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.
After filtering 1,266,009 low-quality sequences, 1,568,052 effective fungal and 987,120 effective bacterial sequences were clustered into 1,278 fungal and 1,583 bacterial operational taxonomic units (OTUs) at 97% sequence similarity. Of the 1,278 fungal OTUs, 196 occurred in all six treatments while 85, 94, 48, 21, 202, and 33 OTUs were found only in the uninoculated condition (NCK), P. putaminum inoculation (NPP), and A. vagum inoculation (NAV) under well-watered and in the three DSE inoculation treatments DCK, DPP, and DAV under drought stress, respectively (Fig. 3). Of the 1,583 bacterial OTUs, 1,082 were detected in all treatments and 31, 45, 71, 36, 64, and 38 existed only in NCK, NPP, NAV, DCK, DPP, and DAV, respectively (Fig. 3).
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 significant 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 significant effect on soil bacterial community diversity and richness. Compared with the uninoculated condition, drought stress decreased the soil bacteria Simpson index.
Rhizosphere microbial community composition
A total of 1,278 fungal OTUs were found in the licorice rhizosphere and classified as Ascomycota, Basidiomycota, Zygomycota, Glomeromycota, and certain unknown fungi. Ascomycota and Basidiomycota were identified in all treatments. Ascomycota was the dominant fungal phylum and its relative abundance range was 89.9–99.6% across the various treatments (Fig. 4). A heatmap based on the top 50 abundant fungal genera revealed that the colonization of certain relatively abundant fungi varied among treatments (Fig. 5). Of the 50 abundant fungal genera, five OTUs (Aspergillus OTU86, Fusarium OTU1748, Gibberella OTU1913, Trichoderma OTU8380 and unclassified_f_Chaetomiacease OTU936) were distributed mainly in NCK, four OTUs ( Acremonium OTU454, Chaetomium OTU1508, Phialophora OTU1296 and Stachybotrys OTU1633) were distributed mainly in NPP, two OTUs (Aspergillus OTU86 and Trichoderma OTU8380) were distributed mainly in NAV, three OTUs (Aspergillus OTU86, Trichoderma OTU8380 and unclassified_f_Microascaceae OTU1963) were distributed mainly in DCK, four OTUs (Chaetomium OTU1508, Pseudalescheria OTU946, unclassified_f_Chaetomiaceae OTU936 and unclassified_ k_Fungi OTU1449) were distributed mainly in DPP, and two OTUs (Aspergillus OTU86 and Trichoderma OTU8380) were distributed mainly in DAV (Fig. 5).
A total of 1,583 bacterial OTUs were detected in the licorice rhizosphere. The dominant bacterial phyla were Proteobacteria, Actinobacteria, Chloroflexi, Firmicutes, Acidobacteria, Cyanobacteria, Bacteroidetes, Gemmatimonadetes, Planctomycetes, Verrucomicrobia, Nitrospirae, Saccharibacteria, and certain unknown bacteria. The relative abundance of these bacterial phyla varied among treatments (Fig. 4). Of the 50 dominant bacterial genera, nine OTUs (Bacillus OTU4110, Devosia OTU1666, Nocardioides OUT5635, Oscillatoria OTU6539, Pseudarthrobacteria OTU3533, Sphingomonas OTU5209, norank-c-Acidobacteria OTU1690, norank-c-Gitt-GS-136 OTU3951 and norank-o-JG30-KF-CM45 OTU6085) were distributed mainly in NCK, six OTUs (Bacillus OTU4110, Pseudarthrobacteria OTU3533, Pseudomonas OTU5762, norank-c-Acidobacteria OTU1690, norank-f- Anaerolineaceae OTU1186 and norank-c-KD4-96 OTU3568) were distributed mainly in NPP, eight OTUs (Bacillus OTU4110, Devosia OTU1666, Ensifer OTU6341, Paenibacillus OTU4751, norank-o-Acidmicrobiales OTU4282, norank-c- Acidobacteria OTU1690, norank-f-Anaerolineaceae OTU1186 and norank-o-JG30- KF-CM45 OTU6085) were distributed mainly in NAV, seven OTUs (Bacillus OTU4110, Nocardioides OTU5635, Paenibacillus OTU4751, norank-c-Acidobacteria 1690, norank-o-Acidmicrobiales OTU4282, norank-c-Gitt-GS-136 OTU3951 and norank-c-KD4-96 OTU3568) were distributed mainly in DCK, six OTUs (Bacillus OTU4110, Lamia OTU4301, Pseudomonas OTU5762, norank-c-Acidobacteria OTU1690, norank-f-Anaerolineaceae OTU1186 and norank-c-KD4-96 OTU3568) were distributed mainly in DPP, and 11 OTUs (Bacillus OTU4110, Lysinibacillus OTU9205, Microbacterium OTU1007, Nocardioides OTU5635, Paenibacillus OTU4751, Sporocytophaga OTU2217, norank-c-Acidobacteria OTU1690, norank- o-Acidmicrobiales OTU4282, norank-f-Anaerolineaceae OTU1186, norank-c-KD4-96 OTU3568 and norank-c-Gitt-GS-136 OTU3951) were distributed mainly in DAV (Fig.5).
Nonmetric multidimensional scaling (NMDS) ordination revealed that the rhizosphere fungal community composition significantly differed between the DSE inoculation and uninoculated treatments under well-watered (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 significantly 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 significantly affected the bacterial community composition relative to the uninoculated condition. In contrast, A. vagum inoculation had no significant impact on the bacterial community composition (Fig. 6). Permutational multivariate analysis of variance (PerMANOVA) indicated that the fungal (F = 7.435, R2= 0.264, P = 0.001; F = 6.224, R2= 0.331, P = 0.001) and bacterial (F = 6.125, R2= 0.392, P = 0.001; F = 5.648, R2= 0.440, P = 0.001) community compositions were significantly 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.
Edaphic factor and rhizosphere microbe preferences
An edaphic factor/microbe preference analysis showed that the fungi and bacteria colonizing the licorice rhizosphere significantly preferred the soil moisture, organic matter content, and available N, P, and K (Fig. 7). Of the 50 relatively abundant fungal OTUs, two (Gibberella OTU1913 and Phialophora OTU1296) had a significant positive preference for soil moisture but OTU575 (Vermispora) presented with a significant negative moisture preference. OTU308 (Guehomyces) showed negative available N preference. Three OTUs (Guehomyces OTU308, unclassified_f_Lasiosphaeriaceae OTU161, and Schizothecium OTU474) showed negative available P preferences. OTU1389 (Humicola) displayed a positive available K preference while OTU1539 (Mycoarthris) displayed a positive soil organic matter preference (Fig. 7). Of the 50 relatively abundant bacterial OTUs, OTU3533 (Pseudarthrobacter) and three OTUs (Actinobacteria OTU1174, Streptomyces OTU5992 and Xanthomonadales OTU7347) showed positive and negative moisture preferences, respectively. Two OTUs (norank-f-Anaerolineaceae OTU1186 and Pseudomonas OTU5762) positively correlated with available N. Seven OTUs (Acidimicrobiales OTU4282, Gaiella OTU 8721, Iamia OTU 4301, Oscillatoria OTU6539, norank-f-Gemmatimonadaceae OTU1174, norank-f-Nitrosomonadaceae OTU732 and Streptomyces OTU5992) had a significant negative correlation with available K. Three OTUs (norank-c-Cynobacteria OTU5893, norank-f- Anaerolineaceae OTU1186 and norank-f-Sandaracinaceae OTU476) and one OTU (Lysinibacillus OTU9205) showed positive and negative organic matter preferences, respectively (Fig.7).
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 beneficial 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 beneficial bacteria but increased the relative abundance of neutral fungi and neutral and pathogenic bacteria. The A. vagum inoculation increased the relative abundance of beneficial fungi and neutral bacteria but decreased the relative abundance of pathogenic fungi and bacteria.