The effects of drought stress on the productions of lobetyolin, syringin, and atractylolide III in different parts of C. pilosula
The contents of lobetyolin, syringin, and atractylolide III in different parts of C. pilosula were detected through HPLC–UV analysis. According to Duncan’s multiple range test, the average contents of lobetyolin and atractylolide III were significantly high in the root part at 1–8 days (5.77 and 0.59 mg·g−1, respectively), in comparison with those in the leaf (3.90 and 0.15 mg·g−1, respectively) and stem parts (1.81 and 0.02 mg g−1, respectively; Fig. 1a, b). Besides, the average contents of lobetyolin, syringin, and atractylolide III were significantly lower in the stem part at 1–8 days (1.81, 0.03, and 0.02 mg·g−1, respectively) than that of the leaf (3.90, 0.34, and 0.15 mg·g−1, respectively) and root parts (5.77, 0.22, and 0.59 mg·g−1, respectively; Fig. 1a–c). Based on Student’s t–test, the lobetyolin contents in root (except for the third day), stem, and leaf parts of C. pilosula subjected to drought treatment for 1, 3, and 8 days markedly increased by 8.47%–23.40%, 26.67%–86.47%, and 26.41%–39.42%, respectively, in comparison with the untreated parts (Fig. 1d). Meanwhile, the atractylolide III content in the root, stem, and leaf parts under drought treatment for 1–8 days was remarkably increased by 32.17%–119.17%, 74.43%–177.86%, and 80.51%–107.50%, respectively, in comparison with those in control parts (Fig. 1e). In addition, the data indicated that the syringin content in the root, stem, and leaf parts after 8 days of drought treatment was observably increased by 56.30%–58.31%, 229.61%–230.98%, and 28.78%–31.31%, respectively, compared with that of the untreatment (Fig. 1f). Collectively, these results indicated that the accumulations of lobetyolin, syringin, and atractylolide III showed tissue specificity in C. pilosula, and their contents were increased in drought–stressed tissues.
The effects of drought stress on the alpha diversity of endophytes in different parts of C. pilosula
A total of 1,057,465, 1,140,973, and 1,121,510 16S rRNA reads, as well as 1,082,687, 1,131,502, and 1,078,567 ITS reads were obtained from the leaf, stem, and root tissues, respectively (Additional file 2: Table S1–S3). The mean sequencing depth per sample was 62,820 for bacteria and 60,977 for fungi, respectively. Chao 1 index showed a higher diversity number of microbial species in the roots than that of leaves and stems (Duncan’s multiple range test, P < 0.05; Fig. 2a, b and Additional file 3). H’ index revealed that the diversity of endophytic bacteria in the roots was the highest, followed by those in the stem and leaf parts, whereas the diversity of endophytic fungi was the highest in the leaf part, followed by the root and stem parts (Duncan’s multiple range test, P < 0.05; Fig. 2c, d and Additional file 3). The bacterial Chao 1 index was higher in the leaf, stem, and root parts under 1-day-drought treatment than in the control parts (Student’s t-test, P < 0.05; Fig. 2e and Additional file 3). The fungal H' index in the drought treatment groups remarkably increased after 8 days compared with that of the untreated groups (Student’s t-test, P < 0.05; Fig. 2f and Additional file 3). In addition, the Chao 1 and H' indices of other drought treatment parts in C. pilosula (except for the Chao 1 of fungi in leaf part) displayed an increasing trend, or were markedly increased in comparison with those in the untreated parts (Fig. 2e–h and Additional file 3). These results revealed that the alpha diversity of endophytes in C. pilosula was tissue-dependent, and that the drought treatment could increase the alpha diversity of endophytes.
The effects of drought stress on the beta diversity of endophytes in different parts of C. pilosula
PCoA based on weighted UniFrac distances revealed that the microbial communities were remarkably different between different parts of C. pilosula (bacteria, R2 = 0.939, P < 0.001; fungi, R2 = 0.452, P < 0.001; Fig. 3a and Additional file 3). The PCoA results showed that the endophytic communities in C. pilosula parts subjected to drought treatment were significantly different from those of the control parts. The first and second principal component axes explained 67.84% and 31.62% of the total fungi changes in the leaf part (R2 = 0.695, P < 0.001; Fig. 3b and Additional file 3: Dataset S1). The bacterial communities in the leaf parts were clustered and negligible change (R2 = 0.098, P = 0.169; Fig. 3b and Additional file 3: Dataset S2). Moreover, the first (92.57% contribution) and second (5.82% contribution) principal component axes differentiated the fungal communities in drought treatment and untreated stem parts (R2 = 0.495, P = 0.045; Fig. 3c and Additional file 3: Dataset S3). The first principal component (77.68% contribution) distinguished the bacterial communities in the stem part treated by drought stress for 1 and 8 days from those in the untreated counterpart (R2 = 0.502, P = 0.032; Fig. 3c and Additional file 3: Dataset S4). Furthermore, the first principal component (79.02% contribution) differentiated the fungal communities in the root part under drought treatment for 1 and 8 days from those in the control treatment for 1 and 3 days; The second principal component (14.52%) highlighted the fungal communities in the root part subjected to drought treatment for 3 days from those in untreated groups (R2 = 0.422, P = 0.034; Fig. 3d and Additional file 3: Dataset S5). The first principal component axis (67.84% contributions) indicated that in the 1 and 8 days of experiment, the bacterial communities in the drought-treated roots were distinctly different from those of the control parts (R2 = 0.476, P = 0.008; Fig. 3d and Additional file 3: Dataset S6). These results demonstrated that the beta diversity of endophytic communities in C. pilosula displayed tissue specificity and remarkable differences between untreated (except for bacteria in the leaf compartment) and drought treatment tissues.
The effects of drought stress on the compositions of endophytes in different parts of C. pilosula
The composition of endophytic communities in different parts of C. pilosula showed remarkable discrepancies (Fig. 4 and Additional file 3). The bacterial communities in different parts of C. pilosula at the order level were dominated by the bacterial OTUs of Rickettsiales (Fig. 4a and Additional file 3). The relative abundances of the OTUs of Rickettsiales were 70.45%–94.47%, 85.66%–99.13%, and 34.45%–55.45% in the leaf, stem, and root parts, respectively (Fig. 4a and Additional file 3). Compared with that of the untreated parts, the relative abundances of bacterial Rickettsiales in the leaf part were remarkably increased by 15.38%–22.08% under drought treatment for 1, 3, and 8 days, whereas those of Rickettsiales were decreased by 6.06%–12.30% and 14.27%–41.82% in the stem and root parts, respectively (Fig. 4a and Additional file 3). The relative abundances of bacterial Rhizobiales were higher by 28.73%–97.93% in the root part treated by drought treatment for 1–8 days than that of the untreated part (Fig. 4a and Additional file 3: Dataset S6).
Moreover, the main orders of fungal communities in different parts of C. pilosula comprised Hypocreales, Pleosporales, Ascomycota, and Glomerellales (Fig. 4b and Additional file 3). Among these orders, the average relative abundance of Hypocreales were 18.69%, 76.95%, and 67.23% in the leaf, stem, and root parts, respectively, while those of Pleosporales were 48.50%, 3.87%, and 10.02%, respectively (Fig. 4b and Additional file 3). The relative abundances of fungal communities were remarkably different in the leaf part between the drought treatment and untreated groups (Fig. 4b and Additional file 3: Dataset S1). The relative abundances of fungal Hypocreales in the stem part subjected to drought treatment for 1, 3, and 8 days were decreased by 25.89%–28.66%, 13.48%–14.34%, and 85.35%–88.97%, respectively, relative to those in the control stem (Fig. 4b and Additional file 3: Dataset S3). The relative abundances of fungal Pleosporales in the root part under drought treatment for 1 and 8 days were lower by 29.64%–58.55% than those in untreated root part, whereas those of Hypocreales were higher by 20.87%–46.31% (Fig. 4b and Additional file 3: Dataset S5). These results revealed that the composition of endophytic community in C. pilosula was tissue-specific and altered by drought stress.
Biomarkers of endophytes within C. pilosula related to drought stress and their correlations with the contents of secondary metabolites
LEfSe was performed to determine the microbiome members that could be used as biomarkers, and then Spearman’s correlation analysis revealed the relationships between the abundances of biomarkers and the contents of pharmacological components. 70 bacterial taxa were enriched as potential biomarkers in different parts of C. pilosula (leaf: 3; root: 67; Fig. 5a). These bacterial taxa belonged to seven phyla, namely Proteobacteria, Actinobacteria, Bacteroidetes, Acidobacteria, Chloroflexi, Planctomycetes, and Cyanobacteria (Fig. 5a). A total of 54, 6, and 67 biomarkers were strongly and positively associated with contents of lobetyolin, syringin, and atractylolide III, respectively; among biomarkers, most were mainly belonging to the orders of Proteobacteria, Chloroflexi, and Planctomycetes (Fig. 5b and Additional file 2: Figure S1a). A total of 61 fungal taxa from Ascomycota and Mucoromycota were identified as potential biomarkers in different parts of C. pilosula (leaf: 33; stem: 18; root: 10; Fig. 5c). Moreover, 21, 45, and 16 biomarkers were substantially and positively correlated with the contents of lobetyolin, syringin, and atractylolide III, respectively (Fig. 5d and Additional file 2: Figure S1a). These biomarkers mainly belonged to Leotiomycetes, Tetracladium, Phaeosphaeriaceae, Cantharellales, Ceratobasidiaceae, and Didymellaceae (Fig. 5d). These results suggested that the distribution of pharmacological components in the different parts of C. pilosula was correlated with the composition of endophytes.
Based on the comparison between untreated and drought treatment parts, 14, 10, and 64 bacterial taxa were enriched as potential biomarkers in the leaf, stem, and root parts, respectively (Fig. 6a-c). These potential biomarkers mainly belonged to Firmicutes, Cyanobacteria, Verrucomicrobia, Planctomycetes, Acidobacteria, Chloroflexi, and Proteobacteria (Fig. 6a–c). Compared with those in the control C. pilosula, the abundances of 11, 5, and 58 potential biomarkers in the leaf, stem, and root parts under drought treatment were increased by 20.00%–95.24%, 52.27%–84.91%, and 10.96%–78.03%, respectively (Additional file 2: Figure S1b–S1d and Additional file 3). The abundances of 9, 10, and 1 bacterial biomarkers in the leaf part were positively associated with the contents of lobetyolin, syringin, and atractylolide III, respectively (Fig. 6d). The abundances of 4, 4, and 2 bacterial biomarkers in the stem part were positively correlated with the contents of lobetyolin, syringin, and atractylolide III, respectively (Fig. 6e). The abundances of 28, 46, and 40 bacterial biomarkers in the root part were positively related to the contents of lobetyolin, syringin, and atractylolide III, respectively (Fig. 6f). These biomarkers mainly belonged to Xanthomonadaceae, Bacillales, Methylobacterium, and Ramlibacter in the leaf part (Fig. 6d); Microbacterium and Pseudomonadales in the stem part (Fig. 6e); and Pseudorhodoferax, Lacunisphaera, Tahibacter, Verrucomicrobiae, Verrucomicrobia, and Chloroflexia in the root part (Fig. 6f).
In accordance with the comparison of the untreated and drought treatment, 48, 10, and 44 fungal taxa were identified as potential biomarkers in the leaf, stem, and root parts, respectively (Fig. 7a–c). The potential biomarkers belonged to Basidiomycota, Mucoromycota, Olpidiomycota, and Ascomycota (Fig. 7a–c). In comparison with those in untreated parts of C. pilosula, the abundances of 44, 10, and 38 potential biomarkers in the leaf, stem, and root parts after drought treatment increased by over 121.00%, 824.00%, and 33.60%, respectively (Additional file 2: Figure S1b–S1d and Additional file 3). The abundances of 14, 14, and 37 fungal biomarkers in the leaf part were positively correlated with the contents of lobetyolin, syringin, and atractylolide III, respectively (Fig. 7d). The abundances of 10, 1, and 8 fungal biomarkers in stem part were positively associated with the contents of lobetyolin, atractylolide III, and syringin, respectively (Fig. 7e). The abundances of 9, 10, and 16 fungal biomarkers in the root part were positively related to the contents of lobetyolin, syringin, and atractylolide III, respectively (Fig. 7f). These biomarkers mainly belonged to Exophiala, Herpotrichiellaceae, Plectosphaerella, Glomerellales, Plectosphaerellceae, Paraphoma, and Phaeosphaeriaceae in the leaf part (Fig. 7d); Chaetomiaceae, Solicoccozyma, and Piskurozymaceae in the stem part (Fig. 7e); and Pyronemataceae, Leotiomycetes, and Fusidium in the root part (Fig. 7f). These results suggested that the endophytes were related to the contents of pharmacological components and might be involved in the accumulations of pharmacological components in C. pilosula parts under drought stress. In the following experiments, we further verified the function of endophytes in metabolite accumulations.
Endophytes participated in the accumulations of pharmacodynamic compounds
Three endophytic bacteria and seven endophytic fungi obtained from the microbiological culture collection library of the laboratory were cultured with sterile C. pilosula powder to verify the functions of endophytes in metabolite accumulations. Following incubation, the bacterium Pseudomonas nitroreducens and the fungi Epicoccum thailandicum, Filobasidium magnum, and Paraphoma rhaphiolepidis were confirmed to be involved in the accumulations of pharmacodynamic compounds (Fig. 8a, b). Lobetyolin, syringin, and atractylolide III were not detected in groups (endophyte + medium). The contents of lobetyolin and atractylolide III was increased by E. thailandicum at the rates of 33.14‒%34.70% and 30.24%‒31.40%, respectively; by F. magnum at the rates of 41.89%‒42.48% and 45.7%‒46.02%, respectively; and by P. rhaphiolepidis at the rates of 39.45%‒40.93% and 11.12%‒11.98%, respectively (Fig. 8b). And P. nitroreducens could increase syringin content at the rates of 118.61%‒119.36% (Fig. 8b).