3.1 Characterization and identification of DSE
Typical DSE hyphae and microsclerotia were observed in the roots of O. japonicus and L. japonica (Supplementary Fig. S3). Brown to yellow-brown septate hyphae with thick lateral walls invaded the epidermal and/or cortical cells (Supplementary Fig. S3A, S3C). Chainlike and conglomerated microsclerotia filled single cortical cells and/or colonised > 1 cell (Supplementary Fig. S3B, S3D).
Five and two DSE colonies isolated from O. japonicus and L. japonica, respectively,were ashen grey to dark brown (Supplementary Fig. S4). DSE1 and DSE4 produced spores but neither conidia nor reproductive structures were observed in the other isolates. A comparative analysis of the fungal sequences in the GenBank database identified Acrocalymma vagum (DSE1), Paraphoma radicina (DSE2), Curvularia pallescens (DSE3), Scytalidum lignicola (DSE4), Paraboeremia putaminum (DSE5),and Phoma herbarum (DSE6, DSE7) (Supplementary Fig. S5). Based on their growth status, we selected S. lignicola (SL, DSE4), P. putaminum (PP, DSE5), and P. herbarum (PH, DSE6) for the pot inoculation experiments.
3.2 Shoot morphological traits of G. uralensis seedlings
After 3 months of growth, all inoculated licorice seedlings were alive, green, and healthy. The roots of all inoculated plants were colonised by DSE4, DSE5, and DSE6 (Supplementary Fig. S6). Relative to the control plants, DSE or MT inoculation significantly increased plantheight whereas HT inoculation decreased it. In contrast, LT inoculation did not significantly modify plant height compared with the control plants (Fig. 1A). Only S. lignicola orHT inoculation significantly increased shoot branch number relative to the control plants. However, no significant differences in shoot branch number were observed between the other inoculated plants and the control plants (Fig. 1B). Compared with the control plants, P. herbarum, LT, and MT inoculation increased the leaf number whereas the other inoculants had no significant effect on this trait (Fig. 1C).
Interactions between DSE species and T. viride density were significant for plant height and shoot branch number (Table 1). Under LT conditions, P. putaminum inoculation increased plant height, S. lignicola decreased it, and P. herbarum had no significant effect on this characteristic compared with the control plants (Fig. 1A). Under LT conditions, only P. herbarum increased shoot branch and leaf numbers relative to the control plants. The other isolates had no significant impact on shoot branch or leaf number compared with the control plants (Fig. 1B, 1C). Under MT conditions, P. putaminum and S. lignicola increased plant height and leaf number, S. lignicola increased shoot branch number, and P. herbarum had no significant influence on plant height and shoot branch or leaf number relative to the control plants (Fig. 1A–1C). Under HT conditions, compared with the control plants, P. herbarum increased plant height and shoot branch and leaf numbers, S. lignicola increased both shoot branch and leaf numbers and decreased plant height, and P. putaminum decreased plant height but had no effect on shoot branch or leaf numbers (Fig. 1A–1C).
3.3 Root morphological traits of G. uralensis seedlings
P. putaminum and S. lignicola inoculation significantly increased licorice root length, diameter, and surface area relative to the control plants. However, P. herbarum only increased the root surface area compared with the control plants (Fig. 1D–1F). Root diameter and surface area wereincreased at various T. viride densities (Fig. 1E, 1F). The LT and HT treatments increased the root length whereas the MT treatment had no significant impact on this trait compared with the control plants (Fig. 1D).
Under LT conditions, DSE increased the root length, diameter, and surface arearelative to the control plants.Under MT conditions,DSE increased the root diameter and surface area, S. lignicola and P. herbarum increased the root length, and P. putaminum had no significant influence on root length compared with the control plants.Under HT conditions, DSE increased the root surface area but S. lignicola and P. herbarum onlyincreased the root length and diameter relative to the control plants (Fig. 1D–F). The interactions between DSE species and T. viride density were significant only for root length and surface area (Table 1).
3. 4 Biomass production of G. uralensis seedlings
The shoot and root biomass and the root:shoot ratio of licoriceweresignificantly and separately influenced by DSE species and T. viride density (Fig. 2A). P. putaminum increased the root biomass and the root:shoot ratio, S. lignicola increased the shoot biomass anddecreased theroot:shoot ratio, and P. herbarum only increased the root biomass compared with the control plants. The LT and MT conditions increased the shoot and root biomass whereas HT increased the root biomass and the root:shoot ratio and decreased the shoot biomass relative to the control plants (Fig. 2A). There were significant positive interactions between DSE and T. viride with respect to shoot and root biomass. However, the interactions between DSE and T. viride on the root:shoot ratio varied with DSE species and T. viride density (Fig. 2B, Table 1).
Nonmetric multidimensional scaling (NMDS) and analysis of similarities (ANOSIM) indicated that licorice root morphology and biomass were significantly separated by DSE species (R = 0.0505, P = 0.046) and T. viride density (R = 0.1223, P = 0.001) (Fig. 3).
3. 5 Correlation analyses
A Mantel test and a structural equation model (SEM) were used to illustrate the effects of DSE, T. viride, and their interaction on the growth parameters of licorice. The Mantel test disclosed significant relationships among DSE, T. viride, plant biomass, root length, diameter, and surface area, leaf number, plant height, and shoot branching (Supplementary Table S1). We used correlation coefficients (R-values) and the SEM to quantify the relative effects of DSE, T. viride, the combination of DSE and T. viride on total root length, diameter, and surface area on plant biomass, plant height, leaf number, and shoot branching (χ2 = 136.933, degrees of freedom (df) = 12, P = 0.005, root mean square error of approximation (RMSEA) = 0.407, goodness-of fit index (GFI) = 0.750, Akaike information criterion (AIC) = 222.933). DSE had significant direct effects on root length and diameter, plant biomass, and shoot branching. T. viride had significant direct effects on root length, surface area, and diameter, plant biomass and height, and leaf number. The combination of DSE and T. viride significantly positively influenced root surface area, plant biomass and height, and shoot branching (Fig. 4).
3. 6 Variation partitioning of plant growth parameters and biomass production
A variance partitioning analysis was performed to quantify the contributions of DSE species and T. viride density to the plant growth parameters and biomass production (Figs. 5–7). The combination of PPandLT explained 38.2% of the variance in shoot biomass (Fig. 5A), 61.2% of the variance in root biomass (Fig. 5B), 16.9% of the variance in shoot growth traits (Fig. 5C), and 58.0% of the variance in root growth traits (Fig. 5D). The pure variances in root biomass and growth traits explained by PP were 45.3% and 39.5%, respectively, whilst LT explained 15.9% and 18.5%, respectively. The simultaneous influence of PP combined with LT on root biomass and growth traits explained 9.9% and 6.7% of the variance, respectively. The combination of PPandMT explained 55.9% of the variance in shoot biomass (Fig. 5E), 70.8% of the variance in root biomass (Fig. 5F), 34.6% of the variance in shoot growth traits (Fig. 5G), and 16.1% of the variance in root growth traits (Fig. 5H). The pure variances in root biomass and growth traits explained by PP were 20.0% and 13.9%, respectively, whilst MT explained 32.7% and 2.2%, respectively. The simultaneous influence of PPcombined with MT on root biomass and growth traits explained 18.1% and 4.3% of the variance, respectively. The combination of PP andHT explained 47.1% of the variance in shoot biomass (Fig. 5I), 13.0% of the variance in root biomass (Fig. 5J), 14.5% of the variance in shoot growth traits (Fig. 5K), and 19.7% of the variance in root growth traits (Fig. 5L). The pure variances in root biomass and growth traits explained by PP were 8.1% and 6.0%, respectively, whilst HT explained 4.9% and 13.7%, respectively. The combination of PP andHT explained 22.1% and 7.1% of the variance in root biomass and growth traits, respectively.
The combination of SLandLT explained 50.2% of the variance in shoot biomass (Fig. 6A), 29.5% of the variance in root biomass (Fig. 6B), 23.3% of the variance in shoot growth traits (Fig. 6C), and 48.2% of the variance in root growth traits (Fig. 6D). The pure variances in root biomass and growth traits explained by SL were 8.7% and 22.1%, respectively, whilst LT explained 20.8% and 26.1%, respectively. The combination of SLand LT explained 5% and 11.1% of the variance in root biomass and growth traits, respectively. The combination of SLandMT explained 12.2% of the variance in shoot biomass (Fig. 6E), 12.6% of the variance in root biomass (Fig. 6F), 59.5% of the variance in shoot growth traits (Fig. 6G), and 12.4% of the variance in root growth traits (Fig. 6H). The pure variance in the root biomass explained by SL was 4.4% whilst MT explained 8.2% and 12.4% of the variance in root biomass and growth traits, respectively. The combination of SLand MT explained 18.9% and 16.6% of the variance in root biomass and growth traits, respectively. The combination of SLandHT explained 77.3% of the variance in shoot biomass (Fig. 6I), 44.1% of the variance in root biomass (Fig. 6J), 24.8% of the variance in shoot growth traits (Fig. 6K), and 26.2% of the variance in root growth traits (Fig. 6L). The pure variances in root biomass and growth traits explained by SL were 16.7% and 5.2%, respectively, whilst HT explained 27.4% and 21.0%, respectively. The combination of SL and HT explained 8.6% of the variance in shoot biomass.
The combination of PHandLT explained 49.0% of the variance in shoot biomass (Fig. 7A), 47.3% of the variance in root biomass (Fig. 7B), 23.3% of the variance in shoot growth traits (Fig. 7C), and 43.4% of the variance in root growth traits (Fig. 7D). The pure variances in root biomass and growth traits explained by PH were 23.2% and 0.9%, respectively, whilst LT explained 24.1% and 42.5%, respectively. The combination of PHand LT explained 9.4% and 11.7% of the variance in root biomass and growth traits, respectively. The combination of PHandMT explained 83.0% of the variance in shoot biomass (Fig. 7E), 20.9% of the variance in root biomass (Fig. 7F), 2.3% of the variance in shoot growth traits (Fig. 7G), and 14.2% of the variance in root growth traits (Fig. 7H). The pure variances in the root biomass and growth traits explained by PH were 9.4% and 4.3%, respectively. MT explained 11.5% and 9.9% of the variance in the root biomass and growth traits, respectively. The combination of PHand MT explained 11.4% and 0.5% of the variance in the root biomass and growth traits, respectively. The combination of PHandHT explained 59.0% of the variance in shoot biomass (Fig. 7I), 15.1% of the variance in root biomass (Fig. 7J), 40.6% of the variance in shoot growth traits (Fig. 7K), and 39.1% of the variance in root growth traits (Fig. 7L). The pure variances in the root biomass and growth traits explained by PH were 0.7% and 0.9%, respectively. HT explained 7.4% and 38.2% of the variance in root biomass and growth traits, respectively. The combination of PHand MT explained 10.0% and 20.4% of the variance in the root biomass and growth traits, respectively.