal and bacterial OTU diversity.Data shown in the gures are means of ≥ 3 replicates.Variations among treatment means were compared using Tukey's honestly signi cant difference (HSD) tests at P < 0.05.Non-metric multidimensional scaling (NMDS) was used to visualize compositional dissimilarities in the rhizosphere fungal and bacterial communities.The metaMDS command in the vegan package v. 2.4-1 was used [70].To evaluate the effects of DSE inoculation on the rhizosphere fungal and bacterial communities, permutational multivariate analysis of variance (PerMANOVA) was run using the adonis command in the vegan package with 999 permutations [70].Rarefaction curves for the bacterial and fungal OTUs were calculated using the specaccum function in the vegan package [70].The edaphic factors such as water condition, soil organic matter, available N, available P, and available K/microbe preference analysis was performed according to Yao et al. [71] and Huang et al. [72].
To visualize the structure of the rhizosphere bacterial and fungal networks among treatments, a network was drawn on the basis of genera with abundance > 200 for OTU-level matrices.To this end, the Prefuse Force Directed OpenCL Layout in CYTOSCAPE v. 3.4.0was used [73].
To identify the bene cial, harmful, and neutral fungi and bacteria, a network analysis was conducted on the abundance of the genera that signi cantly differed among treatments [74,75].The online FUNGuild application (http://www.stbates.org/guilds/app.php) was used to assign trophic status to frequent OTUs [74].The trophic status was assigned with different certainty levels (possible, probable, or highly probable) based on a combination of the aforementioned effects.The community designating the nutrient type as a compound nutrient type was included in "other fungi".The community identi ed as a compound multifunctional method was uni ed into "other pathogens/saprophytic fungi" under the nutrient type [74,75].

Samples Shannon
Ranking by the abundance of the fungal (A) and bacterial (B) operational taxonomic units (OTUs) in licorice rhizosphere.
Biological materials and growth substratesThe DSE strains, Acrocalymma vagum and Paraboeremia putaminum, were isolated from licorice roots naturally growing on the arid arable land of Northern China.They were deposited in the Laboratory of Endangered Species Breeding Engineering of the Institute of Medicinal Plant Development of the Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China [8].Licorice seeds were provided by China National Traditional Chinese Medicine Corporation and stored at 4 °C.
4.0) based on the OTU count and associated taxonomy tables.The Alpha diversity index was calculated in mothur (v.1.30.2).Other statistical analysis was performed in SPSS v. 22.0 (IBM Corp., Armonk, NY, USA) and the remaining graphs were generated with Origin v. 9.0 (OriginLab, Northampton, MA, USA) [67].Abundance and diversity analyses To characterize microbial diversity, the Chao1, Ace, Shannon, and Simpson indices were calculated based on the OTU data.Chao1 and Ace re ect community abundance while Shannon and Simpson indicate community diversity [68].Rank abundance and rarefaction curves generated in QIIME estimate species evenness and evaluate species richness and sequence depth, respectively [69].Fungal or bacterial OTU richness was de ned as the number of fungal or bacterial OTUs per sample.The relative abundance of a speci c fungal or bacterial OTU and class was de ned as the ratio of corresponding sequences and class to the total reads PER sample.Each representative OTU sequence in this study was used for taxonomic identi cation at the phylum, class, order, family, and genus levels.

Figures Figure 1
Figures

Figure 4 Relative
Figure 4

Table 2
Different letters in the same column indicate signi cant differences between different samples (P < 0.05).NCK, non-inoculation under well-watered condition; NPP, inoculation with Paraboeremia putaminum under well-watered condition; NAV, inoculation with Acrocalymma vagum under well-watered condition; DCK, non-inoculation under drought stress; DPP, inoculation with Paraboeremia putaminum under drought stress; DAV, inoculation with Acrocalymma vagum under drought stress.Diversity and richness index of licorice rhizosphere soil bacteria