Effects of root exudates of woody species on the soil anti-erodibility in the rhizosphere in a karst region, China

Materials and Methods

Root exudate extraction

We selected eight of the typical tree species to extract the root exudates in a karst forest of the Qianling Mountains (106°41′–106°42′E, 26°17′–26°22′N, 1,100–1,396 m elevation) in Guiyang, China. These species included Carpinus pubescens, Cladrastis platycarpa, Zanthoxylum planispinum Sieb.et Zucc., Ligustrum lucidum, Itea yunnanensis, Cinnamomum glanduliferum, Cyclobalanopsis gracilis, and Platycarya longipes. We further determined three sample trees for each species based on the similarity in the individual growth and the lack of diseases, pests, and anthropogenic disturbance impacting the individual growth. The litters, humus layers, and soils under the canopy of each sample tree were then removed. After finding the living fibrous roots, we removed the external (thickness >1 cm) soils around the roots and collected the inner soils (0–1 cm thickness; at least 500 g). The subsamples, which equaled 60 g of dried soils, were taken from the 500-g soil samples and placed in a wide mouth bottle. The root exudates of the subsamples were extracted with 200 ml ether under oscillating conditions at 20 °C (for 1 h). The mixture in the wide mouth bottle was then filtered and the filtrate was condensed at 20 °C using a rotary evaporator. Finally, the condensed filtrate was diluted to 10 ml to obtain the mother liquid of the root exudates. The remaining soil samples were sieved through 2-mm and 0.25-mm sieves after open-air drying for a week. The soil samples were then used to test the organic matter of the root exudates. All field experiments of this study have been approved by the Administration Bureau of Two Lakes and One Reservoir in Guiyang.

Soil incubation and AES tests

Approximately 2 kg of samples of rendzina soil were collected from a depth of 0–20 cm in an evergreen broadleaf forest (elevation: 1,220 m) in Guiyang. After open-air drying, the soil samples were sieved through a 2-mm sieve. We then weighed three soils samples of 30 g, which were put into three 100-ml conical flasks. We successively added one, two, and three times the volume (10 ml) of the mother liquid from one tree species to the conical flasks. The different volumes of the mother liquid were replicated three times. The control samples were prepared by adding the same volume of distilled water to three 100-ml conical flasks with 30 g of soil sample. We then adjusted the C/N ratio of the mixtures in all conical flasks to 10 using KNO₃ solution (the amount of KNO₃ solution was calculated based on the C and N contents of the soil samples and the mother liquid of the root exudates, which were determined in advance using the potassium dichromate oxidation method and Kjeldahl determination, respectively). The water content of the mixtures was also adjusted to ∼60% of the field moisture capacity. Subsequently, all conical flasks were closed with rubber stoppers and were incubated at 25 °C in illuminating incubators for 8 h every day for 25 days. The water-stable aggregates and microaggregates of the soils were then tested. The water-stable aggregates were analyzed using the wet-screening method and an aggregate analyzer, which included the five particle diameters: >2mm, 2–1 mm, 1–0.5 mm, 0.5–0.25 mm and <0.25 mm. The microaggregates were measured with the pipette method (0.25–0.05 mm, 0.05–0.02 mm, 0.02–0.002 mm and <0.002 mm). The MWD and GMD were calculated using Eqs. (1) and (2). (1)${\displaystyle \mathrm{MWD\; =}\frac{\left(\sum _{i=1}^n\overline{X_i}{W}_i\right)}{\sum _{i=1}^n{W}_i}}$ (2)${\displaystyle \mathrm{GMD}=exp\left(\frac{\sum _{i=1}^n{W}_{i}ln\overline{X_i}}{\sum _{i=1}^n{W}_i}\right)}$ where $\overline{x_i}$ is the mean diameter (mm) of the aggregates within a particle-size range and W_i is the ratio of the weight (g) of the aggregates within the particle-size range to the total weight (g) of the soil sample.

GC-MS analysis of the organic matter and active biological matter tests

We sieved 40 g of the soil samples from the rhizospheres of each tree species through a 40-mesh sieve and filled the soil in a 500-ml conical flask with a stopper. Subsequently, 150 ml of dichloromethane was decanted into the conical flask. The conical flask was closed with a stopper and continually oscillated for 1 h. Subsequently, the mixture in the conical flask was extracted for 20 min using ultrasonic waves and filtrated. The residue was collected and placed into another 500-ml conical flask. The root exudates of the residue were extracted in the same way. The filtrates from two extractions were mixed, concentrated for 20 min with a rotary evaporator, and dissolved with 5 ml ether that was led through a 0.45-µm filter membrane. Finally, the mixed liquid was filled into a sterile centrifuge tube for GC-MS analysis.

The GC-MS analysis of the organic matter was performed using a HP 6890 gas chromatograph equipped with an Agilent MSD 5975C mass spectrometer (Agilent Technologies) and chromatographic column (AB-5MS 5% phenyl-95% dimethylpolysiloxane; 30 m × 0.25 mm × 0.25 µm; elastic quartz capillary). The temperature in the vaporization chamber was maintained at 250 °C. Highly pure helium was used as the carrier gas, with a flow rate of 1.0 ml min⁻¹. The inlet pressure was 7.62 psi; the split ratio was 20:1. The solvent delay time was set to 1.5 min (Müller et al., 2008; Hutzler et al., 2014). The identification of the organic matter and relative contents was conducted with a mass spectrometry data system. Specifically, the different peaks of the total ion spectrum were first compared with the standard spectrum of the NIST05 and Wiley275 databases to determine the volatile constituents of the root exudates. The peak area normalization method was then used to measure the relative mass fraction of the volatile constituents.

We tested the active biological matter of the rhizosphere soils, including total sugar, total amino acids, phenolic compounds, and free amino acid, using anthracenone colorimetry (Abdelhamid et al., 2013), tri-ketone colorimetry (Song et al., 2009a; Song et al., 2009b), and Folin-ciocalteu colorimetry (Song et al., 2009a; Song et al., 2009b; Faujdar, Prasad & Paliwal, 2012).

Tests and analysis of additional plant species

We selected 34 additional tree species (different from the eight tree species) of the karst forests of Guiyang to validate the effect of the root exudates. Specifically, we used the same methods as described above to collect the rhizosphere soils of three sample trees for each species, extract the root exudates, and identify the contents of the organic matter with GC-MS (only 13 tree species showed satisfactory flow diagrams). The active biological matter was detected with biochemical methods. The four comprehensive AES indices, aggregate status, degree of aggregation, dispersion ratio, and dispersion coefficient, of the rhizosphere soils of the 34 species and the previous 8 plants were quantified using the Eqs. (3)–(6) (Wang et al., 2014a; Wang et al., 2014b). Finally, the relationship between the AES indices and the organic matter in the root exudates was analyzed. (3)${\displaystyle \text{Aggregate status}\left(\text{\%}\right)=\left(\text{the micro-aggregate at a}>50\text{µm particle diameter},\text{\%}\right)-\left(\text{soil mechanical components at a}>50\text{µm particle diameter},\text{\%}\right)}$ (4)${\displaystyle \text{Degree of aggregation}\left(\text{\%}\right)=\frac{\text{Aggregate status}\times 100}{\text{The micro-aggregates at a}>50\text{µm particel diameter}}}$ (5)${\displaystyle \text{Dispersion ratio}\left(\text{\%}\right)=\frac{\text{The micro-aggreate at a}<50\text{µm particle diameter}\times 100}{\text{Soil mechanical components at a}<50\text{µm particle diameter}}}$ (6)${\displaystyle \text{Dispersion coefficient}\left(\text{\%}\right)=\frac{\text{The micro-aggreate at a}<2\text{µm particle diameter}\times 100}{\text{Soil mechanical components at a}<2\text{µm particle diameter}}}$

Data analysis

The t-test was used to measure the differences between the control samples (i.e., test of single population) and the water-stable aggregates, microaggregates, MWD, and GMD, respectively. Specifically, we used the formula t =  $\left(\bar{\mu }-{\mu }_0\right)\sqrt{n-1}∕s$ where $\bar{\mu }$ and μ₀ were the mean indices of the AES incubated with the root exudates and the distilled water (controls), respectively; s is the standard deviation; and n is the number of samples. A variation coefficient (CV) was used to test the interspecific variation of the individual indices of the AES (F-and T-tests could not be applied); $\mathit{CV}\left(\text{\%}\right)=s\times 100∕\bar{\mu }$. When CV⩾30%, it was statistically defined that there was a significant statistical difference among plant species; when CV < 30%, the significance level was determined by CV_u. The CV_u is an upper confidence limit of the CV. When CV < CV_u, no significant statistical variation between the plant species was observed; Here, the ${\mathit{CV}}_u=\left\{\left(\right.X_{1-\alpha }^2\left(n-1\right)\left[\right.1+{CV}^2\left(n-1\right)∕n\left.\right)\left.\right]\right\}∕\left[\left(n-1\right){CV}^2\right]$, where $X_{1-\alpha }^2\left(n-1\right)$ was obtained by searching the quantiles of the chi-squared distribution when the free degree =n − 1 and probability 1 − α (Standardization Administration of PRC, 2009).

The interspecific differences of the relative contents of the organic matter identified by GC-MS and the active biological matter were also tested using CV. The AES of the rhizosphere and non-rhizosphere soils was compared using the t-test of a double-population. The relationship between the four comprehensive AES indices and the organic compounds was described with Pearson’s correlation coefficients. The significance levels were also tested with a t-test.

Effects of the root exudates on the AES

The comparison with the control samples shows that the amount of water-stable aggregates (>2 mm and 2–1 mm) of the soils incubated with 1–3 times of the mother liquid of the root exudates increased, except for a few of the soil samples (Table 1). The average increase is 15.52% and 21.39% (1×), 13.33% and 35.58% (2×), and 19.25% and 40.65% (3×) for the two aggregate diameters, respectively. The rhizosphere soils of C.platycarpa, C.gracilis,I.yunnanensis and P.longipes contain more water-stable aggregates (>2 mm and 2–1 mm) than other plants. However, the incubation of the soils with 1–3 times of the mother liquid resulted in the decrease of the concentration of water-stable aggregates (<0.25 mm) of 41.3% (1×), 51.34% (2×) and 58.30% (3×), respectively (Table 1). The water-stable aggregates of the soils incubated with 2–3 times of the mother liquid did not always show a higher percentage. The t-test indicated a significant difference between the water-stable aggregates (>2 mm, 2–1 mm, and <0.25 mm) and the control samples. The water-stable aggregates with diameters of 0.5–0.25 mm show a relatively small change compared to the control samples, although the t-test implied a significant difference. The water-stable aggregates with diameters of 1–0.5 mm did not indicate a notable difference between the incubated soils and control samples (Table 1). The CV and CV_u revealed that the water-stable aggregates of all diameters significantly differ among the tree species; the aggregates with a diameter of 1–0.5 mm or 0.5–0.25 mm show a greater difference than the aggregates with other diameters (Table 1).

The MWD of the soils incubated with 1–3 times of the mother liquid increased on average by 8.58%, 11.34%, and 13.52%, respectively, compared with the control samples (Fig. 2A). The t-test indicated significant differences (1 × :t = 11.58, p < 0.005, n = 8; 2 ×: t = 5.86, p < 0.005, n = 8; 3 ×: t = 10.03, p < 0.005, n = 8). The MWDs of the soils incubated with the root exudates extracted from the rhizosphere soils of C.platycarpa, I. yunnanensis and C. gracilis are relatively greater than that of the other tree species. The GMD of the soils increased by 10.16%, 13.87%, and 16.41%, respectively, compared with the control samples (Fig. 2B).The increasing rates are higher than that of the MWD. The t-test indicated significant differences (1 ×: t = 13.13, p < 0.005, n = 8; 2 ×: t = 7.09, p < 0.005, n = 8; 3 ×: t = 11.08, p < 0.005, n = 8). The GMD of the soils incubated with the root exudates from the rhizospheres of C.platycarpa, I. yunnanensis and C. gracilis are greater than that of the other plant species (Fig. 2B). However, the values of the MWD and GMD did not always increase with increasing volume of the mother liquid.

Compared with the control samples, the microaggregates (0.05–0.02 mm) of the soils incubated with 1–3 times the volume of the mother liquid decreased by 46.42% (1×), 54.72% (2×), and 36.01% (3×), respectively. However, the microaggregates (>0.002 mm) only decreased by 10.70%, 15.34%, and 21.59%, respectively (Table 2). Conversely, the microaggregate concentration (0.02–0.002 mm) strongly increased; the increasing rate is 129.85%, 135.68%, and 157.1%, respectively. The t-test showed no significant differences between the microaggregates with a diameter of 2–0.25 mm or 0.25–0.05 mm and the control samples (Table 2). Comparatively, the differences are more significant between the microaggregates with diameters of 0.05–0.02 mm, 0.02–0.002 mm, or <0.002 mm and the control samples. Based on CV and CV_u, the microaggregates with different diameters significantly differ among the tree species, except for the microaggregate (2–0.25 mm) of the soils incubated with two times the volume of the mother liquid. The CV of the microaggregates with a diameter of 0.05–0.02 mm or 0.02–0.002 mm is relatively large.

Active biological matter and organic matter of the root exudates of the incubated soils

The total sugar content is the highest and lowest in the rhizosphere soils of C. pubescens and P. longipes, respectively (Table 3). The highest and lowest total amino acid contents are observed in the rhizosphere soils of C. platycarpa and L. lucidum and I. yunnanensis, respectively. However, the highest concentrations of phenolic compounds and free amino acid are determined in the rhizosphere soils of C. platycarpa and I. yunnanensis, respectively. The contents of all active biological matter are closely related to the MWD and GMD of the rhizosphere soils of these eight plants; the total amino acid content shows the highest significance. We detected high contents of free amino acid or total amino acid in the rhizosphere soils of C.platycarpa, I.yunnanensis C. gracilis, and P.longipes. The rhizosphere soils of the four plants contain a high concentration of water-stable aggregates (>2 mm and 2–1 mm, Table 1) and show high MWD and GMD values (Fig. 2).

The organic matter in the root exudates identified by GC-MS primarily includes hydrocarbon, amide, alcohol, phenolic ether, aldehyde, acid, ketone, and ester (low concentrations, Table 3). Each type also includes many specific compounds (Supplemental Information 1 and Supplemental Information 2). These types of organic matter account for more than 80% of the total organic matter of the root exudates except for C. glandulifer um and C. gracilis. Hydrocarbon shows the highest percentage of all types.

The amount of specific compounds of hydrocarbon is also the highest. The amide, phenolic ether, alcohol, and ester percentages are relatively lower than that of hydrocarbon. Only four types of organic matter are closely related to MWD and GMD. Amide, aldehyde, and ester showed a higher correlation than other organic matter types. The rhizosphere soils of C.platycarp and C.gracilis contain relatively high contents of amide and ester. Correspondingly, the rhizosphere soils of the two plants comprise a high concentration for water-stable aggregates (>2 mm and 2–1 mm, Table 1) and have high MWD or GMD values (Fig. 2).

AES and root exudates in the rhizosphere soils of additional plants

The status and degree of aggregation of the rhizosphere soils of the additional plant species are greater than that of the non-rhizosphere soils (Table 4). Conversely, the dispersion ratio and coefficient are smaller than those of the non-rhizosphere soils. The paired t-test indicates that the four comprehensive AES indices of the rhizosphere and non-rhizosphere soils are significantly different, except for the degree of aggregation. The status and degree of aggregation and dispersion ratio and coefficient of the additional plant species show a high variation; the CVs of the four AES indices are greater than the CV_u values. The CVs of the dispersion ratio and coefficient are higher than 30% (Table 5).

Based on the CV, the variation of the active biological matter is smaller than that of the organic matter detected by CG-MS. Other statistical quantities such as the variance, standard deviation, mean, maximum, and minimum also vary. On average, the total soluble sugar and phenolic compounds are higher than the total amino acids in the rhizosphere soils of the additional plant species (Table 5). The free amino acid content is the lowest. The CV s of the active biological matter contents are not only greater than the CV_u, but also higher than 30%, indicating a significant interspecific difference. The relative hydrocarbon content is the highest of all nine organic matter types identified by GC-MS. The relative alcohol and ester contents rank second and third, respectively. The CV and CV_u also indicate significant differences in the relative contents of these organic matter compounds of the additional 19 plant species; most of the CVs are significantly higher than 30%. Only hydrocarbon and the total in a relative content are slightly lower than 30%. However, both are larger than the CV_u (Table 5). Comparatively, the interspecific variation of the organic matter identified by GC-MS is more significant than that of the active biological matter (Table 5).

Most of the relative organic matter contents (identified by GC-MS) of the root exudates of the rhizosphere soils of the additional plant species do not significantly correlate with the comprehensive AES indices (Table 6). Only hydrocarbon shows a significant correlation with the two comprehensive AES indices, the aggregation status and dispersion ratio. If a 90% confidence level is considered to be a weak correlation, the relative content of the phenolic ether shows significant correlation. However, almost all active biological matter correlates highly with the four comprehensive AES indices (Table 6).

The four comprehensive AES indices indicate different changes with varying contents of organic matter; the organic matter significantly correlates with AES, at least at a 90%- confidence level (Fig. 3 and Table 6). The aggregation status shows an increase with increasing hydrocarbon content, while the dispersion ratio displays a significant decrease (Fig. 3A). The values of the dispersion ratio slightly scatter as a function of the phenolic ether content; however, the dispersion ratio increases, indicating the negative effect of the phenolic ether on the AES (Fig. 3B). The comprehensive AES indices correlate relatively strongly with the active biological matter contents (Figs. 3C–3E). Specifically, both the status and degree of aggregation present logarithmic or linear growth with increasing active biological matter content. Consequently, the dispersion ratio and coefficient show a logarithmic decrease or change of the power function. These organic matter compounds explain 20%–76% of the variation in the total effect of the root exudates on the AES based on the different R² values. The phenolic compound can explain the aggregation status the best. It is worth noting that although Fig. 3F was used to describe the relationship between the free amino acid contents and comprehensive AES indices, it also represents the varying characteristics of other insignificant comprehensive AES indices corresponding to the organic matter contents (Table 6).