Dark Septate Endophytes Isolated From Wild Licorice Roots Grown in the Desert Regions of Northwest China Enhance the Growth of Host Plants Under Water Deficit Stress

Abstract

This study aimed to explore dark septate endophytes (DSE) that may improve the cultivation of medicinal plants in arid ecosystems. We isolated and identified eight DSE species (Acremonium nepalense, Acrocalymma vagum, Alternaria chartarum, Alternaria chlamydospora, Alternaria longissima, Darksidea alpha, Paraphoma chrysanthemicola, and Preussia terricola) colonizing the roots of wild licorice (Glycyrrhiza uralensis) in the desert areas of northwest China. Moreover, we investigated the osmotic stress tolerance of the DSE using pure culture, along with the performance of licorice plants inoculated with the DSE under drought stress in a growth chamber, respectively. Here, five species were first reported in desert habitats. The osmotic-stress tolerance of DSE species was highly variable, A. chlamydospora and P. terricola increased the total biomass and root biomass of the host plant. All DSE except A. vagum and P. chrysanthemicola increased the glycyrrhizic acid content; all DSE except A. chartarum increased the glycyrrhizin content under drought stress. DSE × watering regimen improved the glycyrrhizic acid content, soil organic matter, and available nitrogen. Structural equation model analysis showed that DSE × watering regimen positively affected soil organic matter, and total biomass, root length, glycyrrhizic acid, and glycyrrhizin (Shapotou site); and positively affected soil organic matter, available phosphorus, and glycyrrhizin (Minqin site); and positively affected the root length (Anxi site). DSE from the Shapotou site accounted for 8.0, 13.0, and 11.3% of the variations in total biomass, root biomass, and active ingredient content; DSE from the Minqin site accounted for 6.6 and 8.3% of the variations in total biomass and root biomass; DSE from the Anxi site accounted for 4.2 and 10.7% of the variations in total biomass and root biomass. DSE × watering regimen displayed a general synergistic effect on plant growth and active ingredient contents. These findings suggested that the DSE-plant interactions were affected by both DSE species and DSE originating habitats. As A. chlamydospora and P. terricola positively affected the total biomass, root biomass, and active ingredient content of host plants under drought stress, they may have important uses as promoters for the cultivation of licorice in dryland agriculture.

Keywords: dark septate endophytes; desert ecosystem; drought stress; inoculation; licorice.

FIGURE 1

Colonies of endophytic fungi isolated from the roots of wild licorice (A–H). Microscopic morphology of endophytic fungi (a–h) (Bars = 50 μm). (A) a: Acrocalymma vagum (DSE1); (B) b: Paraphoma chrysanthemicola (DSE2); (C) c: Alternaria longissima (DSE3); (D) d: Darksidea alpha (DSE4); (E) e: Alternaria chlamydospora (DSE5); (F) f: Acremonium nepalense (DSE6); (G) g: Preussia terricola (DSE7); (H) h: Alternaria chartarum (DSE8). Arrows indicate: Hy, DSE hyphae; S, DSE spores.

FIGURE 2

Maximum parsimony tree generated from ITS (ITS4 and ITS5) sequences of the isolate strains and their closest matches, followed by GenBank accession number.

FIGURE 3

Response of the biomass (A), superoxide dismutase (SOD) activity (B), and malondialdehyde (MDA) (C), and melanin content (D) in the eight dark septate endophytes (DSE) to different concentrations of polyethylene glycol (PEG) 6000. Different letters above the error bars indicate significant differences at P < 0.05.

FIGURE 4

(A–E) Effects of dark septate endophytes (DSE) and water treatment on the morphological parameters of licorice plants. Different letters above the error bars indicate significant difference at P < 0.05. CK indicates non-inoculated plants. DSE1–DSE8, indicate plants inoculated with different DSE species. WW, DS, indicate well-watered and drought stress treatment, respectively.

FIGURE 5

(A–D) Effects of dark septate endophytes (DSE) and water treatment on the biomass production of licorice plants. Different letters above the error bars indicate significant difference at P < 0.05. CK indicates non-inoculated plants. DSE1–DSE8, indicate plants inoculated with different DSE species. WW, DS, indicate well-watered and drought stress treatment, respectively.

FIGURE 6

(A,B) Effects of dark septate endophytes (DSE) and water treatment on the active ingredient content of licorice plants. Different letters above the error bars indicate significant difference at P < 0.05. CK indicates non-inoculated plants. DSE1–DSE8, indicate plants inoculated with different DSE species. WW, DS, indicate well-watered and drought stress treatment, respectively.

FIGURE 7

(A–C) Effects of dark septate endophytes (DSE) and water treatment on soil parameters. Different letters above the error bars indicate significant difference at P < 0.05. CK indicates non-inoculated plants. DSE1–DSE8, indicate plants inoculated with different DSE species. WW, DS, indicate well-watered and drought stress treatment, respectively.

FIGURE 8

Structural equation model showing the causal relationships among DSE from different sites, water condition, DSE × water, soil parameters, and growth indicators and active ingredients. The final model fitted the data well: maximum likelihood, Shapotou site (A) X² = 43.513, df = 10, P = 0.014, root mean square error of approximation = 0.253, goodness-of-fit index = 0.613, Akaike information criteria = 148.468; Minqin site (B) X² = 69.482, df = 12, P = 0.005, root mean square error of approximation = 0.267, goodness-of-fit index = 0.0.571, Akaike information criteria = 161.992; Anxi site (C) X² = 82.476, df = 13, P = 0.001, root mean square error of approximation = 0.323, goodness-of-fit index = 0.538, Akaike information criteria = 172.965. Solid lines and dashed lines indicate significant and non-significant pathways, respectively. The width of the solid lines indicates the strength of the causal effect, and the numbers near the arrows indicate the standardized path coefficients (^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001). DW, combination of DSE and water; SOM, soil organic matter; SAP, soil available P; SAN, soil available N; e, the values of residuals; TB, total biomass; TRL, root length; GAC, glycyrrhizinic acid content; GC, glycyrrhizin content.

FIGURE 9

Variation partitioning of DSE from different sites, water condition, and soil parameters on the total biomass (A,D,G), root biomass (B,E,H), and active ingredient contents (C,F,I) of licorice plants. DSE, DSE species. Soil nutrient, nutrient content in soil (including soil organic matter, available N, and available P). Values below 0 are not shown. (A–C) DSE from Shapotou; (D–F) DSE from Minqin; (G–I) DSE from Anxi.