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Article

Differential and Interactive Effects of Scleroderma sp. and Inorganic Phosphate on Nutrient Uptake and Seedling Quality of Castanea henryi

by
Ronghua Zuo
1,
Feng Zou
1,
Shiyi Tian
1,
Joseph Masabni
2,
Deyi Yuan
1 and
Huan Xiong
1,*
1
Key Laboratory of Cultivation and Protection for Non-Wood Forest Trees, Ministry of Education, College of Forestry, Central South University of Forestry and Technology, Changsha 410004, China
2
Texas A&M AgriLife Research and Extension Center, 17360 Coit Rd., Dallas, TX 75252, USA
*
Author to whom correspondence should be addressed.
Agronomy 2022, 12(4), 901; https://doi.org/10.3390/agronomy12040901
Submission received: 5 March 2022 / Revised: 6 April 2022 / Accepted: 6 April 2022 / Published: 8 April 2022
(This article belongs to the Special Issue Soil Nutrient Cycling and Management of Agronomy)
Browse Figures
Versions Notes

Abstract

:
Both ectomycorrhizal fungi (ECMF) and inorganic phosphate (Pi) can improve plant growth. However, the relationship between Pi levels and mycorrhizal colonization rate is divergent, and information on the differential and interactive effects of Pi levels and ECMF on nutrient uptake and seedling quality is lacking. We conducted a study on 4-week-old Castanea henryi ‘You Zhen’ seedlings by inoculating with Scleroderma sp. (+ECM) to compare with uninoculated (-ECM), under low Pi level (-Pi) and high Pi level (+Pi). The mycorrhizal colonization rate, seedlings morphological and physiological parameters, nutrient uptake content, and the rhizosphere soil enzymatic activities and chemical properties were investigated. Results indicated that the mycorrhizal colonization rate at low Pi level (84 ± 2%) was higher than that at high Pi level (71 ± 2.52%). No matter the Pi level, inoculating with ECMF significantly improved seedling dry weight, height, diameter, and seedling quality index (SQI). The +ECM+Pi and +ECM-Pi treatments significantly increased SQI by 282.76% and 232.76% in comparison to the -ECM-Pi treatment, respectively. Applied Pi had no significant effect on SQI. ECMF inoculation significantly increased nitrogen (N), phosphorus (P), and potassium (K) uptake in roots, stems, and leaves, while Pi application increased the P uptake in roots only. There was no significant interaction between ECMF and Pi levels on seedling quality and nutrient uptake (except P uptake in leaf). Results indicated that ECMF is a suitable alternative to the use of phosphorus fertilizer in nursery production of C. henryi seedlings in terms of protecting the environment, saving resources, and reducing production costs.

1. Introduction

Castanea henryi, a member of the Fagaceae family, is an economically and ecologically important species, and is widely cultivated in the mountains of southern China, especially in Hunan, Fujian, Jiangxi, and Zhejiang Provinces [1,2]. Its nut is one of the most popular foods in China for its high nutritious value, which contains starch, soluble sugars, amino acids, proteins, lipids, and so on [3,4]. To date, planting C. henryi for its nut harvest has been the main source of economic income for growers in the mountains of southern China. In addition, C. henryi forests play an important role in protecting the environment [5]. However, the low survival rate of seedlings immediately after transplanting has been a major hurdle for C. henryi afforestation [6]. This is due to the prevalence of insect and disease pressure [7] and to the low levels of available phosphorus (P) in soils of this region due to their low pH (4.25–5.7) [8]. Low soil pH results in large quantities of inorganic phosphate (Pi) remaining insoluble in the soil, but can easily be fixed [9].
High-quality seedlings have a high tolerance to abiotic and biotic stresses in afforestation [10]. Seedling quality index (SQI) is used in evaluating seedling quality; a higher index value signifies better plant quality [11]. Sáenz et al. [12] proposed that seedlings with SQI < 1.0 are of low quality, while those with SQI > 1.0 are high-quality seedlings. To improve seedling quality, the most common management program in nurseries is to increase fertilizer application [13]. P fertilization can enhance plant biomass [14] and nutrient content [15,16]; however, the long-term intensive application of chemical fertilizer increases the risk of P loss from soil to water causing toxic algae blooms in water bodies and a deleterious effect on the environment [17,18]. Moreover, P is a non-renewable resource, and its availability is becoming depleted over time [19]. Finally, P fertilizer significantly increases the cost of seedlings production in nurseries [20].
Ectomycorrhizal fungi (ECMF) are considered as a promising alternative in reducing fertilizer use in seedling production [21]. Previous studies showed that Scleroderma is a common ECM fungus associated with C. henryi [22], C. mollissima [23], and C. dentata [24]. The role of ECMF in improving plant growth has been evaluated extensively in Castanea species [25,26]. ECMF symbiosis with the host plant resulted in a positive effect on nutrient uptake through the mycelium by the host plant [27]. ECMF also enhanced root length and surface area of the host plant, resulting in increased nutrient uptake [28]. Several studies have indicated a high enzymatic activity near the mycorrhiza, which directly influences nutrient cycling in the rhizosphere soil and nutrient uptake for the host plant [29,30,31].
The assessment of seedling quality is based on the level of mycorrhizal colonization of seedlings [32]. The mycorrhizal colonization of roots can be affected by soil nutrient content, in particularly, soil Pi status [33]. The relationship between Pi level and mycorrhizas colonization rate is contradictory. Graham et al. [34] and Song et al. [35] indicated that a higher mycorrhizal colonization rate can be achieved in low soil Pi levels than high soils Pi levels. However, Gosling et al. [36] and Lin et al. [37] did not support this result, and they indicated that a lower mycorrhizal colonization rate is achieved at low Pi levels in the soil than in soils with high Pi levels.
Several reports demonstrated the interactive effects between mycorrhizal fungi and Pi levels on root growth [38,39]; however, little information is available on the interactive effects between ECMF and Pi on nutrient uptake and seedling quality in general.
Therefore, the following questions were addressed in this research study: (1) is mycorrhizal colonization affected by Pi levels in the soil? (2) What are the effects of ECMF and Pi levels on nutrient uptake and seedling quality? (3) Is there an interactive effect between ECMF and Pi levels on nutrient uptake and seedling quality?

2. Materials and Methods

2.1. Seedling Production

Seeds of C. henryi ‘You Zhen’ were dipped in 0.5% carbendazim solution for 20 min and rinsed in distilled water 5 times. They were then placed in pots (25 cm in height and 16 cm in diameter) containing 1 kg of media substrate. The substrate consisted of acidic red soil, peat (Klasmann, Niedersachsen, Germany), and perlite mixed at a ratio of 3:2:1 (v/v/v). The substrate pH was 5.5, total nitrogen (TN) was 188 mg·kg−1, total phosphorus (TP) was 115 mg·kg−1, total potassium (TK) was 433 mg·kg−1, and available phosphorus (AP) was 2.06 mg·kg−1. The substrate was sterilized with 37% formaldehyde and covered with a plastic film for 1 week, then was placed for 2 weeks under the sun, before filling the pots.

2.2. Inoculum Production and Seedling Inoculation

Fruiting bodies of wild fungus from a C. henryi plantation were used as explants. The wild fungus was separated, purified and sequenced [40]. The ITS sequences were analyzed by DNAMAN software and compared using BLSAT (http://www.ncbi.nlm.nih.gov/BLAST. accessed on 25 November 2019). Thus, the fungus species was identified as Scleroderma sp.
The fungus was maintained as subcultures at 25 °C for about 15 d in Melin–Norkrans (MMN) medium. To produce the inoculation agent, two fungal-activated blocks of 40-mm diameter were placed in a 250 mL flask and were inoculated with 50 mL liquid MMN medium (pH 5.5) for 21 d at 25 °C, then the flasks were placed in the dark and agitated at 180 r·min−1. At the end of the 21 days, we transferred 10 mL of liquid substrates inoculate into 200 g of sterile solid substrate consisting of peat, perlite, and sand at a ratio of 8:1:1 (v/v/v) and were left at 25 °C for 45 d. C. henryi seedlings of approximately 15 cm in height were inoculated on 10 June 2020. A 20-g sample of the solid substrate was buried near the root tips. A 20-g solid substrate without fungal inoculum was used in the control treatment.

2.3. Experimental Design

A completely randomized design was used to evaluate the effects of ECMF and Pi on the nutrient uptake and seedling quality of C. henryi seedlings. Seedlings were randomly separated into four treatment groups: -ECM-Pi treatment (uninoculated and low Pi level), -ECM+Pi treatment (uninoculated and high Pi level), +ECM-Pi treatment (inoculated and low Pi level), and +ECM+Pi treatment (inoculated and high Pi level). Each treatment had three replications with each replication consisting of 15 seedlings for a total of 180 seedlings. Starting on 10 July 2020, the seedlings were supplied weekly with 150 mL of nutrient solution (pH 5.5) for 8 weeks. Each pot was watered twice per week using tap water [41]. The high Pi nutrient solution consisted of 2 mM NH4Cl, 0.5 mM KH2PO4, 2 mM KNO3, 0.6 mM MgSO4, 0.5 mM CaCl2, 0.2 mM NaCl, and micronutrients at 0.2 mL L−1 [42]. In order to maintain a constant K concentration, 0.5 mM KH2PO4 was replaced with KCl in the low Pi nutrient solution. The high Pi nutrient solution delivered the equivalent of 0.14 mM Pi per seedling at the end of the experiment.

2.4. Root Colonization Rate and Seedling Morphological Parameters

At the end of the experiment on 10 November 2020, ten pots were selected from each treatment, and 20 lateral root segments were randomly collected from each pot. The roots colonization rate of mycorrhizal colonization was calculated as the proportion of the number of root segments colonized by ECMF relative to the total number of the sampled root segments [43]. Ectomycorrhizal (ECM) morphology was photographed using a stereoscope (SZX16, Olympus, Tokyo, Japan), and the anatomical characteristics were observed by paraffin sectioning using an optical microscope (BX-51, Olympus, Tokyo, Japan) [44].
Plant height was measured using a band tape and the base diameter was measured using vernier calipers (Mitutoyo, Kawasaki, Japan). Root architecture was analyzed using a scanner (WinRHIZO PRO 2013, Regent Instruments, Quebec, QC, Canada) [45]. After scanning, shoots (stem and leaf) and roots were dried at 70 °C for 48 h. Then, the seedling quality index (SQI) of each treatment was calculated using the following formula [46].
SQI = s h o o t   d r y   w e i g h t + r o o t   d r y   w e i g h t h e i g h t d i a m e t e r + s h o o t   d r y   w e i g h t r o o t   d r y   w e i g h t

2.5. Physiological Parameters of Seedlings

After drying, each part of the plant tissue (root, stem, and leaf) was ground to a fine powder to measure its nutrient content. Nitrogen content (N) was measured using the SN-1965 Kjeldahl method [47]. Phosphorus (P) was assayed using the molybdenum anti-colorimetric method [48]. Potassium (K) was measured using the flame photometry method [49]. N, P, and K uptake was calculated by multiplying the N, P, and K content by the plant dry weight, as described by Husna et al. [50]. The acid phosphatase activity and alkaline phosphatase activity of root tips were measured using the kit by Solarbio Science and Technology Co. (Beijing, China) [51].

2.6. Rhizosphere Soil Chemical Properties and Enzymatic Activities

Each soil sample was collected from a pooled sample of 5 seedlings and each treatment consisted of 3 soil sample replicates. The soil sample was collected near the plant roots using sterile brushes and sieved through a 0.25-mm sieve. The pH was measured with a pH meter (PHS-3E, Shanghai, China). TN, TP, and TK content of the rhizosphere soils was determined using the same method as plant tissues [47,48,49]. Available P was analyzed using the method of Maluf et al. [52]. The catalase, urease, sucrase, and acidic phosphatase activities of each sample were measured using the kit by Solarbio Science and Technology Co. (Beijing, China) [53].

2.7. Statistical Analysis

The data were analyzed using a one-way ANOVA and mean comparisons were done using Duncan’s Multiple Range Test at p < 0.05 level using SPSS software version 21.0 (SPSS Inc., Chicago, IL, USA), with each treatment consisting of 3 replicates. The Origin 8.5 software (Origin Laboratory, Northampton, MA) was used to draw figures. The effect of physiological and biochemical indices of C. henryi seedlings were collectively evaluated using the principal component analysis, and the relationships between SQI and N, P, and K uptake (in root, stem, and leaf) were evaluated by linear regression using the statistical program R3.6.3 [54]. A two-way ANOVA was used to evaluate the significance of ECMF, Pi, and ECMF × Pi effects on SQI and N, P, and K uptake (in root, stem, and leaf) of C. henryi seedlings.

3. Results

3.1. Morphology of ECM and Mycorrhizal Colonization Rate

The +ECM treatments resulted in symbiotic associations between ECMF and C. henryi roots (Figure 1a,b). The root colonization rate differed with Pi treatments. The mycorrhizal colonization rate of +ECM-Pi treatment ranged from 82% to 86% with an average of 84% ± 2% and was higher than +ECM+Pi treatment at 68% to 73% with an average of 71% ± 2.52% (Figure 1c). The ECM was white in color, and there was an abundant epitaxial mycelium winding around the ECMF-colonized root tips (Figure 1a). No mycorrhizal colonization was observed in -ECM treatments.

3.2. Morphological Parameters of C. henryi Seedlings

Inoculating with ECMF significantly improved dry weight, height, base diameter, and SQI of C. henryi seedlings, and there were no differences at low or high Pi levels (Figure 2). On the other hand, applying Pi alone (-ECM+Pi treatment) had no significant effect on dry weight, height, base diameter, and SQI (Figure 2). Leaf dry weight of +ECM-Pi and +ECM+Pi treatments was 3.16 times and 3.33 times greater than -ECM-Pi treatment, respectively. Stem dry weight of +ECM-Pi and +ECM+Pi treatments was 3.37 times and 4.68 times greater than -ECM-Pi treatment, respectively. Root dry weight of +ECM-Pi and +ECM+Pi treatments was 3.34 times and 3.58 times greater than -ECM-Pi treatment, respectively (Figure 2b). The +ECM-Pi and +ECM+Pi treatments significantly increased seedling height by 79.78% and 94.81%, and base diameter by 47.31% and 69.94% compared to the -ECM-Pi treatment, respectively (Figure 2c,d). The +ECM+Pi and +ECM-Pi treatments significantly increased SQI by 282.76% and 232.76% in comparison to the -ECM-Pi treatment, respectively (Figure 2e).
Inoculating with ECMF significantly increased total length, surface area, average diameter, and volume of roots of C. henryi seedlings. There were no observed differences between low Pi and high Pi levels (Table 1). Applying Pi alone (-ECM+Pi treatment) had no significant effect on root total length, root surface area, root average diameter, and root volume. The +ECM-Pi and +ECM+Pi treatments significantly increased root total length by 11.39% and 11.55% compared to the -ECM-Pi treatment, respectively (Table 1). Root surface area of +ECM-Pi and +ECM+Pi treatments was significantly higher than -ECM+Pi and -ECM-Pi treatments (Table 1). The +ECM+Pi treatment significantly increased root average diameter by 21.28% compared to the -ECM-Pi treatment (Table 1). Root volume of +ECM-Pi and +ECM+Pi treatments was at least four times greater than -ECM-Pi treatment (Table 1).

3.3. Analysis of Seedling Physiological Parameters

Inoculating with ECMF significantly increased N, P, and K uptake in leaf, stem, and root of C. henryi seedlings; however, there were no significant differences between low Pi and high Pi except for P content in leaves (Figure 3). Applying Pi alone (-ECM+Pi treatment) significantly increased P uptake in roots of C. henryi seedlings (Figure 3b). The promotional effect of ECMF inoculation on P uptake in roots was higher than N and K, while the promotional effect of ECMF inoculation on N uptake in stems was higher than P and K (Figure 3).
The +ECM-Pi and +ECM+Pi treatments significantly increased root N uptake by 252.64% and 270.28%, and stem N uptake by 348.33% and 486.25% in comparison to the -ECM-Pi treatment, respectively. The treatment of +ECM-Pi and +ECM+Pi significantly increased leaf N uptake by 245.30% and 223.73% over the -ECM-Pi treatment, respectively (Figure 3a).
The +ECM-Pi, +ECM+Pi and -ECM+Pi treatments significantly increased root P uptake by 473.18%, 557.95% and 252.32% in comparison to the -ECM-Pi treatment, respectively. The +ECM-Pi and +ECM+Pi treatments significantly increased stem P uptake by 184.72% and 388.89% and leaf P uptake by 260.04% and 176.86% compared to the -ECM-Pi treatment, respectively (Figure 3b).
The +ECM-Pi and +ECM+Pi treatments significantly increased root K uptake by 129.41% and 170.59% and stem K uptake by 160.71% and 289.29% compared to the -ECM-Pi treatment, respectively. The +ECM-Pi and +ECM+Pi treatments significantly increased leaf K uptake by 176.71% and 221.91% over the -ECM-Pi treatment, respectively (Figure 3c).
Inoculating with ECMF significantly increased root acid and alkaline phosphatase activities; however, there were no observed differences between low Pi and high Pi levels (Figure 4). Applying Pi alone (-ECM+Pi treatment) had no effect on root acid and alkaline activities (Figure 4). The root alkaline phosphatase activity of +ECM-Pi and +ECM+Pi treatments was 1.60 times and 1.53 times greater than -ECM-Pi treatment, respectively (Figure 4a). The root acid phosphatase activity of +ECM-Pi and +ECM+Pi treatments were 5.13 times and 5.88 times greater than -ECM-Pi treatment, respectively (Figure 4b).

3.4. Rhizosphere Soil Chemical Properties and Enzymatic Activities

Inoculating with ECMF significantly increased AP, TN, TP, and TK levels. There were significant differences between low Pi and high Pi in AP and TP (Table 2). Applying Pi alone (-ECM+Pi treatment) also significantly increased AP and TP (Table 2). The +ECM-Pi, +ECM+Pi and -ECM+Pi treatments significantly increased AP by 261.59%, 673.19%, and 523.19% and TP by 38.82%, 130.88% and 116.18% in comparison to the -ECM-Pi treatment, respectively. The +ECM-Pi and +ECM+Pi treatments significantly increased TN by 13.68% and 16.14% in comparison to the -ECM-Pi treatment, respectively. The +ECM-Pi treatment significantly increased TK by 6.22% over the -ECM-Pi treatment, respectively. There were no obvious differences in pH among the four treatments. There were no obvious differences in pH among the four treatments (Table 2).
Inoculating with ECMF significantly increased the rhizosphere soil catalase, urease, sucrase, and acid phosphatase activities. There were significant differences between low Pi and high Pi in the urease and acid phosphatase activities (Figure 5). Applying Pi alone (-ECM+Pi treatment) significantly increased urease activity (Figure 5). The catalase activity of +ECM-Pi and +ECM+Pi treatments was 1.55 times and 1.49 times greater than -ECM-Pi treatment, respectively (Figure 5a). The urease activity of +ECM-Pi treatment was significantly higher than +ECM+Pi treatment, and both were 1.20 and 1.15 times greater than -ECM-Pi treatment, respectively (Figure 5b). The +ECM-Pi and +ECM+Pi treatments significantly increased sucrase activity by 17.02% and 13.83% in comparison to the -ECM-Pi treatment, respectively (Figure 5c). Acid phosphatase activity of +ECM-Pi treatment was significantly higher than +ECM+Pi treatment, and both were 1.12 and 1.05 times greater than -ECM-Pi treatment, respectively (Figure 5d).

3.5. Principal Component Analysis (PCA)

PCA analysis is illustrated using a bi-dimensional plot in which components PC1 and PC2 explain 98.35% of data variability of nutrient uptake, and root and seedling growth parameters (Figure 6a). Components PC1 and PC2 explain 84.47% of the data variability of root tip enzyme activities, rhizosphere soil enzyme activities, and chemical properties (Figure 6b). Results indicated that ECMF inoculation had a high positive correlation with nutrient uptake, and root and seedling growth parameters (Figure 6a), while applying Pi alone (-ECM+Pi treatment) had no correlation with nutrient uptake, and root and seedling growth parameters (Figure 6a). Similarly, there was a high positive correlation with root tip enzyme activities, the rhizosphere soil enzyme activities, and chemical properties in the +ECM+Pi treatment and +ECM-Pi treatment (Figure 6b). Applying Pi alone (-ECM+Pi) treatment had no correlation with root tip enzyme activities, the rhizosphere soil enzyme activities, and chemical properties (Figure 6b).

3.6. Linear Regression Analysis

There was a significant positive correlation between SQI, and N, P, and K uptake (in root, stem, and leaf) of C. henryi seedlings (Figure 7). For N uptake, the coefficients of determination (R2) of root, stem, and leaf were 0.91 (p < 0.001), 0.94 (p < 0.001), and 0.79 (p < 0.01), respectively. For P uptake, R2 values for root, stem, and leaf were 0.88 (p < 0.001), 0.84 (p < 0.001), and 0.64 (p < 0.01), respectively. For K uptake, R2 values for root, stem, and leaf were 0.90 (p < 0.001), 0.92 (p < 0.001), and 0.87 (p < 0.01), respectively.

4. Discussion

4.1. Effect of Pi Levels on ECMF Colonization Rate of C. henryi Seedlings

In this study, the colonization rate differed with Pi levels (Figure 1). Soil available phosphorus (AP) at high Pi (10.67 mg·kg−1) was 2.14 times that at low Pi (4.99 mg·kg−1) (Table 2), and both higher than the wild ecological niche where C. henryi grows (3 mg·kg−1) [55]. The colonization rate at low Pi (84%) was higher than at high Pi (71%) (Figure 1). Our results agree with Bougher et al. [56] who observed that the introduction of a low Pi rate significantly stimulated ECMF colonization rate in karri seedlings, and the colonization rate decreased at high Pi. When more phosphate is available, plants could be less dependent on the ECMF and high Pi levels will have an inhibitory effect on ECMF colonization [57,58]. However, when phosphate is scarce, plants could be more dependent on absorbed phosphate through the mycelium and will have a facilitating effect on ECMF colonization [59]. Therefore, the ectomycorrhizal colonization rate increased under low Pi levels, as indicated by our results.

4.2. ECMF and Pi Effects on Seedling Quality and Nutrient Uptake of C. henryi Seedlings

ECMF inoculation is regarded as a good management practice [60], especially in increasing growth and nutrient uptake of seedlings [61]. In this study, inoculating with ECMF significantly improved the growth parameters (Table 1, Figure 2) and nutrient uptake (Figure 3) of C. henryi seedlings. These results are consistent with the study on Quercus ilex [62], Eucalyptus globulus, and Pinus radiata [63]. However, applying Pi alone (-ECM+Pi treatment) had minimal effect on nutrient uptake (except P uptake in roots) and growth (Figure 2 and Figure 3). Additionally, according to the seedling quality standards by Sáenz et al. [12], inoculating with ECMF, with or without additional Pi, resulted in high-quality seedlings, while plants not inoculated with ECMF, with or without Pi, were of low quality (Figure 2). Therefore, we have demonstrated that ECMF could be used as a replacement of P fertilization in nursery production of C. henryi seedlings.
Pi application in this study seemed to have no effect on improving the growth of C. henryi seedlings, which is consistent with the study on Mesua ferrea [64] and coffee [65]. Still, there are many studies indicating that a large application rate of Pi could significantly promote the growth of seedlings [66,67,68]. In our study, application of P alone (-ECM+Pi) treatment resulted in 0.14 mM Pi content in the pot by the end of this experiment, which is 3.36 times lower than that observed in a study on Populus trichocarpa (0.47 mM Pi) [69], indicating that the Pi level in this study might be insufficient to improve the growth of C. henryi seedlings. Additionally, the promoting effect of Pi was not obvious, probably due to the short period of Pi application or availability in our study. A similar tendency was reported by Liu et al. [70] who observed that Pi application hardly enhanced the nutrient uptake of dwarf bamboo in the short term. In general, application of Pi alone (-ECM+Pi treatment) hardly had any effect on soil TN and TK content of C. henryi seedlings (Table 2). However, only a certain proportion of TN, TP, and TK can better promote plant nutrient uptake [71,72], for there was a synergistic regulation mechanism among TN, TP, and TK during plant nutrient uptake [73]. However, due to low levels of TN and TK in -ECM+Pi treatment, the effect of synergistic regulation mechanism in nutrition uptake might not be obvious. Therefore, in order to obtain high-quality seedlings, it may be necessary to use a high rate of P fertilizer over a long period. However, this is contrary to our original goal which is to prevent water and soil eutrophication and reduce the use of phosphate rock and the nursery cost.
Inoculating with ECMF had improved N, P, and K uptake and SQI. In this study, ECMF inoculation significantly increased the root acid and alkaline phosphatase activities (Figure 4), and the rhizosphere soil catalase, urease, sucrase, and acid phosphatase activities (Figure 5). The increases of these enzyme activities might significantly improve AP, TP, TN, and TK [74,75,76], especially for AP and TP of the +ECM treatments which were at least six times and two times greater than -ECM-Pi treatment, respectively (Table 2). In addition, the hyphae also translocate nonlimiting elements, contributing to N, P, and K uptake, which is an advantageous strategy for long-term plant success [77]. Thus, the seedlings’ quality was improved, for there was a significant positive correlation between SQI, and N, P, and K uptake in root, stem, and leaf (Figure 7).

4.3. Interaction between ECMF and Pi on Seedling Quality and Nutrient Uptake of C. henryi Seedlings

In this study, there was no significant interaction between ECMF and Pi levels on seedling quality and nutrient uptake, except for P uptake in leaf (Table 3, p < 0.05). The role of ECMF was determined to be more important than that of Pi on seedling quality and nutrient uptake (Figure 6). These results are consistent with a study on P. koraiensis seedlings [78]. The main reason could be that ECMF exhibits substantial flexibility in its nutrient-acquisition strategies at different Pi levels [79]. On the one hand, in severely P-impoverished soils, ECMF usually plays increasingly important roles in P uptake [80] by increasing the mycorrhizal colonization rate [81], and the acid phosphatase activity [82]. In our study, the mycorrhizal colonization rate in +ECM-Pi treatment (84 ± 2%) was higher than that of +ECM+Pi treatment (71 ± 2.52%). More epitaxial mycelium extended outward into the soil, increasing the surface area to accumulate more soluble P in +ECM-Pi treatment compared to +ECM+Pi treatment. In addition, the acid phosphatase activity of +ECM-Pi treatment (68.41 U·mg−1) was significantly higher than +ECM+Pi treatment (64.42 U·mg−1). The acid phosphatase was conducive to the hydrolysis of organic P, which might increase the absorption and utilization of insoluble P [83]. On the other hand, a relatively low Pi level (only 0.14 mM) was applied per seedling by the end of the experiment. We think there might be a “tipping point of P uptake” in the +ECM+Pi treatment [84].

5. Conclusions

The mycorrhizal colonization rate of C. henryi seedlings was higher at low Pi level than at high Pi level, which is in the range of Pi levels evaluated in this study. The effect of ECMF inoculation was greater than that of Pi application on seedling quality and nutrient uptake. Results indicated that ECMF is a suitable alternative to the use of phosphorus fertilizer in nursery production of C. henryi seedlings, in terms of protecting the environment, saving resources, and reducing production costs.

Author Contributions

R.Z. and H.X. conceived the experiment. R.Z., F.Z. and S.T. developed and carried out the specific methodology. R.Z. contributed to the data analysis. F.Z. and D.Y. supervised the research. H.X. secured funding for the research and was responsible for overall project administration. R.Z., H.X. and J.M. contributed to the preparation of the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financially supported by the National Natural Science Foundation of China (Grant No. 32001309), the Hunan Provincial Department of Education Fund Project (Grant No. 21B0266) and the Central Finance Forestry Science and Technology Promotion Demonstration Fund Project (Grant No. [2021] XT01).

Data Availability Statement

Data sharing not applicable.

Conflicts of Interest

The authors declare that they have no conflict of interest.

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Figure 1. Morphology and anatomy of ECM and root colonization levels of C. henryi seedlings. (a) ECM observed by Stereomicroscope (bar scale = 2 mm); (b) cross section of ECM. Red arrowhead points to Hartig net (bar scale = 50 µm); (c) colonization rate of each treatment in % of the number of root segments colonized by ECMF relative to the total number of the sampled root segments. -ECM-Pi: uninoculated and low Pi; -ECM+Pi: uninoculated and high Pi; +ECM-Pi: inoculated and low Pi; +ECM+Pi: inoculated and high Pi. The error bars indicate standard deviation. Different letters indicate significant differences among treatments (p < 0.05) according to Duncan’s multiple range test.
Figure 1. Morphology and anatomy of ECM and root colonization levels of C. henryi seedlings. (a) ECM observed by Stereomicroscope (bar scale = 2 mm); (b) cross section of ECM. Red arrowhead points to Hartig net (bar scale = 50 µm); (c) colonization rate of each treatment in % of the number of root segments colonized by ECMF relative to the total number of the sampled root segments. -ECM-Pi: uninoculated and low Pi; -ECM+Pi: uninoculated and high Pi; +ECM-Pi: inoculated and low Pi; +ECM+Pi: inoculated and high Pi. The error bars indicate standard deviation. Different letters indicate significant differences among treatments (p < 0.05) according to Duncan’s multiple range test.
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Figure 2. The effects of ECMF and Pi levels on dry weight, height, base diameter, and SQI of the C. henryi seedlings. -ECM-Pi: uninoculated and low Pi; -ECM+Pi: uninoculated and high Pi; +ECM-Pi: inoculated and low Pi; +ECM+Pi: inoculated and high Pi. (a) Visual comparison of C. henryi seedlings growth status of shoot (stem and leaf) and root; (b) dry weight of leaf (green), stem (yellow) and root (grey) of each treatment; (c) plant height of each treatment; (d) plant base diameter of each treatment; (e) SQI (seedling quality index) of each treatment. The error bars refer to standard deviation. For each figure, different letters indicate significant differences (p < 0.05) according to Duncan’s multiple range test.
Figure 2. The effects of ECMF and Pi levels on dry weight, height, base diameter, and SQI of the C. henryi seedlings. -ECM-Pi: uninoculated and low Pi; -ECM+Pi: uninoculated and high Pi; +ECM-Pi: inoculated and low Pi; +ECM+Pi: inoculated and high Pi. (a) Visual comparison of C. henryi seedlings growth status of shoot (stem and leaf) and root; (b) dry weight of leaf (green), stem (yellow) and root (grey) of each treatment; (c) plant height of each treatment; (d) plant base diameter of each treatment; (e) SQI (seedling quality index) of each treatment. The error bars refer to standard deviation. For each figure, different letters indicate significant differences (p < 0.05) according to Duncan’s multiple range test.
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Figure 3. The effects of ECMF and Pi levels on nutrient uptake in leaves, stems, and roots of C. henryi seedlings. -ECM-Pi: uninoculated and low Pi; -ECM+Pi: uninoculated and high Pi; +ECM-Pi: inoculated and low Pi; +ECM+Pi: inoculated and high Pi. (a) The N uptake (g·plant−1) of each treatment; (b) the P uptake (g·plant−1) of each treatment; (c) the K uptake (g·plant−1) of each treatment. Color denotes, green (leaf), yellow (stem) and grey (root). The error bars refer to standard deviation. Different letters indicate significant differences among treatments (p < 0.05) according to Duncan’s multiple range test.
Figure 3. The effects of ECMF and Pi levels on nutrient uptake in leaves, stems, and roots of C. henryi seedlings. -ECM-Pi: uninoculated and low Pi; -ECM+Pi: uninoculated and high Pi; +ECM-Pi: inoculated and low Pi; +ECM+Pi: inoculated and high Pi. (a) The N uptake (g·plant−1) of each treatment; (b) the P uptake (g·plant−1) of each treatment; (c) the K uptake (g·plant−1) of each treatment. Color denotes, green (leaf), yellow (stem) and grey (root). The error bars refer to standard deviation. Different letters indicate significant differences among treatments (p < 0.05) according to Duncan’s multiple range test.
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Figure 4. The effects of ECMF inoculation and Pi levels on root acid and alkaline phosphatase activities of the C. henryi seedlings. -ECM-Pi: uninoculated and low Pi; -ECM+Pi: uninoculated and high Pi; +ECM-Pi: inoculated and low Pi; +ECM+Pi: inoculated and high Pi. (a) Root alkaline phosphatase activity of each treatment; (b) root acid phosphatase activity of each treatment. The error bars refer to standard deviation. Different letters among treatments indicate significant differences (p < 0.05) according to Duncan’s multiple range test.
Figure 4. The effects of ECMF inoculation and Pi levels on root acid and alkaline phosphatase activities of the C. henryi seedlings. -ECM-Pi: uninoculated and low Pi; -ECM+Pi: uninoculated and high Pi; +ECM-Pi: inoculated and low Pi; +ECM+Pi: inoculated and high Pi. (a) Root alkaline phosphatase activity of each treatment; (b) root acid phosphatase activity of each treatment. The error bars refer to standard deviation. Different letters among treatments indicate significant differences (p < 0.05) according to Duncan’s multiple range test.
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Figure 5. The effects of ECMF and Pi levels on the rhizosphere soil enzymatic activities of C. henryi seedlings. -ECM-Pi: uninoculated and low Pi; -ECM+Pi: uninoculated and high Pi; +ECM-Pi: inoculated and low Pi; +ECM+Pi: inoculated and high Pi. (a) Soil catalase activity; (b) soil urease activity; (c) soil sucrase activity; (d) soil acid phosphatase activity. The error bars refer to standard deviation. Values followed by different letters among treatments indicate significant differences (p < 0.05).
Figure 5. The effects of ECMF and Pi levels on the rhizosphere soil enzymatic activities of C. henryi seedlings. -ECM-Pi: uninoculated and low Pi; -ECM+Pi: uninoculated and high Pi; +ECM-Pi: inoculated and low Pi; +ECM+Pi: inoculated and high Pi. (a) Soil catalase activity; (b) soil urease activity; (c) soil sucrase activity; (d) soil acid phosphatase activity. The error bars refer to standard deviation. Values followed by different letters among treatments indicate significant differences (p < 0.05).
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Figure 6. Principal component analysis (PCA) describing the responses of nutrient uptake, and root and seedling growth parameters (a), and the root tip enzyme activities, the rhizosphere soil enzyme activities, and chemical properties (b). -ECM-Pi: uninoculated and low Pi; -ECM+Pi: uninoculated and high Pi; +ECM-Pi: inoculated and low Pi; +ECM+Pi: inoculated and high Pi; H: height (cm); D: diameter (mm); DW: dry weight (g); SQI: seed quality index; TSA: root total surface area (cm2); TL: root total length (cm); RV: root volume (cm3); AD: root average diameter (mm); NU: whole plant (sum of roots, stems and leaves) N uptake (g·plant−1); PU: whole plant (sum of roots, stems and leaves) P uptake (g·plant−1); KU: whole plant (sum of roots, stems and leaves) K uptake (g·plant−1); ALP: root alkaline phosphatase (U·g−1); ACP: root acid phosphatase (U·g−1); S-ACP: soil acid phosphatase (U·mg−1); S-CAT: soil catalase activity (U·g−1); S-SC: soil sucrase activity (U·g−1); S-UE: soil urease activity (U·g−1); AP: available phosphorus (mg·kg−1); TN: soil total nitrogen (mg·kg−1); TP: soil total phosphorus (mg·kg−1); TK: soil total potassium (mg·kg−1); PH, pH.
Figure 6. Principal component analysis (PCA) describing the responses of nutrient uptake, and root and seedling growth parameters (a), and the root tip enzyme activities, the rhizosphere soil enzyme activities, and chemical properties (b). -ECM-Pi: uninoculated and low Pi; -ECM+Pi: uninoculated and high Pi; +ECM-Pi: inoculated and low Pi; +ECM+Pi: inoculated and high Pi; H: height (cm); D: diameter (mm); DW: dry weight (g); SQI: seed quality index; TSA: root total surface area (cm2); TL: root total length (cm); RV: root volume (cm3); AD: root average diameter (mm); NU: whole plant (sum of roots, stems and leaves) N uptake (g·plant−1); PU: whole plant (sum of roots, stems and leaves) P uptake (g·plant−1); KU: whole plant (sum of roots, stems and leaves) K uptake (g·plant−1); ALP: root alkaline phosphatase (U·g−1); ACP: root acid phosphatase (U·g−1); S-ACP: soil acid phosphatase (U·mg−1); S-CAT: soil catalase activity (U·g−1); S-SC: soil sucrase activity (U·g−1); S-UE: soil urease activity (U·g−1); AP: available phosphorus (mg·kg−1); TN: soil total nitrogen (mg·kg−1); TP: soil total phosphorus (mg·kg−1); TK: soil total potassium (mg·kg−1); PH, pH.
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Figure 7. Correlation between SQI and nutrient uptake in the leaf, stem, and root of C. henryi seedlings. NU: N uptake; PU: P uptake; KU: K uptake. Figures represent correlation between SQI and root N uptake (a); stem N uptake (b); leaf N uptake (c); root P uptake (d); stem P uptake (e); leaf P uptake (f); root K uptake (g); stem K uptake (h); and leaf K uptake (i).
Figure 7. Correlation between SQI and nutrient uptake in the leaf, stem, and root of C. henryi seedlings. NU: N uptake; PU: P uptake; KU: K uptake. Figures represent correlation between SQI and root N uptake (a); stem N uptake (b); leaf N uptake (c); root P uptake (d); stem P uptake (e); leaf P uptake (f); root K uptake (g); stem K uptake (h); and leaf K uptake (i).
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Table
Table 1. The effects of ECMF and Pi levels on root architecture of C. henryi seedlings.
Table 1. The effects of ECMF and Pi levels on root architecture of C. henryi seedlings.
Treatment zRoot Total Length (cm)Root Surface Area (cm2)Root Average
Diameter (mm)		Root Volume (cm3)
-ECM-Pi606 ± 11.4 b y31.2 ± 0.2 b0.47 ± 0.03 b1.58 ± 0.50 b
+ECM-Pi675 ± 15.6 a32.9 ± 0.3 a0.53 ± 0.07 ab7.33 ± 2.85 a
-ECM+Pi613 ± 46.2 b32.0 ± 0.4 b0.51 ± 0.01 ab1.81 ± 0.27 b
+ECM+Pi676 ± 16.0 a32.8 ± 0.7 a0.57 ± 0.04 a6.52 ± 0.28 a
z -ECM-Pi: uninoculated and low Pi; -ECM+Pi: uninoculated and high Pi; +ECM-Pi: inoculated and low Pi; +ECM+Pi: inoculated and high Pi. y Within columns, different letters denote significant differences (p < 0.05) between treatments according to Duncan’s multiple range test. Values in the table represent Mean ± SE.
Table
Table 2. Influence of ECMF and Pi levels on the chemical properties of the rhizosphere soil.
Table 2. Influence of ECMF and Pi levels on the chemical properties of the rhizosphere soil.
TreatmentAP (mg·kg−1)TN (mg·kg−1)TP (mg·kg−1)TK (mg·kg−1)pH
-ECM-Pi1.38 ± 0.09 d285 ± 2.5 b68.0 ± 9.5 c386 ± 5.4 b5.44 ± 0.18 a
+ECM-Pi4.99 ± 0.56 c324 ± 15.3 a94.4 ± 16.5 b410 ± 11.5 a5.72 ± 0.21 a
-ECM+Pi8.60 ± 0.07 b296 ± 3.8 b147.0 ± 2.2 a392 ± 17.3 ab5.71 ± 0.26 a
+ECM+Pi10.67 ± 0.11 a331 ± 8.0 a157.0 ± 10.5 a408 ± 9.9 ab5.76 ± 0.20 a
Note: -ECM-Pi: uninoculated and low Pi; -ECM+Pi: uninoculated and high Pi; +ECM-Pi: inoculated and low Pi; +ECM+Pi: inoculated and high Pi; AP: available phosphorus (mg·kg−1); TN: total nitrogen (mg·kg−1); TP: total phosphorus (mg·kg−1); TK: total potassium (mg·kg−1). Different letters denote significant differences (p < 0.05) between the four treatments according to Duncan’s multiple range test. Values in the table represent Mean ± SE.
Table
Table 3. Two-way ANOVA (p values) for SQI and nutrient uptake (N, P, and K) in stem, root, and leaf of C. henryi seedlings.
Table 3. Two-way ANOVA (p values) for SQI and nutrient uptake (N, P, and K) in stem, root, and leaf of C. henryi seedlings.
Variable SourceECMFPiECMF × Pi
SQI0.002 *0.2940.909
N uptakeRoot0.001 *0.4230.682
Stem0.001 *0.2210.540
Leaf0.001 *0.6580.329
P uptakeRoot0.001 *0.035 *0.243
Stem0.001 *0.027 *0.194
Leaf0.000 *0.4420.013 *
K uptakeRoot0.007 *0.2180.849
Stem0.001 *0.1090.284
Leaf0.001 *0.0800.624
Note: p values below 0.05 are highlighted with asterisk (*).
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Zuo, R.; Zou, F.; Tian, S.; Masabni, J.; Yuan, D.; Xiong, H. Differential and Interactive Effects of Scleroderma sp. and Inorganic Phosphate on Nutrient Uptake and Seedling Quality of Castanea henryi. Agronomy 2022, 12, 901. https://doi.org/10.3390/agronomy12040901

AMA Style

Zuo R, Zou F, Tian S, Masabni J, Yuan D, Xiong H. Differential and Interactive Effects of Scleroderma sp. and Inorganic Phosphate on Nutrient Uptake and Seedling Quality of Castanea henryi. Agronomy. 2022; 12(4):901. https://doi.org/10.3390/agronomy12040901

Chicago/Turabian Style

Zuo, Ronghua, Feng Zou, Shiyi Tian, Joseph Masabni, Deyi Yuan, and Huan Xiong. 2022. "Differential and Interactive Effects of Scleroderma sp. and Inorganic Phosphate on Nutrient Uptake and Seedling Quality of Castanea henryi" Agronomy 12, no. 4: 901. https://doi.org/10.3390/agronomy12040901

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