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Article

Karst Soil Patch Heterogeneity with Gravels Promotes Plant Root Development and Nutrient Utilization Associated with Arbuscular Mycorrhizal Fungi

by
Qing Li
1,
Muhammad Umer
1,2,
Yun Guo
1,
Kaiping Shen
1,
Tingting Xia
1,
Xinyang Xu
1,
Xu Han
1,
Wenda Ren
1,
Yan Sun
1,
Bangli Wu
1,
Xiao Liu
3 and
Yuejun He
1,2,*
1
Forestry College, Research Center of Forest Ecology, Guizhou University, Guiyang 550025, China
2
Institute for Forest Resources & Environment of Guizhou, Guizhou University, Guiyang 550025, China
3
Forestry Survey and Planning Institute of Guizhou Province, Guizhou University, Guiyang 550003, China
*
Author to whom correspondence should be addressed.
Agronomy 2022, 12(5), 1063; https://doi.org/10.3390/agronomy12051063
Submission received: 25 March 2022 / Revised: 21 April 2022 / Accepted: 26 April 2022 / Published: 28 April 2022
(This article belongs to the Special Issue Emerging Research on Adaptive Plants in Karst Ecosystems)
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Abstract

:
Arbuscular mycorrhizal (AM) fungi associated with plant roots play an essential role in the belowground ecological process in karst habitats with high spatial and substrate heterogeneity. However, the effects of AM fungi on root morphology and nutrient uptake under different soil patch sizes and gravel content in karst habitats are still unclear. A controlled experiment was conducted using a square device divided into 16 grid patches. This experiment had three treatments, including the mycorrhizal fungal treatment inoculated with (M+) or without Glomus etunicatum Becker & Gerd (M−), the patch heterogeneity treatment through the homogeneous patch (Homo), heterogeneity-large patch (Hetl) and heterogeneity-small patch (Hets), and substrate heterogeneity treatment through the gravel-free substrate (GF), gravel-low substrate (GL), and gravel-high substrate (GH). Root traits and nutrients of Bidens pilosa L were analyzed, and the result showed the AM fungi significantly increased the dry weight, length, surface area, average diameter, volume, tips, branching points, and N, P, and K acquisitions of B. pilosa roots, but significantly decreased the specific root length. The Hets with soil and gravel increased the dry weight, length, surface area, tips, branching points, and N, P, and K acquisitions of B. pilosa roots compared with Hetl regulated by AM fungi. The GL and GH treatments also increased the dry weight, length, surface area, tips, branching points, and N, P, and K acquisitions of B. pilosa roots compared with GF regulated by AM fungi. These results indicate that the B. pilosa roots’ nutritional acquisition benefits were higher in Hets mixed with gravel for its root morphological development regulated by AM fungi in karst soil. In conclusion, we suggest that soil patch heterogeneity with gravels promotes root morphological development and nutrient utilization to karst plants associated with arbuscular mycorrhizal fungi.

1. Introduction

Karst landforms mainly developed through carbonate rocks and are a crucial terrestrial ecosystem with the most fragile ecological landscape [1]. The karst landform in southwest China, having an area of approximately 530,000 km2, is the largest in the world [2], and is characterized by soil and water loss, low vegetative coverage, bedrock exposure, and rocky terrain desertification [3]. Karst landscape disintegration induces a higher spatial heterogeneity by the shallow and discontinuous soil layer crossing with the exposed rocks through various heterogeneous habitats such as rocky fissures, rocky gullies, soil faces, and rocky pores [4,5]. Additionally, the karst soil’s natural habitat composition is usually mixed with different gravels due to the differential weathering of rock through physicochemical disintegration [6], which presents soil resource heterogeneity in terms of mechanical components. Therefore, the karst plants always suffer from the spatial patch heterogeneity induced by different microhabitats, such as the soil face, stone face, and swallet. Meanwhile, the substrate heterogeneity of soil substrate is caused by mechanical composition.
Habitat spatial heterogeneity and substrate heterogeneity affect biodiversity and further change the stability and sustainability of the karst ecosystem [7]. The heterogeneous soil resource heterogeneity has long been theoretically recognized as an essential driver of plant species’ coexistence and community diversity [8,9], including soil nutrients and water-induced spatial-scale heterogeneity [10,11], which significantly affects plants and community [12,13,14]. Liu et al. [15] found that soil resource availability was much more important than soil resource heterogeneity in determining the species diversity of plant communities of karst, and it was supported by the resources availability hypothesis that the species diversity among the community was lower in general under low resource availability because a few species could possibly grow and survive [16]. The spatial heterogeneity in essential resource availability can affect placement and growth of leaves and roots, the growth of whole plants, the intensity of inter-plant competition, and the yield and structure of plant populations [17,18,19,20]. For instance, Tamme et al. [20] found that species that segregate along the heterogeneity niche axis grow better in heterogeneous soil, and species’ responses to soil heterogeneity would fluctuate depending on the size of the soil patches. Hutchings et al. [21] demonstrated that plants are most advantaged in heterogeneous conditions, and the proportion of roots developed in rich and poor patches closely matches the relative quality of each patch type.
Furthermore, Qian et al. [22] also revealed that different patch sizes significantly affect biomass; plants’ plastic responses can effectively utilize nutrient-rich patches at larger spatial scales, in contrast to minor spatial scales. Mi et al. [23] found that plants grown in soils containing rock debris were shorter in heterogeneous substrates, and had smaller basal stem diameters and lower root biomass, than plants grown in rock-free soils. Similarly, Masoni et al. [24] indicated that the increase in soil gravel decreased the biomass, N, and P of durum wheat. Therefore, patch and substrate heterogeneity are crucial for plant growth and community establishment under karst habitat conditions. Physiological and morphological responses of plants to habitat heterogeneity initially occur at plant root systems [25]. Plant roots play an essential role in the underground ecological process and maintain aboveground productivity. Plant root developments also affect nutrient uptake [3,26]. The root system is jointly affected by spatial and resource heterogeneity [23,24]. For example, Wijesinghe et al. [27] showed that plants in nutrient-rich patches produced significantly more root biomass than in nutrient-poor patches when patch size was kept large. Hutchings et al. [21] also discovered that plants can respond with great effectiveness to the heterogeneous substrate, closely matching the mass of roots produced in different patches to the relative quality of each patch. Day et al. [28] illustrated that when plants grow in heterogeneous conditions, the plant yield strongly depends on the patch scale, and plant roots obtained more biomass at the larger patch scale than at the smaller patch scale. Alagna et al. [29] stated that soil–rock ratio has a significant impact on root growth, morphological establishment, the gravel contents determining soil pore status, and the hydrological process [30,31]. The gravel contents further affect root growth [32], compared with gravel-free soil, and gravel forms more pores in the soil, which reduce mechanical resistance, thereby encouraging the extension of roots [31,33]. Therefore, patch heterogeneity and substrate heterogeneity jointly affect plant growth, root development, and nutritional uptakes.
Changes in soil physicochemical properties have a response regulation for a plant with the participation of microorganisms [34], and soil microorganisms are essential drivers of plant growth and the establishment of the community [35,36,37]. Changes in soil physicochemical properties also affect plant growth, root development, and nutrient uptake in heterogeneous environments. As an example, arbuscular mycorrhizal (AM) fungi can have symbiotic relationships with most land plants to form mycorrhizae, which have slender hyphae and strong branching ability, and regulate the morphological development of the root system and improve nutrient uptakes [38,39,40,41]. Facelli et al. [18] found that the arbuscular mycorrhizal symbiotic relationship can strongly influence plant growth in heterogeneous soil by facilitating the pre-emption of limiting resources. Plants preferentially obtain resources from soil patches through plant–AM fungi symbionts instead of root foraging because the metabolic cost of mycelial growth is lower than that of root growth [42,43]. Specifically, AM fungal hyphae can respond to heterogeneous soil in which plant roots cannot obtain more soil resources; thus, AM fungi significantly improved biomass, N, and P of plants [42,44]. Furthermore, the extensive underground mycelial network can redistribute the nitrogen and phosphorus in heterogeneous soil patches and enhance plant ability to obtain optimal growth [45,46,47]. Liang et al. [48] found that heterogeneous karst habitats maintain high AM fungal diversity. The karst soil contains significant amounts of detritus and gravel in natural heterogeneous karst habitats, resulting in soil substrate heterogeneity and patch heterogeneity. Many studies have reported the effects of AM fungi on plant growth and nutrient acquisition in karst ecosystems. In particular, He and Xia [49,50] suggested that the external root mycelia of AM fungi can obtain soil resources outside the rhizosphere to increase the biomass, N, and P acquisitions of karst plants. Zhang et al. [51] also showed that AM fungi have a positive effect on the growth of karst plants in heterogeneous soil. However, AM fungi jointly regulate the soil, and patch heterogeneity, which affects plant root development, remains unclear. Therefore, we hypothesized that: (1) AM fungi can promote root growth and nutrient uptakes of plants in heterogeneous karst soil; (2) heterogeneous patches promote more root growth and nutrient uptake of plants than homogeneous patches regulated by AM fungi; and (3) soil mixed with gravels promotes more root growth and nutrient uptake of plants than soil substrate through AM fungi.

2. Materials and Methods

2.1. Experimental Treatments

A controlled experiment was conducted using a square microcosm composed of polypropylene plastic (26 cm × 26 cm × 15 cm, length × width × height) divided into 16 small grid cells through a movable grid plate for forming heterogeneous patches by quantitatively filling with growth substrates in each grid (Figure 1). There were 4 round holes of 0.5 cm diameter at the bottom of the device to prevent water accumulation. This experiment included three treatments, namely, AM fungi, patch heterogeneity, and substrate heterogeneity. Specifically, the AM fungi treatment was inoculated with (M+) or without (M−) AM fungus Glomus etunicatum Becker & Gerd. The patch heterogeneity treatment was conducted through three patch sizes in a microcosm, e.g., homogeneous patch (Homo), heterogeneity-large patch (Hetl), and heterogeneity-small patch (Hets) (Figure 1). The substrate heterogeneity treatment was conducted through three different substrates: gravel-free substrate of 100% soil (GF) for Hetl and Hets patches, the gravel-low substrate mixed with 80% soil and 20% gravel (GL) for Homo patch, and the gravel-high substrate mixed with 60% soil and 40% gravel (GH) for Hetl and Hets patches. Although there were three different substrates, the total amount of soil and gravel components was the same in each treatment microcosm (An equal amount of soil and gravel was placed in each square microcosm used a measuring cylinder), except for the patch size. Three seeds of B. pilosa were sown into each grid patch in each microcosm. A 50 g inoculum of G. etunicatum was added into the M+ treatment, whereas, to the M− treatment was added 50 g of autoclaved G. etunicatum inoculum; this treatment also received an additional 10 mL filtrate by weighing 50 g of inoculum with double-layer filter paper, in order to ensure that M+ and M− treatments had a consistent microflora, except for the target AM fungus. One plant each cell was retained after germination, and there was a total of 16 seedlings in each microcosm.
The soil and gravel were collected from a typical karst habitat near Guiyang. Nutrient concentrations were calculated per kilogram of soil sample using the Bao [52] method, as 0.622 g of total nitrogen, 0.315 g of available nitrogen (AN), 1.274 g of total phosphorus (TP), 0.163 g of available phosphorus (AP), 37.794 g of total potassium (TK), and 0.532 g of available potassium (AK). For one hour, the collected soil and gravel were air dried and sterilized at 0.14 Mpa at 126 °C. In particular, experimental gravels diameter were approximately 2~4 mm when sieved alternatively and developed from the weathering of carbonate rock. The seed of B. pilosa was collected from a typical karst habitat as the pioneer species in the primary succession stage in Huaxi District, Guiyang, Guizhou, China. Additionally, the G. etunicatum inoculum purchased from the Institute of Nutrition Resources, Beijing Academy of Agricultural and Forestry Sciences, BGA0046, was propagated with Trifolium repens through a sterilized limestone soil substrate for four months, which included approximately 150 spores per 10 g of soil, hyphal pieces, and colonized root segments. The eight replicates of treatments included a total of 48 microcosms containing 468 plants in this experiment. Treatments were incubated in a greenhouse for 12 weeks at the western campus of Guizhou University (106°22′ E, 29°49′ N, 1120 m above the sea level), and then were harvested for measurement.

2.2. Measurements of Mycorrhizal Colonization Rate, Dry Weight, and Traits of Root

We adopted the method described by He et al. [38] to determine the mycorrhizal colonization rate. Specifically, fresh plant roots were selected and stained by fuchsin after cleaning the roots in 10% KOH. After staining, the percentage of AM colonization was determined with the gridline intersection method. The root dry weight of B. pilosa was determined by weighing the root material after drying at 80 °C for constant weight. Plant nitrogen concentration was determined by the Kjeldahl method, and phosphorus was determined by Molybdenum-Antimony colorimetry [52]. Plant root morphological traits were measured using a root scanning analysis system (STD 1600 Epsom USA; Winrhigo -Version 410 B) to obtain root length, average diameter, surface area, volume, branching points, and tips of roots. The specific root length, the root area, and the root volume were calculated by the root length, area, and volume divided by the root dry weight, respectively [53].

2.3. Statistical Analysis

Three-way ANOVA was applied for the effects of mycorrhizal fungus (M+ vs. M−), patch heterogeneity (Homo vs. Hetl vs. Hets), and substrate heterogeneity (GL vs. GF vs. GH), and their interactions on root traits and nutrients of nitrogen and phosphorus. Statistical analyses were performed using the SPPS 18.0 software. All graphs were drawn using the Origin 8.0 software, and data were tested for normality and homogeneity of variance before analysis.

3. Results

3.1. Mycorrhizal Colonization Rate for B. pilosa Seedling Roots in Different Heterogeneous Patches

The root mycorrhizal colonization rates of B. pilosa under M+ treatment were different, but the root mycorrhizal colonization rates were zero under all M− treatments (Table 1), for which the AM mycelium and spores were not observed. Under the substrate heterogeneity treatments, the root mycorrhizal colonization rates of the gravel-free substrate of 100% soil (GF) in Hetl and Hets treatments were significantly lower than that of the gravel-high substrate mixed with 60% soil and 40% gravel (GH); however, the colonization of gravel-low substrate mixed with 80% soil and 20% gravel (GL) was significantly greater than GF of Hetl, but not for Hets. The mycorrhizal colonization of Hets was significantly greater than that of Hetl, for both GF and GH treatments, indicating the heterogeneity-small patch could facilitate mycorrhizal colonization of B. pilosa in our study. In addition, the interaction of patch heterogeneity and substrate heterogeneity significantly affected root mycorrhizal colonization rates of B. pilosa. This study showed that the patch heterogeneity in spatial size and substrate heterogeneity with gravel content increased root mycorrhizal colonization of plant seedlings.

3.2. The Dry Weight of B. pilosa Seedling Roots in Different Heterogeneous Patches

The mycorrhizal fungus treatments significantly affected the root dry weight of B. pilosa (Table 2). The root dry weight in the M+ treatment was significantly greater than in M− treatment (Figure 2). The patch heterogeneity treatments significantly affected the root dry weight (Table 2). Under the M+ treatment, the root dry weight in Homo was significantly greater than that in Hets and Hetl of GF; under the M− treatment, there was no significant difference among treatments of different patches (Figure 2). The substrate heterogeneity treatments significantly affected the dry weight of roots (Table 2). Under the M+ treatment, the dry weight of roots in GL and GH was found to be higher than in GF, for both Hetl and Hets; under the M− treatment, GH in Hetl and Hets was significantly greater than GF in Hets (Figure 2). The interaction of patch heterogeneity and substrate heterogeneity significantly affected the dry weight of B. pilosa roots (Table 2). Overall, AM fungi significantly improved the dry weight of B. pilosa roots. The dry weight of roots in the heterogeneity-small patch was greater than that in the homogeneous and heterogeneity-large patches under the gravel-low and gravel-high substrates regulated by AM fungi; the dry weight of roots in the gravel-low and gravel-high substrates was higher than that of the gravel-free substrate regulated by AM fungi.

3.3. B. pilosa Roots Length, Surface Area, Average Diameter, and Volume in Different Heterogeneous Patches

The mycorrhizal fungus treatments significantly affected the morphological traits of B. pilosa roots (Table 3). The length, surface area, volume, and average diameter in roots of the M+ treatment were significantly more than those of M− under all heterogeneous treatments (Figure 3a–d). The patch heterogeneity treatments significantly affected roots’ length and surface area (Table 3). The M+ treatment showed that the root length and surface area of Homo were significantly higher than those of Hetl in GF and GH, and those of Hets in GF. The average root diameter of Hetl and Hets in GH was significantly more prominent than that of Homo, and the root volume of Hets in GH was significantly more than that of Hets in GH. The M− treatment showed that the root length and surface area of Hetl in GH were higher than those of Hets in GH and GF, and the root average diameter and volume of Hets in GH were significantly more than those of Homo (Figure 3a–d). The substrate heterogeneity treatments significantly affected the surface area, volume, and average diameter of roots (Table 3). Under M+ treatment, the root length and surface area of GL were significantly greater than those of GF and GH under Hetl, and of GF under Hets; the root volume of GH under Hets was significantly larger than that of GF under Hetl and Hets; and the root average diameter of GH was significantly more than that of GL and GF, for both Hetl and Hets (Figure 3a-d). Under M− treatment, the root surface area of GH under Hetl was significantly greater than that of GL, and the root volume and average diameter of GH under Hets were significantly greater than those of GF under Hets and GL (Figure 3b–d). The interaction of M × P significantly affected the root length and surface area; and the interaction of M × S significantly affected the surface area, average diameter, and volume of roots. However, the interactions of S × P and M × S × P did not significantly affect the root’s phenotypic traits (Table 3). Overall, AM fungi significantly increased the length, surface area, average diameter, and volume of B. pilosa roots. These root morphological indexes were higher in the homogeneous and heterogeneity-small patches than in the heterogeneity-large patch under the gravel-low and gravel-high substrates, and were more prominent in gravel-low and gravel-high substrates than in gravel-free substrate.

3.4. Root Tips and Branching Points of B. pilosa

The mycorrhizal fungals treatments significantly affected the numbers of root tips and branching points of B. pilosa (Table 4). The results showed that AM fungus significantly improved the numbers of root tips and branching points in all heterogeneous treatments (Figure 4a–b). The patch heterogeneity treatments significantly affected the numbers of root tips and branching points (Table 4). Under the M+ treatment, the numbers of root tips and branching points of Homo and Hets in GH were significantly higher than those of Hetl in GF and GH; under the M− treatment, there were no significant differences among patches of different sizes (Figure 4a–b). For the substrate heterogeneity treatments, under M+, the numbers of root tips and branching points of GL and GH under Hets were greater than those of GF and GH under Hetl, and of GF under Hets treatment; under M−, there were no significant differences in root tips and branching points among different soil substrates (Figure 4a–b). In addition, the interaction of M × P significantly affected the numbers of root tips and branching points (Table 4). Altogether, AM fungi significantly increased the number of root tips and root branching points. The root tips and branching points were more remarkable in the homogeneous and heterogeneity-small patches than in the heterogeneity-large patches under the gravel-low and gravel-high substrates. They were higher in gravel-low and gravel-high substrates under homogeneous and heterogeneity-small patches than in gravel-free substrates.

3.5. B. pilosa Specific Root Traits in Different Heterogeneous Treatments

The mycorrhizal fungals treatments significantly affected the specific root traits of B. pilosa (Table 5). AM fungus significantly improved the specific root volume while decreasing the specific root length and surface area in all heterogeneous treatments (Figure 5). For patch heterogeneity treatments, under the M+ treatment, the specific root length and surface area in Homo were significantly greater those in Hets and Hetl under GH treatment (Figure 5a,b), and the specific root volume in Hetl under GH treatment was significantly greater than those in Homo under GL treatment and in Hets under GH treatment (Figure 5c). Under the M− treatment, the specific root length in Homo was significantly greater than that in Hets under the GH treatment (Figure 5a). The specific root surface area and volume had no significant difference among these patches (Figure 5b,c). The soil substrate heterogeneity treatments significantly affected the specific root traits (Table 5). Unambiguously, under the M+ treatment, the specific root length and surface area in GL under Homo and GF under Hetl were significantly higher than those in GH under Hetl and GH under Hetl (Figure 5c). Under the M− treatment, the specific root length in GL under Homo and GF under Hets were significantly greater than that in GH under Hets (Figure 5a), whereas the specific root surface area and volume had no significant difference among these substrate treatments (Figure 5b,c). In addition, the interactions of M × S and S × P significantly affected specific root volume, and the interaction of M × S × P significantly affected specific root length and volume (Table 5). The results showed that the AM fungi significantly improved the specific root volume while decreasing the specific root length and area. The specific root length, specific root area, and specific root volume in the heterogeneity-large patch were prominently higher than in the heterogeneity-small patch under the gravel-free substrate, and in the heterogeneity-small patch were higher than in the heterogeneity-large patch under gravel-low and gravel-high substrates; the specific root length and specific root area in a gravel-low substrate for the homogeneous patch were greater than those in gravel-free substrate for the heterogeneity-large patch and heterogeneity-small patch.

3.6. N, P and K Acquisitions of B. pilosa Roots

The mycorrhizal fungus treatments affected N, P, and K acquisitions through B. pilosa roots (Table 6). The AM fungus significantly enhanced the root’s ability to acquire the N, P, and K acquisitions in all heterogeneous treatments (Figure 6). The patch heterogeneity treatments significantly affected the root N, P, and K acquisitions of B. pilosa (Table 6). Under the M+ treatment, the root N acquisition in Homo was significantly greater than that in Hetl under GF treatment, whereas that in Homo under GL treatment and in Hetl under GH treatment was significantly lower than that in Hets under GH treatment (Figure 6a). The root P acquisition in Homo was significantly greater than that in Hets and in Hetl under GF treatments (Figure 6b). The root K acquisition in Homo was significantly greater than that in Hets and Hetl under GF treatments, and that in Homo under GL treatment and in Hets under GH treatment was significantly greater than that in Hetl under GF treatment (Figure 6c). Under the M− treatment, the root N acquisition in Hets under GH treatment was significantly greater than that in Homo under GL treatment and in Hetl under GH treatment (Figure 6a). The root P acquisition in Hetl under GH treatment was significantly greater than that in Homo (Figure 6b). The soil substrate heterogeneity treatments significantly affected the root N, P, and K acquisitions of B. pilosa (Table 6). Specifically, under the M+ treatment, the root N, P, and K acquisitions in GF were significantly lower than those in GH and in GL under Hetl, Hets, and Homo (Figure 6). Under the M− treatment, the root N acquisition in GL was significantly greater than that in GH under Hetl, and that in GH under Hets was significantly greater than that in GL (Figure 6a); the root P acquisition in GH under Hetl was significantly higher than that in GL (Figure 6b); the root K acquisition in GF was significantly more than that in GH under Hets (Figure 6c). In addition, the interactions of M × S and M × P significantly affected the root N, P, and K acquisitions of B. pilosa (Table 6). In conclusion, AM fungi significantly improved plant root N, P, and K acquisitions and promoted plant root accumulation of nutrients in heterogeneity-small patches compared to in heterogeneity-large patches, and in gravel-low and gravel-high substrates compared to in gravel-free substrate.

4. Discussion

4.1. Effect of AM Fungi on Root Dry Weight, Morphology, and Nutrients Uptakes of B. pilosa in Heterogeneous Soil

In our experiment, AM fungi significantly affected the dry weight, morphology, and nutrients of B. pilosa roots (Table 2, Table 3, Table 4 and Table 5). It has been well demonstrated that AM fungal mycelia can accompany plant roots to obtain soil resources in a broader range to improve plant nutrients [50,54]. For example, AM fungi can enhance the N and P uptake of host plants by transporting soil nutrients outside the rhizosphere [55,56] and can improve the K uptake of plant roots [57]. He et al. [58] also showed that AM fungi significantly promoted the N and P content of karst plant roots. These studies suggest that AM fungi play a crucial role in facilitating nutrient uptake by host plants, and the results of our experiment verified that AM fungi significantly increased the N, P, and K acquisitions of B. pilosa roots under all heterogeneous treatments. Many studies have documented that AM fungi can promote plant growth by improving plant nutrient acquisition [55,56]. In the present study, AM fungi significantly promoted the growth and development of B. pilosa roots, manifested as an increase in dry weight of the M+ treatment compared with the M− treatment (Figure 2). This is consistent with the results of Hussain et al. [59], who found that the root biomass of tea plant was significantly increased via AM fungi. Studies have shown that AM fungi can change root morphology due to the strong plasticity of plant roots [50]. In the current experiment, AM fungi significantly increased the length, surface area, average diameter, volume, tips, and branching points of B. pilosa roots (Figure 3 and Figure 4); this result verified our first hypothesis that AM fungi can promote root growth and nutrient uptakes of plants in heterogeneous karst soil. Our findings are similar to those of He et al. [38], in that AM fungi significantly changed the root morphology by increasing the length, surface area, and volume of host plant roots in karst soil. The specific root length can reflect the absorption capacity of the root systems [50], and the larger the specific root length, the stronger the ability of plants to absorb water and nutrients; however, it is less dependent on mycorrhiza [60]. AM fungi significantly reduced the specific root length of B. pilosa in our study (Figure 5a), consistent with the results of He et al. [61], who found that inoculation with AM fungi decreased the specific root length of host plants. This may be due to the belowground mycorrhizal network formed by AM fungi replacing part of the functions of plant roots [62].

4.2. Effects of Heterogeneous Patch Size on Plant Root Traits and Nutrients Uptake Regulated by AM Fungi

Roots are critical functional organs of plants, and the root system has strong plasticity, which can be affected by the patch scale of environmental spatial heterogeneity [62,63]. Studies have shown that root traits of plants can have different responses along with the separation of heterogeneous niche axis, despite these responses depending on the matching of root systems and nutrient patches [62,63]. Our study showed that the dry weight, length, surface area, volume, tips, and branch points of B. pilosa roots in Hetl of GH were higher than those in Hets and Homo under the M− treatment (Figure 2, Figure 3 and Figure 4). Day et al. [28] and Hutchings and Wijesinghe [64] also verified that plants growing in heterogeneous soil patches have greater root biomass, length, and surface area in large patches rather than small patches. This could be because the smaller soil patches lead to the appropriate morphological response of plant roots to local conditions not being completed before plants grow beyond a given patch [21]. Therefore, the opportunities for plants to obtain resources in small patches may not be maximized; conversely, plants growing in large patches may obtain more resources, resulting in more remarkable growth in root systems. However, in this experiment, the N, P, and K acquisitions of B. pilosa roots treated with M− showed no significant difference among different patch treatments (Figure 6a–c). This result is inconsistent with our second hypothesis. Cui and Caldwell [65] and Hodge et al. [66] also found that soil patch size did not affect plants’ N and P uptakes, although it differentially affected the root morphology. This may be because the smaller patch differences in heterogeneous treatments cannot affect plant nutrient acquisition [66]. Previous studies have shown that the physiological and morphological plastic response of plant roots to a heterogeneous soil patch is affected by the action of AM fungi [46,50,67]. In this study, AM fungi reversed the root response of B. pilosa to soil patches of different sizes compared with the M− treatment. Specifically, the dry weight, and N, P, and K acquisitions of B. pilosa roots of Homo and Hets in GH were greater than those of Hetl under the M+ treatment (Figure 1, Figure 2, Figure 3 and Figure 4). The epitaxial root hyphae of AM fungi can extend to the soil area outside the root systems to obtain more resources and promote plant growth [55,56]. Furthermore, in small-scale patches with a higher degree of heterogeneity, AM hyphae can contact more soil patches, which stimulates the proliferation and extension of hyphae [47,68]. These may effectively explain the greater dry weight and nutrient acquisitions of B. pilosa roots in homogeneous and heterogeneous small patches with the soil–gravel mixed substrate in this experiment. In addition, the study showed that AM fungi will inevitably cause changes in plant phenotype while promoting plant nutrients [69]. In this experiment, the length, surface area, volume, tips, and branching points of B. pilosa roots of Homo and Hets in GH were greater than those in Hetl under the M+ treatment (Figure 3 and Figure 4). These results were similar to those of Xia et al. [50], suggesting that the epitaxial root mycelia absorbed more nutrients to promote the growth and development of host plant root phenotypes in karst soil. Root length and root surface area can represent the capacity of plants to absorb soil resources, and the greater the length and surface area in roots, the stronger the absorption capacity of plants [70,71]. The increase in the number of root tips can improve the ability of plants to utilize soil resources in situ, and the growth in lateral roots can increase the total root length and expand the utilization area of soil resources by plants [72,73]. Research suggests plants in heterogeneous patches with higher fragmentation need higher root growth to absorb soil resources from adjacent patches, especially in the patches mixed with soil and gravel [62,74]. Mycorrhizal fungi have been widely recognized to promote root development in heterogeneous soils [62,68]. These may effectively explain the greater morphological growth and development of B. pilosa roots in homogeneous and heterogeneity-small patches with the soil–gravel mixed substrate through AM fungi.

4.3. Effects of Substrate Heterogeneity on Plant Root Morphology and Nutrients Uptake Regulated by AM Fungi

In this study, the substrate heterogeneity significantly affected the root dry weight, root morphology, and root N, P, and K acquisitions of B. pilosa (Table 2, Table 3, Table 4 and Table 5). This may be because AM fungi may regulate the effect of gravel content on the root dry weight and nutrient acquisition. Previous research showed that the gravel content significantly affects the root biomass and nutrients uptake [24,75,76] by changing the hydrological process (e.g., water use efficiency) and nutrient mobility in soil [30,31]. For example, the presence of gravel increases the soil porosity and affects root growth [31,32]; in addition, the high gravel content in the soil causes the deficiency of soil water and nutrients [32]. Therefore, excessive gravel content can inhibit plant root growth and nutrient acquisition [77,78]. This is consistent with our results showing that the root dry weight, root N, P, and K acquisitions of B. pilosa in the gravel-free substrate under Hetl were more than those in GL substrate under Homo when not inoculated with AM fungi; whereas those in GL substrate under Homo were higher than those in GF substrate under Hetl when inoculated with AM fungi (Figure 2 and Figure 6a–c). One possible reason for this is that AM fungi can promote plant root growth and accumulate nutrients in the nutrient-poor area with higher gravel content. Many studies showed that the benefits of mycorrhizal fungi are presumably meager in the nutrient-rich area while being more active in enhancing plant root growth and nutrient acquisition in the nutrient-poor area [79,80]. Another possible reason is that it is difficult for the plant roots to access nutrients in soil with higher gravel content; in contrast, the mycelium of AM fungi is thinner than the root system, and therefore more able to access nutrients than the root system, thus promoting the growth of plant roots [62,81].
In addition, root morphology can reveal the ability to absorb nutrients for plants [62,82], and AM fungi may also regulate the effect of gravel content on the root morphology. In this study, the root length, root surface area, root tips, and root branching points in GH were greater than those in GF under the M+ treatment (Figure 3a,b and Figure 4a,b), which indicates that, under high gravel content conditions, the root system has a greater nutrient acquisition capacity via combination with AM fungi than in the gravel-free substrate condition. One possible reason for this is that plant roots have a foraging behavior [83]. Therefore, under high gravel content conditions within heterogeneous patches, plant roots adapt to the heterogeneous resource environment via high root length, root surface area, root tips, and root branching points to access nutrients in neighboring or even more distant soil substrate patches [74]. Moreover, the specific root traits can indicate the trade-off between plant benefits and costs [84]. In the present study, under the Hetl condition, the specific root length and specific root surface area in GH were higher than those in GF under the M− treatment, whereas in GH they were lower than those in GF under the M+ treatment (Figure 5a,b). High specific root length can enhance nutrient acquisition by permitting the exploration of higher soil volume per unit carbon investment in root length [85]; thus, the plants have higher specific root length and area to access more resources under high gravel content conditions than under soil substrate conditions. However, after inoculation with the AM fungi, the mycelium partially replaced the role of the root system [54]; thus, plants may be more dependent on mycelium to uptake nutrients under high gravel content conditions with lower specific root length and area than under soil substrate conditions. Therefore, this study result verified our third hypothesis that the root systems of plants have greater nutrient uptake, and phenotypic growth in the gravel-low and gravel-high substrates was higher than that in gravel-free substrate through AM fungi.

5. Conclusions

In our study, AM fungi enhanced the root development and nutrient uptake of B. pilosa by significantly increasing its dry weight, length, surface area, average diameter, volume, tips, branching points, and N, P, and K acquisitions. The heterogeneity-small patch with soil and gravel increased the dry weight, length, surface area, tips, branching points, and N, P, K acquisitions of B. pilosa roots compared with heterogeneity-large patch regulated by AM fungi; and the gravel-low substrate and gravel-high substrate treatments also increased these root indexes of B. pilosa compared with gravel-free substrate regulated by AM fungi. Overall, the B. pilosa roots have higher growth and nutritional acquisition benefits in heterogeneity-small patches and soil with gravels regulated by AM fungi in karst soil. In conclusion, soil patch heterogeneity with gravels promotes root morphological development and nutrient utilization to karst plants associated with arbuscular mycorrhizal fungi. These results will contribute to studies on ecosystem and vegetation restoration in degraded karst areas.

Author Contributions

Conceptualization, Q.L. and Y.H.; Methodology, X.X.; Software, T.X.; Validation, K.S. and M.U.; Formal Analysis, Y.G. and B.W.; Investigation, Q.L. and X.L.; Resources, W.R.; Data curation, Y.S.; Writing—Original Draft Preparation, Q.L.; Writing—Review and Editing, Q.L.; Visualization, X.H.; Supervision, Y.H.; Project Administration, Y.H.; Funding Acquisition, Y.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (NSFC: 31660156; 31360106), the First-class Disciplines Program on Ecology of Guizhou Province (GNYL [2017] 007), the Guizhou Hundred-level Innovative Talents Project (Qian-ke-he platform talents [2020] 6004), the Science and Technology Project of Guizhou Province ([2021] General-455; [2016] Supporting-2805), the Talent-platform Program of Guizhou Province ([2017] 5788; [2018] 5781).

Data Availability Statement

Not applicable.

Acknowledgments

We thank the Institute of Nutrition Resources, Beijing Academy of Agricultural and Forestry Sciences (NO. BGA0046) for providing Glomus etunicatum for use in our experiments.

Conflicts of Interest

The authors declare no conflict of interest.

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Agronomy 12 01063 g001 550
Figure 1. Schematic representation of experimental design grid patch in the square microcosm. The (a–c) represent homogeneous patch, heterogeneous-large patch and heterogeneous-small patch, respectively. The experiment was conducted using a square device divided into 16 patches. Homo = homogeneous patch. Hetl = heterogeneous-large patch. Hets = heterogeneous-small patch. The GL substrate was filled with a mixed substrate of 80% soil + 20% gravel, and the GF substrate was filled with a substrate of 100% soil; the GH substrate was filled with a mixed substrate of 60% soil + 40% gravel.
Figure 1. Schematic representation of experimental design grid patch in the square microcosm. The (a–c) represent homogeneous patch, heterogeneous-large patch and heterogeneous-small patch, respectively. The experiment was conducted using a square device divided into 16 patches. Homo = homogeneous patch. Hetl = heterogeneous-large patch. Hets = heterogeneous-small patch. The GL substrate was filled with a mixed substrate of 80% soil + 20% gravel, and the GF substrate was filled with a substrate of 100% soil; the GH substrate was filled with a mixed substrate of 60% soil + 40% gravel.
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Figure 2. The dry weight of B. pilosa roots. Abbreviations: M+ = B. pilosa was inoculated with a mycorrhizal fungus. M− = B. pilosa was not inoculated with a mycorrhizal fungus. Homo = homogeneous patch. Hetl = heterogeneous-large patch. Hets = heterogeneous-small patch. GL = gravel-low substrate. GF = gravel-free substrate. GH = gravel-high substrate. The different lowercase letters (x, y) indicate significant differences between M+ and M− treatments of B. pilosa (p ≤ 0.05). The different lowercase letters (a–c) indicate significant differences between Homo, Hetl, and Hets patches (p ≤ 0.05); The different lowercase letters (α–γ) indicate significant differences between GL, GF, and GH substrates (p ≤ 0.05). The error bars represent the standard deviation (SD).
Figure 2. The dry weight of B. pilosa roots. Abbreviations: M+ = B. pilosa was inoculated with a mycorrhizal fungus. M− = B. pilosa was not inoculated with a mycorrhizal fungus. Homo = homogeneous patch. Hetl = heterogeneous-large patch. Hets = heterogeneous-small patch. GL = gravel-low substrate. GF = gravel-free substrate. GH = gravel-high substrate. The different lowercase letters (x, y) indicate significant differences between M+ and M− treatments of B. pilosa (p ≤ 0.05). The different lowercase letters (a–c) indicate significant differences between Homo, Hetl, and Hets patches (p ≤ 0.05); The different lowercase letters (α–γ) indicate significant differences between GL, GF, and GH substrates (p ≤ 0.05). The error bars represent the standard deviation (SD).
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Agronomy 12 01063 g003 550
Figure 3. Phenotypic traits of B. pilosa roots. (a–d) represent the root length, surface area, volume and average diameter, respectively. See Figure 2 for an explanation of M+, M−, Homo, Hetl, Hets, GL, GF, and GH, lowercase letters (x, y) and (a, b, c) and Greek alphabet (α, β, γ).
Figure 3. Phenotypic traits of B. pilosa roots. (a–d) represent the root length, surface area, volume and average diameter, respectively. See Figure 2 for an explanation of M+, M−, Homo, Hetl, Hets, GL, GF, and GH, lowercase letters (x, y) and (a, b, c) and Greek alphabet (α, β, γ).
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Agronomy 12 01063 g004 550
Figure 4. Numbers of root tips and branching points of B. pilosa. (a,b) represent the root tips and branching points, respectively. See Figure 2 for an explanation of M+, M−, Homo, Hetl, Hets, GL, GF and GH, lowercase letters (x, y) and (a, b, c) and Greek alphabet (α, β, γ).
Figure 4. Numbers of root tips and branching points of B. pilosa. (a,b) represent the root tips and branching points, respectively. See Figure 2 for an explanation of M+, M−, Homo, Hetl, Hets, GL, GF and GH, lowercase letters (x, y) and (a, b, c) and Greek alphabet (α, β, γ).
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Agronomy 12 01063 g005 550
Figure 5. Specific traits of B. pilosa roots. (a–c) represent specific root length, surface area and volume, respectively. See Figure 2 for an explanation of M+, M−, Homo, Hetl, Hets, GL, GF, and GH, lowercase letters (x, y) and (a, b, c) and Greek alphabet (α, β, γ).
Figure 5. Specific traits of B. pilosa roots. (a–c) represent specific root length, surface area and volume, respectively. See Figure 2 for an explanation of M+, M−, Homo, Hetl, Hets, GL, GF, and GH, lowercase letters (x, y) and (a, b, c) and Greek alphabet (α, β, γ).
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Agronomy 12 01063 g006 550
Figure 6. Acquisitions of N, P, and K through B. pilosa roots. (a–c) represent the nitrogen, phosphorus and potassium acquisition of plant roots, respectively. See Figure 2 for an explanation of M+, M−, Homo, Hetl, Hets, GL, GF, and GH, lowercase letters (x, y) and (a, b, c) and Greek alphabet (α, β, γ).
Figure 6. Acquisitions of N, P, and K through B. pilosa roots. (a–c) represent the nitrogen, phosphorus and potassium acquisition of plant roots, respectively. See Figure 2 for an explanation of M+, M−, Homo, Hetl, Hets, GL, GF, and GH, lowercase letters (x, y) and (a, b, c) and Greek alphabet (α, β, γ).
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Table
Table 1. The root mycorrhizal colonization rates (%) of Bidens pilosa.
Table 1. The root mycorrhizal colonization rates (%) of Bidens pilosa.
TreatmentsHomoHetlHets
GLGFGFGFGH
M+58.75 ± 0.66xbcβγ48.13 ± 0.97xdΔ48.13 ± 0.97xdΔ58.21 ± 0.67xcγ63.79 ± 0.49xaα
M−0 yaα0 yaα0 yaα0 yaα0 yaα
M+ = B. pilosa was inoculated with a mycorrhizal fungus. M− = B. pilosa was not inoculated with a mycorrhizal fungus. Homo = homogeneous patch. Hetl = heterogeneous-large patch. Hets = heterogeneous-small patch. GL = gravel-low substrate. GF = gravel-free substrate. GH = gravel-high substrate. The different lowercase letters (x, y) indicate significant differences between M+ and M− treatments of B. pilosa (p ≤ 0.05). The different lowercase letters (a–d) indicate significant differences between Homo, Hetl, and Hets patches (p ≤ 0.05); The different lowercase letters (α–Δ) indicate significant differences between GL, GF, and GH substrates (p ≤ 0.05). The values are “mean ± SD”.
Table
Table 2. Three-way ANOVA for the effect of mycorrhizal fungus (M+ vs. M−), patch heterogeneity (Homo vs. Hetl vs. Hets), and soil substrate heterogeneity (GL vs. GF vs. GH) on the dry weight of roots.
Table 2. Three-way ANOVA for the effect of mycorrhizal fungus (M+ vs. M−), patch heterogeneity (Homo vs. Hetl vs. Hets), and soil substrate heterogeneity (GL vs. GF vs. GH) on the dry weight of roots.
TreatmentsDfRoot Dry Weight
Fp
Mycorrhizal fungi (M)1760.150<0.001 ***
Patch heterogeneity (P)24.4690.037 *
Substrate heterogeneity (S)245.832<0.001 ***
M × P24.6580.034 *
M × S244.903<0.001 ***
P × S41.2060.275
M × P × S41.2690.263
Abbreviations: M = mycorrhizal fungus treatments; P = patch heterogeneity treatments; S = substrate heterogeneity treatments; * or *** indicates a significant difference at p ≤ 0.05 or p ≤ 0.001.
Table
Table 3. Three-way ANOVA for the effect of mycorrhizal fungus (M+ vs. M−), patch heterogeneity (Homo vs. Hetl vs. Hets), and soil substrate heterogeneity (GL vs. GF vs. GH) on root length, surface area, and volume, and average diameter.
Table 3. Three-way ANOVA for the effect of mycorrhizal fungus (M+ vs. M−), patch heterogeneity (Homo vs. Hetl vs. Hets), and soil substrate heterogeneity (GL vs. GF vs. GH) on root length, surface area, and volume, and average diameter.
TreatmentsdfRoot Length
(cm)		Root Surface Area
(cm2)		Root Volume
(cm3)		Average Diameter
(mm)
FpFpFpFp
Mycorrhizal fungi (M)1545.125<0.001 ***518.377<0.001 ***501.696<0.001 ***1346.734<0.001 ***
Patch heterogeneity (P)29.9430.002 **6.6120.012 *0.7030.4040.2430.623
Substrate heterogeneity (S)23.8050.0547.0610.009 **7.126<0.001 ***28.376<0.001 ***
M × P210.970<0.001 **6.9550.010 **0.7140.4011.7550.189
M × S23.7470.0566.6830.011 *6.9810.010 **15.542<0.001 ***
P × S40.5500.4610.0670.7961.7210.1930.4700.495
M × P × S40.8720.3530.0720.7891.7010.1962.9920.087
Abbreviations: M = mycorrhizal fungus treatments; P = patch heterogeneity treatments; S = substrate heterogeneity treatments; * or ** or *** indicates a significant difference at p ≤ 0.05 or p ≤ 0.01 or p ≤ 0.001.
Table
Table 4. Three-way ANOVA for the effect of mycorrhizal fungus (M+ vs. M−), patch heterogeneity (Homo vs. Hetl vs. Hets), and soil substrate heterogeneity (GL vs. GF vs. GH) on root tips and branching points.
Table 4. Three-way ANOVA for the effect of mycorrhizal fungus (M+ vs. M−), patch heterogeneity (Homo vs. Hetl vs. Hets), and soil substrate heterogeneity (GL vs. GF vs. GH) on root tips and branching points.
TreatmentsdfRoot TipsRoot Branching Points
FpFp
Mycorrhizal fungi (M)1800.275<0.001 ***459.121<0.001 ***
Patch heterogeneity (P)27.0120.010 **16.386<0.001 ***
Substrate heterogeneity (S)21.9800.1632.3080.132
M × P27.6610.007 **17.632<0.001 ***
M × S21.6040.2092.2090.141
P × S41.7480.1901.4370.234
M × P × S41.5250.2201.6020.209
Abbreviations: M = mycorrhizal fungus treatments; P = patch heterogeneity treatments; S = substrate heterogeneity treatments; ** or *** indicates a significant difference at p ≤ 0.01 or p ≤ 0.001.
Table
Table 5. Three-way ANOVA for the effect of mycorrhizal fungus (M+ vs. M−), patch heterogeneity (Homo vs. Hetl vs. Hets), and soil substrate heterogeneity (GL vs. GF vs. GH) on specific root traits.
Table 5. Three-way ANOVA for the effect of mycorrhizal fungus (M+ vs. M−), patch heterogeneity (Homo vs. Hetl vs. Hets), and soil substrate heterogeneity (GL vs. GF vs. GH) on specific root traits.
TreatmentsdfSpecific Root Length (cm/g)Specific Root Area (cm2/g)Specific Root Volume (cm3/g)
FpFpFp
Mycorrhizal fungi (M)1231.635<0.001 ***11.610<0.001 ***163.225<0.001 ***
Patch heterogeneity (P)20.0170.8980.0020.9683.4250.068
Substrate heterogeneity (S)27.1430.009 **4.3410.040 *7.5680.007 **
M × P20.1800.6730.6130.4363.6460.060
M × S20.1260.7242.7880.0999.0350.003 **
P × S42.4400.1220.0410.8408.2730.005 **
M × P × S47.5330.007 **2.3190.1317.8970.006 **
Abbreviations: M = mycorrhizal fungus treatments; P = patch heterogeneity treatments; S = substrate heterogeneity treatments; * or ** or *** indicates a significant difference at p ≤ 0.05 or p ≤ 0.01 or p ≤ 0.001.
Table
Table 6. Three-way ANOVA for the effect of mycorrhizal fungus (M+ vs. M−), patch heterogeneity (Homo vs. Hetl vs. Hets), and soil substrate heterogeneity (GL vs. GF vs. GH) on the N and P acquisitions.
Table 6. Three-way ANOVA for the effect of mycorrhizal fungus (M+ vs. M−), patch heterogeneity (Homo vs. Hetl vs. Hets), and soil substrate heterogeneity (GL vs. GF vs. GH) on the N and P acquisitions.
TreatmentsdfRoot N AcquisitionRoot P AcquisitionRoot K Acquisition
FpFpFp
Mycorrhizal fungi (M)1549.296<0.001 ***594.266<0.001 ***594.687<0.001 ***
Patch heterogeneity (P)29.2260.003 **7.5400.007 **13.492<0.001 ***
Substrate heterogeneity (S)230.304<0.001 ***33.139<0.001 ***31.153<0.001 ***
M × P28.9050.004 **8.2700.005 **13.390<0.001 ***
M × S230.042<0.001 ***31.502<0.001 ***31.481<0.001 ***
P × S40.2790.5990.0280.8680.0500.824
M × P × S40.1600.6900.0320.8580.0510.821
Abbreviations: M = mycorrhizal fungus treatments; P = patch heterogeneity treatments; S = substrate heterogeneity treatments; ** or *** indicates a significant difference at p ≤ 0.01 or p ≤ 0.001.
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Li, Q.; Umer, M.; Guo, Y.; Shen, K.; Xia, T.; Xu, X.; Han, X.; Ren, W.; Sun, Y.; Wu, B.; et al. Karst Soil Patch Heterogeneity with Gravels Promotes Plant Root Development and Nutrient Utilization Associated with Arbuscular Mycorrhizal Fungi. Agronomy 2022, 12, 1063. https://doi.org/10.3390/agronomy12051063

AMA Style

Li Q, Umer M, Guo Y, Shen K, Xia T, Xu X, Han X, Ren W, Sun Y, Wu B, et al. Karst Soil Patch Heterogeneity with Gravels Promotes Plant Root Development and Nutrient Utilization Associated with Arbuscular Mycorrhizal Fungi. Agronomy. 2022; 12(5):1063. https://doi.org/10.3390/agronomy12051063

Chicago/Turabian Style

Li, Qing, Muhammad Umer, Yun Guo, Kaiping Shen, Tingting Xia, Xinyang Xu, Xu Han, Wenda Ren, Yan Sun, Bangli Wu, and et al. 2022. "Karst Soil Patch Heterogeneity with Gravels Promotes Plant Root Development and Nutrient Utilization Associated with Arbuscular Mycorrhizal Fungi" Agronomy 12, no. 5: 1063. https://doi.org/10.3390/agronomy12051063

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