Light Drought Stress Positively Influenced Root Morphological and Endogenous Hormones in Pinus massoniana Seedlings Inoculated with Suillus luteus

Abstract

Aims An ectomycorrhizal fungus (ECMF) may enhance plant drought resistance. However, there is limited information regarding the effects of ECMFs on drought resistance in Pinus massoniana Lamb., a native species representing an afforestation pioneer tree in subtropical regions of China. Methods In this study, a pot experiment was conducted to determine the effects of ECMF Suillus luteus inoculation on the root morphology and endogenous hormones of P. massoniana, including roots, leaves, and stems, under various water treatment conditions. Four water levels (regular, light, moderate, and severe drought) and three inoculations (inoculated Suillus luteus, numbered S12 and S13, and non-ECMF-inoculated) were compared using a factorial design. Results Under drought stress, P. massoniana seedlings inoculated with S12 and S13 had significantly increased root morphology development (p < 0.05). Light drought positively influenced root development, resulting in a more than twofold increase in root length and root surface area compared to non-inoculated seedlings. Concentrations of gibberellic acid (GA), zeatin riboside (ZR), and indole-3-acetic acid (IAA) in roots, stems, and leaves of inoculated S12 and S13 plants were elevated, whereas abscisic acid (ABA) concentrations were significantly lower, compared to non-inoculated seedlings. The ABA concentrations in the roots of S12 and S13 inoculated seedlings under light drought stress were 1.5 times lower than those in non-inoculated controls. Moreover, root development was positively correlated with plant total GA, IAA, and ZR but negatively correlated with ABA. Conclusions S. luteus can promote the root growth and development of P. massoniana seedlings, notably by regulating the balance in the concentration of endogenous hormones, thus improving the drought resistance of P. massoniana seedlings.

1. Introduction

Drought stress can restrict the survival and growth of plants while changing their structure and function [1]. Water scarcity is one of the most critical factors in arid areas, causing plant mortality [2]. Plants adapt to drought by changing their morphology and physiology [3]. One of the strategies used to enhance plant drought resistance, improve soil condition and forestry productivity, and reduce water consumption is inoculation with an ectomycorrhizal fungus (ECMF) [4,5,6]. The ECMF, which is associated with the roots of Pinaceae plants, can be introduced during planting to stimulate growth and contribute to an enhanced tolerance to abiotic stress [7]. Numerous studies have shown that ECMFs can significantly improve the resistance of seedlings under drought stress [8]. An ectomycorrhizal fungus can build a symbiotic relationship with root plants, which enhances their ability to absorb water and nutrients under drought stress [9]. The ectomycorrhiza is surrounded by the mycelia of mycorrhizal fungi, which affects seedling nutrition absorption and root formation, thus playing an essential role in forest production. The mycorrhizal hyphae significantly increase the contact surface of roots to the soil, thus helping roots absorb moisture and mineral nutrients from the soil [10]. Although the Guizhou province of China has a subtropical humid monsoon climate with abundant rainfall, it has serious karst rocky desertification [11]; many areas are prone to periodic droughts due to shallow soil layers and substantial water leakage in mountain areas.

The root is a vital organ that can help the plant absorb water and nutrients and produce signal substances, such as abscisic acid (ABA), which regulate plant growth. As plant roots are susceptible to drought resistance [12], their growth and development can be an essential index for evaluating drought. Studies have found that mycorrhizal fungi changed the morphology of plant roots [12,13], which may be related to the status of plant nutrients and the balance of endogenous hormones [12]. Endogenous hormones, essential chemical signal substances regulating plant growth and root development, play an important role in alleviating environmental stress [14]. Studies have shown that ECMF inoculation can alter the concentration of endogenous hormones such as indole-3-acetic acid (IAA), gibberellic acid (GA), zeatin riboside (ZR), and abscisic acid (ABA), resulting in an enhanced ability to regulate endogenous hormones under drought stress, thereby helping plants to absorb nutrients and enhancing their resistance to stress from the external environment [12,15].

P. massoniana (Pinus massoniana Lamb.), which grows in subtropical regions of China, is a native pioneer tree species [16]. It is characterized by its strong adaptability, rapid growth, high yield, and wide distribution [17]. P. massoniana has an extremely high ecological and economic value and is commonly used for afforestation and timber, lipid, rosin, and turpentine production [18]. It is also a typical ectomycorrhizal tree species [19,20]. In recent years, mycorrhizal afforestation research has attracted increasing attention. The utilization of drought-resistant mycorrhizal P. massoniana for erosion control, ecological restoration, and afforestation in arid regions reveals its high scientific value and crucial practical significance. So far, studies on the effects of ECMFs on P. massoniana have notably focused on its symbiotic characteristics [21], root architecture [22], nutrient absorption [23,24,25], photosynthetic effects [26], stress resistance [27,28,29], and biological information analysis [19,30]. Suillus luteus is a fungus with essential ecological functions widely distributed in temperate forest ecosystems. It can form a symbiotic relationship with plants such as pine trees and promote plant uptake of water and nutrients [31,32]. However, the effect of ectomycorrhizal fungi on the endogenous hormones of P. massoniana under drought stress remains largely unclear. Therefore, based on previous research, in the current study, we used Suillus luteus in inoculating P. massoniana seedlings, which were treated with various drought stress levels, to assess the impact of the ECMF on P. massoniana root morphology and endogenous hormones, and their relationship. The results provide more theoretical basis and technical support for applications using mycorrhizal P. massoniana seedlings.

2. Materials and Methods

2.1. Pre-Experimental Preparations

The fungal strains of Suillus luteus, designated as S12 and S13, were supplied by Professor Huang Jianguo of Southwest University, China. Although these strains exhibit no significant differences in their culture pattern and general function, they have been selected for this study based on preliminary observations of their positive effects on plant growth under drought stress conditions. The strains were cultured at 28 °C on modified melin-norkrans solid medium (Geruien Biotechnology Co., Ltd., Guizhou, China) 9 cm culture plates until the mycelium covered the plates. The agar mycelia disks were next subcultured in 500 mL cone flasks filled with 300 mL of improved potato dextrose liquid medium (Geruien Biotechnology Co., Ltd., Guizhou, China) and cultured in the dark at 25 °C for 20 d. When the plate was covered with mycelia, a mixture of the fungi mycelium and growth medium was used as the inoculum [33].

The seeds of P. massoniana used in this experiment were obtained from the national base of P. massoniana at the Maanshan Forest Farm, Duyun City, Guizhou Province of China (E 107°31′, N 26°15′). Chosen plump P. massoniana seeds were sterilized with 0.5% KMnO₄ for 2 h and washed four times with sterile water. Subsequently, the seeds were sown in vermiculite and watered using sterile water. After germination, the seedlings were transferred to plastic pots (25 cm in diameter, 28 cm in depth, three seeds per pot), containing 5 kg sterile culture substrate, yellow soil (sifted through a 1 cm × 1 cm sieve), and a clean river sand (V/V = 9:2) mixture that was sterilized in a high-temperature autoclave for 2 h under 121 °C. The basic physical and chemical soil properties are provided in Table S1. The seedlings were simultaneously inoculated, with each seedling inoculated with 10 mL S12 or S13 inoculum. The control group was treated with the same amount of sterilized liquid medium. The seedlings were grown in a greenhouse with a day/night mean temperature of approximately 24 °C/17 °C, a light/dark photoperiod of 14 h/10 h, and relative humidity of 76%. Four-month-old seedlings were used as test materials.

2.2. Design of Drought Stress Experiments

A pot experiment was conducted in the greenhouse of Guizhou Minzu University, using a factorial design involving water and inoculation treatments. Twelve treatments were performed, with 15 replicates, each pot being a replicate with three plants per pot. We obtained reliable measurement of seedling root development parameters and endogenous hormone levels by averaging these three replicate assays. This replication method was implemented to ensure the consistency and dependability of the hormone assay data presented in our study. Water treatments were divided into four categories: no drought stress (ND, ≥70%), light drought stress (LD, 55%–70%), moderate drought stress (MD, 45%–55%), and severe drought stress (SD, 30%–45%), wherein the percentage means the relative moisture content of the soil during 1.5 months. The inoculation treatments were performed with Suillus luteus (numbered S12 and S13), while non-inoculated seedlings served as controls. After four months of seedling growth, three seedlings’ root samples were randomly selected to detect ECMF colonization (≥90%) before drought stress. After successful colonization, the water treatments were applied, and the soil water concentration was controlled using the weighing method for 1.5 months. The seedlings were harvested after 1.5 months of drought stress.

2.3. Measurement of Experimental Parameters

Three plant roots of the fresh seedlings were scanned using an Epson digital scanner (Expression 10000 XL1.0) after the treatments and the images (resolution 300 dpi and black-and-white color mode) of the roots were digitally stored. Morphological parameters, such as total root length, projection area, total root surface area, total root volume, root tip number, connection count, average number of first-order lateral roots, and angle of first-order lateral root were calculated using WinRhizo 2013E root analysis system software [34]. The endogenous hormones GA, IAA, ZR, and ABA were detected and analyzed using a Gas Chromatography-Mass Spectrometer (GC-MS Agilent-7890A/5975C, Agilent Technologies, Santa Clara, CA, USA). According to the manufacturer’s instructions, 3 g of P. massoniana test samples (root [those less than 2 mm in diameter], stem, and leaf) were taken and ground into powder in liquid nitrogen. Then, 20 mL of precooled 80% methanol was added to 100 µL of 1 mmol/L ascorbic acid, which was stored at 4 °C overnight. On the next day, the samples were extracted in an ultrasonic ice bath for 2 h in the dark. The supernatant was separated by centrifugation at 10,000 r/min at −10 °C for 15 min. Then, 2 mL of precooled 80% methanol was added to the precipitate, followed by ultrasonic extraction at 4 °C for 2 h. The supernatant was separated by centrifugation at 10,000 r/min at −10 °C for 15 min. Next, the supernatants were combined. The samples were purified on a polyvinyl polypyrrolidone (PVPP) column and a DEAE Sephadex A-25 column, then dried with nitrogen gas, and finally dissolved in chromatographic pure methanol to 1 mL. The samples were filtered using a 0.22 µm organic membrane [35].

2.4. Statistical Analysis

Data were analyzed using SPSS 22.0 software. All data first underwent tests for normality and homogeneity [36]. A one-way ANOVA was initially used, followed by least significant difference (LSD) and Duncan tests at p < 0.05 for comparisons between treatments, with the Bonferroni correction applied to control for multiple comparisons. Subsequently, a two-way ANOVA was conducted to evaluate the significance of drought and inoculation treatments and their interaction. Post-hoc analyses with the Bonferroni correction were performed to identify specific differences between treatments. Pearson correlation coefficients between parameters were also analyzed. All data are reported as the mean ± standard error (SE). The process of drawing figures was implemented utilizing OriginPro 2021 software. The graphical representation of the correlation analysis was conducted using the ggplot2 package in R version 4.3.1 (https://cran.r-project.org/).

3. Results

3.1. Root Morphology

As shown in Figure 1, two-way ANOVA showed that the degree of drought stress significantly impacted root growth, with ECMF inoculation proving to be an effective method for promoting root development (p < 0.01). Regardless of the inoculation status, all root morphological parameters (except the average number of first-order lateral roots) showed a “rising–falling” trend, with an increase in drought stress and a maximum reached under LD. This finding indicates that LD is more conducive to the growth of P. massoniana seedlings and confers a stronger drought resistance to the plant. All root morphological parameters of the inoculated seedlings were significantly higher than for non-inoculated seedlings (p < 0.05). Specifically (Table S2), compared to the non-inoculated control, plants inoculated with S12 and S13 strains showed an increase in total root length of over 2.5 times, an increase in total root surface area of over 2.9 times, and an increase in total root volume of over 3.1 times under LD conditions. Under SD conditions, total root length increased by over 2.3 times, total root surface area increased by over 1.7 times, and total root volume increased by over 3.1 times.

3.2. Gibberellic Acid Concentration

As shown in Figure 2, two-way ANOVA showed that drought stress had a significant effect on plant GA concentration (p < 0.01). In the roots and leaves, the GA concentration of P. massoniana seedlings varied similarly with the aggravation of drought stress, showing a slowly “falling” trend (Figure 2a,c). In contrast, in the stems, the GA concentration from inoculated seedlings displayed a “falling” trend and a “falling–rising–falling” trend for the non-inoculated seedlings (Figure 2b). In the roots, GA concentrations in inoculated S13 plants were slightly higher than in NM under ND and LD; inoculated S12 plants had slightly higher concentrations than in NM under MD and SD (Figure 2a). In the stems, the concentration in S13 plants was higher than non-inoculated seedlings under ND, LD, and SD, but lower than non-inoculated seedlings under MD. Furthermore, the concentration in S12 plants was lower than non-inoculated seedlings under ND, LD, and MD conditions but slightly higher than non-inoculated seedlings under SD, without significant difference (Figure 2b). In the leaves, the concentration in S12 plants was slightly higher than non-inoculated seedlings and S13 under ND, LD, and MD conditions. However, S12 was statistically significantly higher than non-inoculated seedlings (178.81%) and S13 (115.46%) (p < 0.05) under SD, while S13 was higher than non-inoculated seedlings (Figure 2c).

3.3. Zeatin Riboside Concentration

As shown in Figure 3, two-way ANOVA showed that ectomycorrhizal colonization had a significant effect on plant stem ZR concentration (p < 0.01). Regardless of the inoculation status, the ZR concentration in roots, stems, and leaves showed a slow decreasing trend, which remained constant across the four watering conditions. In the roots, the ZR concentration in non-inoculated seedlings was higher compared to seedlings inoculated with S13 and lower compared to seedlings inoculated with S12 during the stress period (Figure 3a). In the stem, the ZR concentration was higher for seedlings inoculated with S12 compared to S13 and non-inoculated seedlings. Under ND and LD conditions, the concentration in S12 plants was significantly higher than non-inoculated seedlings (p < 0.05). Specifically, the concentration of the hormone ZR in the stems of inoculated S12 plants increased to more than 1 times that of uninoculated plants. In the leaves, the ZR concentrations with S12 and S13 were higher than non-inoculated seedlings during the stressor exposure (Figure 3c).

3.4. Indole-3-Acetic Acid Concentration

As shown in Figure 4, two-way ANOVA showed that exotic mycorrhizal colonization and drought stress had significant (p < 0.01) effects on plant stem and leaf IAA concentrations. The IAA concentration in root stems and leaves showed a similar “falling” trend. In the roots, the IAA concentration of seedlings inoculated with S12 and S13 was higher than that of non-inoculated seedlings under the four drought stress conditions (Figure 4a). In the stem, the IAA concentration in seedlings inoculated with S13 was significantly higher than non-inoculated seedlings and seedlings inoculated with S12 (p < 0.05), reaching a maximum under SD. The IAA concentration in the stem of seedlings inoculated with S13 increased more than 1.6 times compared to non-inoculated seedlings and was more than 22.2 times higher than in seedlings inoculated with S12. Under ND and LD conditions, the IAA concentration in the stem of non-inoculated seedlings was also significantly higher than those inoculated with S12 (p < 0.05). At the same time, the difference did not reach statistical significance under MD and SD (p > 0.05, Figure 4b). In the leaves, the IAA concentration of inoculated seedlings was significantly higher than non-inoculated seedlings (p < 0.05) and reached a maximum under SD stress, where in S12 plants, it was increased by approximately 5.3 times, and in S13 plants by approximately 5.8 times, compared to non-inoculated seedlings (Figure 4c).

3.5. Abscisic Acid Concentration

As shown in Figure 4, two-way ANOVA showed that exotic mycorrhizal colonization and drought stress had significant effects on plant stem ABA concentrations (p < 0.01). The ABA concentration in roots and leaves showed a “rising” trend with the aggravation of drought stress (Figure 5a,c). There was no significant change in the inoculated seedlings. The accumulation rate in non-inoculated seedlings was the fastest. The ABA concentration in roots and leaves increased rapidly under MD and more than 0.29 times and 0.22 times compared with LD, respectively (Figure 5a,c). The ABA concentration in stems of non-inoculated seedlings showed a trend of “rising–falling–rising” and reached the lowest under MD, which decreased 0.85 times compared with LD, and reached the highest under SD stress, which increased 9.12 times compared with MD. The ABA concentration in stems of seedlings inoculated with S12 and S13 showed a slow trend of “rising” with the stress (Figure 5b). Under the same drought stress, the ABA concentration in roots and leaves of non-inoculated seedlings was significantly higher than that of inoculated seedlings (p < 0.05) and reached the maximum under SD. Under SD, the ABA concentration in roots and leaves of non-inoculated seedlings was 2.73 times and 3.38 times higher than that of S12 and 6.57 times and 4.25 times higher than that of S13, respectively (Figure 5a,c). Except under MD, the ABA concentration in non-inoculated seedlings was higher than in seedlings inoculated with S12 and S13, and the difference was highly significant (p < 0.01). Under SD, the ABA concentration in stems of non-inoculated seedlings was 1369.33 ng·g⁻¹, which was 0.81 times and 0.85 times higher than that of seedlings inoculated with S12 and S13, respectively (Figure 5b). Except for stems under MD, the sequence of ABA concentration in roots, stems, and leaves of seedlings under the same drought stress followed the order NM > S12 > S13.

3.6. Ratio of Endogenous Hormones

The ratio of IAA/ABA, ZR/ABA, and (IAA + ZR + GA)/ABA hormones was decreased with the aggravation of drought stress, and the decrease was the largest under SD for non-inoculated seedlings, reaching 1.7 times, 1.38 times, and 1.61 times, respectively (Table 1). During the stress process, the decreasing range was less for inoculated seedlings compared to non-inoculated seedlings, and the ratios of IAA/ABA, ZR/ABA, GA/ABA, and (IAA + ZR + GA)/ABA in non-inoculated seedlings were significantly lower than those in inoculated seedlings (p < 0.05). Under SD, the IAA/ABA ratio of seedlings inoculated with S12 and S13 was 88.18% and 94.64% higher than non-inoculated seedlings, the ZR/ABA ratio was 88.15% and 83.74% higher than non-inoculated seedlings, the GA/ABA ratio of seedlings inoculated with S12 and S13 was 89.25% and 89.56% higher than non-inoculated seedlings, and the (IAA + ZR + GA)/ABA ratio was 88.30% and 90.68% higher than non-inoculated seedlings, respectively. Thus, hormone proportions showed significant differences between inoculated and non-inoculated seedlings (p < 0.05) under drought stress.

3.7. Root Morphology and Endogenous Hormones Correlation Analysis

In this study, correlation analyses were carried out using the mean concentrations of endogenous hormones (GA, ZR, IAA, and ABA) obtained in the plant and the mean values of seedling root development parameters (total root length, projected area, total root surface area, etc.). Correlation analysis showed that total root length, projected area, total root surface area, total root volume, connection count, root tip number, average number of first-order lateral roots, and angle of first-order lateral root were positively correlated with GA, IAA, ZR, and highly significantly positively correlated with IAA (p < 0.001, Figure 6a,b), as well as significant negatively correlated with ABA (p < 0.001). These results indicate that the increase of total root length, projected area, total root surface area, total root volume, connection count, root tip number, average number of first-order lateral roots, and angle of first-order lateral root were associated with an accumulation of GA, IAA, and ZR, especially IAA, together with an inhibition of ABA synthesis signaling. The correlation coefficient between root morphology, GA, IAA, and ABA was high. However, the correlation coefficient between root morphology and ZR was low, indicating that root morphology is strongly related to GA, IAA, and ABA accumulation. The correlation coefficients between GA and the angle of first-order lateral root (0.58, p < 0.01), ZR and the root tip number (0.36, p < 0.05), IAA and the total root length (0.67, p < 0.001), as well as ABA and the average number of first-order lateral roots (−0.78, p < 0.001), were the largest.

4. Discussion

The root system is the main organ for the absorption of water and nutrients and is one of the important indexes for evaluating the drought resistance of plants [37]. Tsuji et al. [38] found that under drought conditions, higher root length density, root growth angle, and lateral root development were conducive to water and nutrient uptake by roots [39]. Our study revealed significantly higher total root length, surface area, projection area, volume, root tip number, connection count, average number of first-order lateral roots, and angle of first-order lateral root in ECMF-inoculated P. massoniana seedlings under drought conditions. The structure adapted to the distribution of soil water, allowing the plant to maintain a normal life vitality, thus enhancing the drought resistance of P. massoniana seedlings.

GA is a hormone that promotes plant growth and development. This study showed that under different drought stress conditions, the GA concentration in seedlings’ roots, stems, and leaves decreased, with the lowest concentration measured under SD stress. In contrast, the inoculated seedlings maintained higher levels of GA. These findings are supported by those from a previous study [40]. The decrease of GA concentration is considered an ecological strategy used by P. massoniana to resist drought stress. The decrease in GA concentration can induce bud dormancy as a response to drought stress [41]. In our study, plants inoculated with S13 under LD and ND had slightly higher root and stem GA values than S12 and NM; under MD and SD, plants inoculated with S13 had slightly higher root and leaf GA values than S13 and NM. This indicates that the inoculated fungi might enhance drought acclimatization by modulating the levels of endogenous hormones in plants. A study found that pre-drought treatment helped wheat plants maintain grain development under drought stress, which may be related to the regulatory role of ZR [42]. Our results indicate that inoculation with S12 could increase the ZR content of seedling stems and allow the plants to resist the damage caused by drought effectively. IAA is a crucial signaling substance that is vital to plant growth [43]. Our results showed that with the aggravation of drought stress, the IAA concentration in roots, stems, and leaves of P. massoniana seedlings showed a decreasing trend, with a lower rate in inoculated seedlings compared to non-inoculated seedlings. This indicates that inoculation with S. luteus could slow down the decrease of IAA concentration in seedlings, alleviate the damage caused by drought, and improve drought resistance. The IAA concentration in roots, stems, and leaves of the plant was increased by inoculating with S. luteus, which is consistent with the research results of Ghosh et al. [44] and Wang et al. [45]. ABA can be used as a signaling molecule to induce and initiate the defense response to adversity in plants. The present study’s results revealed the ABA concentration in the roots, stems, and leaves of mycorrhizal seedlings and non-inoculated seedlings continued to increase under drought stress. The studies by Pirasteh et al. [46], Alwhibi et al. [40], and Quan et al. [47] also showed that the ABA concentration of different plants continued to accumulate under drought stress. In this study, the ABA concentration in non-inoculated seedlings was significantly higher under continuous drought than in inoculated seedlings. It reached a maximum under SD, indicating that the non-mycorrhizal seedlings had an accelerated stress defense response to the damage caused by drought. In contrast, the mycorrhizal seedlings could still maintain the regular physiological activity of the P. massoniana plants. This is consistent with previous reports in Catalpa bungee [47,48]. Interestingly, ECMF colonization resulted in different distributions of hormone levels of GA, IAA, ZR, and ABA in roots, stems, and leaves, e.g., GA levels were not high, and ABA levels were high in plant seedlings under the better-growing LD. Under SD, the distribution of ZR concentration in S13 was slightly lower compared to NM; this indicates that the increase and decrease of the different hormones did not inhibit growth as usually expected, possibly due to the mitigating effect of the mycorrhizal inoculation on the physiological response of the plant to drought stress [49,50].

The research indicates that ectomycorrhizal fungi may alter plant physiology, thereby modulating hormone distribution within the plant and leading to changes in the host response to abiotic stresses [51]. Under drought stress, plants regulate their development by regulating the concentration of hormones in the body, which also affect their own growth by changing the ratio of different hormones [52]. The results showed that the ratios of IAA/ABA, ZR/ABA, and (IAA + ZR + GA)/ABA in P. massoniana seedlings decreased with the aggravation of drought stress, but the ratios of inoculated seedlings were significantly increased compared to non-inoculated seedlings (p < 0.05), and the growth parameters of seedling roots were significantly increased (p < 0.05), indicating that mycorrhizal seedlings could regulate their endogenous hormone levels, thus alleviating the inhibition of plant growth by drought. Additionally, the observed decrease in IAA/ABA, ZR/ABA and (IAA + ZR + GA)/ABA ratios in Suillus luteus-inoculated seedlings suggests that the fungus may affect ABA biosynthesis or promote ABA catabolism. This could contribute to the maintenance of open stomata and improved photosynthesis in drought-stressed seedlings, further enhancing their drought resistance [53].

Furthermore, a parallel has been proposed between the root modifications observed and phytohormone production [54]. Correlation analysis showed that the seedling root development parameters were positively correlated with GA, ZR, and IAA, but negatively correlated with ABA. These results reveal that the increase of total root length, projected area, total root surface area, volume, connection count, root tip number, average number of first-order lateral roots, and angle of first-order lateral root could contribute to promoting the accumulation of GA, IAA, and ZR, inhibit the synthesis of ABA, and thus enhance the drought tolerance of plants.

5. Conclusions

To sum up, S. luteus significantly increased the connection count, root tip number, average first-order lateral roots, angle of first-order lateral root, projection area, total root surface area, and total root volume of P. massoniana before light drought stress (LD), and its distribution structure played an essential role in determining drought resistance. In addition, the root morphology was positively correlated with GA, ZR, and IAA and negatively correlated with ABA. S. luteus increased the concentration of GA, ZR, and IAA, especially IAA, and the angle of the first-order lateral root, total root length, and root tip number of P. massoniana seedlings. In contrast, it significantly reduced ABA concentration and the average number of first-order lateral roots. This endocrine normalization was associated with a strong drought resistance, which could ensure the survival rate and quality of P. massoniana seedlings under stressful conditions.

Author Contributions

Conceptualization: Y.W.; Data curation: Y.W., Y.R. and G.T.; Formal analysis: Y.W. and G.T.; Investigation: Y.W. and Y.R.; Methodology: Y.W. and G.T.; Resources: Y.W. and Z.Z.; Software: Y.R. and X.L.; Supervision: Y.W.; Validation: Y.W., Y.R., G.T., X.L. and Z.Z.; Writing—original draft: Y.R., G.T. and X.L.; Writing—review & editing: Y.W., Y.R., G.T., X.L. and Z.Z. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

Data is contained within the article or Supplementary Material.

Acknowledgments

We would like to thank Maanshan Forest Farm for providing the P. massoniana seeds and Huang Jianguo of Southwest University for providing the fungus strains of Suillus luteus.

Conflicts of Interest

The authors declare no conflicts of interest.

Footnotes

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Figures and Table

Figure 1. Effect of Suillus luteus on root morphology, assessed as (a) total root length, (b) projection area, (c) total root surface area, (d) total root volume, (e) root tip number, (f) connection count, (g) average number of first-order lateral roots, and (h) angle of first-order lateral root of Pinus massoniana seedlings under drought stress. CK, non-ECMF-inoculated; ND, no drought stress; LD, light drought stress; MD, moderate drought stress; SD, severe drought stress; DS, drought stress. Different lowercase letters above the bars indicate significant differences between the treatments under the same drought stress condition (Duncan test at p [less than] 0.05). Data are mean ± SE of three replicates (n = 3). Two-way ANOVA output: ns, not significant; ** p [less than] 0.01.

Figure 2. Effects of Suillus luteus on gibberellic acid concentration in roots (a), stems (b), and leaves (c) of Pinus massoniana seedlings under drought stress. NM, non-ECMF-inoculated; ND, no drought stress; LD, light drought; MD, moderate drought; SD, severe drought; DS, drought stress. Different lowercase letters above the bars indicate significant differences between the treatments under the same drought stress condition (Duncan test at p [less than] 0.05). Data are mean ± SE of three replicates (n = 3). Two-way ANOVA output: ns, not significant; ** p [less than] 0.01.

Figure 3. Effects of Suillus luteus on zeatin riboside concentration in roots (a), stems (b), and leaves (c) of Pinus massoniana seedlings under drought stress. NM, non-ECMF-inoculated; ND, no drought stress; LD, light drought; MD, moderate drought; SD, severe drought; DS, drought stress. Different lowercase letters above the bars indicate significant differences between the treatments under the same drought stress condition (Duncan test at p [less than] 0.05). Data are mean ± SE of three replicates (n = 3). Two-way ANOVA output: ns, not significant; ** p [less than] 0.01.

Figure 4. Effects of Suillus luteus on indole-3-acetic acid concentration in roots (a), stems (b), and leaves (c) of Pinus massoniana seedlings under drought stress. NM, non-ECMF-inoculated; ND, no drought stress; LD, light drought; MD, moderate drought; SD, severe drought; DS, drought stress. Different lowercase letters above the bars indicate significant differences among the treatments under the same drought stress condition (Duncan test at p [less than] 0.05). Data are mean ± SE of three replicates (n = 3). Two-way ANOVA output: ns, not significant; ** p [less than] 0.01.

Figure 5. Effects of Suillus luteus on abscisic acid concentration in roots (a), stems (b), and leaves (c) of Pinus massoniana seedlings under drought stress. NM, non-ECMF-inoculated; ND, no drought stress; LD, light drought; MD, moderate drought; SD, severe drought; DS, drought stress. Different lowercase letters above the bars indicate significant differences between the treatments under the same drought stress condition (Duncan test at p [less than] 0.05). Data are mean ± SE of three replicates (n = 3), two-way ANOVA output: ns, not significant; * p [less than] 0.05; ** p [less than] 0.01.

Figure 6. Pearson correlation coefficients between root morphology and endogenous hormone levels in mycorrhizal colonization of Pinus massoniana seedlings under drought stress (n = 36). (a) Correlation heat map; (b) key index correlation matrix. Note: GA, gibberellic acid; ZR, zeatin riboside; IAA, indole-3-acetic acid; ABA, abscisic acid. *** p [less than] 0.001, ** p [less than] 0.01, * p [less than] 0.05.

Table 1

Changes of IAA/ABA, ZR/ABA, GA/ABA, and (IAA + ZR + GA)/ABA ratios of Suillus luteus under drought stress.

Drought Treatment	Strains	IAA/ABA(ng·g−1)	ZR/ABA(ng·g−1)	GA/ABA(ng·g−1)	(IAA + ZR + GA)/ABA(ng·g−1)
ND	NM	0.073 ± 0.011 b	0.066 ± 0.008 b	0.037 ± 0.007 b	0.176 ± 0.025 b
	S12	0.356 ± 0.105 ab	0.355 ± 0.020 a	0.147 ± 0.010 b	0.857 ± 0.134 ab
	S13	0.932 ± 0.350 a	0.425 ± 0.109 a	0.298 ± 0.063 a	1.655 ± 0.521 a
LD	NM	0.053 ± 0.008 c	0.055 ± 0.003 b	0.022 ± 0.002 b	0.131 ± 0.013 c
	S12	0.238 ± 0.009 b	0.296 ± 0.023 a	0.105 ± 0.016 a	0.639 ± 0.047 b
	S13	0.563 ± 0.025 a	0.302 ± 0.050 a	0.156 ± 0.034 a	1.021 ± 0.081 a
MD	NM	0.050 ± 0.003 c	0.073 ± 0.015 b	0.023 ± 0.007 b	0.146 ± 0.018 c
	S12	0.202 ± 0.006 b	0.277 ± 0.002 a	0.090 ± 0.015 a	0.569 ± 0.018 b
	S13	0.479 ± 0.064 a	0.259 ± 0.014 a	0.101 ± 0.015 a	0.839 ± 0.093 a
SD	NM	0.019 ± 0.002 c	0.031 ± 0.006 b	0.007 ± 0.000 b	0.056 ± 0.008 b
	S12	0.157 ± 0.049 b	0.257 ± 0.049 a	0.063 ± 0.011 a	0.478 ± 0.109 a
	S13	0.347 ± 0.028 a	0.188 ± 0.017 a	0.065 ± 0.008 a	0.599 ± 0.019 a
Significance	ECMF	**	**	**	**
	DS	*	**	**	**
	ECMF × DS	ns	ns	**	ns

NM, non-ECMF-inoculated; ND, no drought stress; LD, light drought; MD, moderate drought; SD, severe drought. Different lowercase letters in the same column indicate a statistically significant difference under the same drought stress condition (Duncan test at p < 0.05). Values indicate mean ± SE (n = 3). Two-way ANOVA output: ns, not significant; * p < 0.05; ** p < 0.01.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/f15111997/s1, Table S1 Basic physical and chemical properties of the soil used in the experiment. Table S2 Table of soil root growth and development parameters.

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