EFFECTS OF CAO ON THE CLONAL GROWTH AND ROOT ADAPTABILITY OF CYPRESS IN ACIDIC SOILS
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Article
Effects of CaO on the Clonal Growth and Root Adaptability of
Cypress in Acidic Soils
Zhen Zhang, Guoqing Jin *, Tan Chen and Zhichun Zhou
Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Daqiao Rd 73, Fuyang Area,
Hangzhou 311400, China; zhenzh19860516@163.com (Z.Z.); pinus@csf.org.cn (T.C.); zczhou_risf@163.com (Z.Z.)
* Correspondence: jgqin@163.com; Tel.: +86-0571-6331-6172
Abstract: Cypress (Cupressus funebris Endl.) is a major tree species planted for forestland restoration
in low-fertility soil and in areas where rocky desertification has occurred. Calcium (Ca) fertilizer can
adjust the pH of soil and has an important effect on the growth of cypress. Soil and water losses
are serious in Southern China, and soil acidification is increasing, which results in high calcium
loss. However, the adaptability of cypress clones to different concentrations of calcium in acidic
soils has not been studied. In this investigation, a potted-plant experiment was set up with three
concentrations of calcium oxide (CaO) fertilizer (0, 3, and 6 g·kg−1 ) added under local soil conditions
with 0 and 3 g·kg−1 nitrogen (N), phosphorus (P), and potassium (K) fertilizer. The effects of CaO
on the growth, root development, and nutrient uptake and utilization efficiency of cypress clones
were analyzed. The growth, root development, and nutrient absorption and utilization of cypress
differed when calcium fertilizer was applied to acidic soils with different degrees of fertility. In the
soil with 0 g·kg−1 NPK fertilizer, the 3 and 6 g·kg−1 CaO treatments significantly increased the clonal
growth of cypress seedling height, basal diameter, and dry-matter weight. In addition, the length,
surface area, and volume of the roots less than 2.0 mm of root diameter also significantly increased,
indicating that the fine cypress roots were somewhat able to adapt to differing Ca levels under
Citation: Zhang, Z.; Jin, G.; Chen, T.; lower fertility conditions. Moreover, the efficiency of N, P, and Ca accumulation was highest in the
Zhou, Z. Effects of CaO on the Clonal
3 g·kg−1 CaO treatment. After adding 3 g·kg−1 CaO fertilizer to the soil with 3 g·kg−1 NPK fertilizer,
Growth and Root Adaptability of
only the root dry-matter weight increased significantly, indicating that root development (including
Cypress in Acidic Soils. Forests 2021,
root length, surface area, and volume) in the D1–D3 diameter classes (≤1.5 mm in diameter) was
12, 922. https://doi.org/10.3390/
significantly elevated. When CaO application reached 6 g·kg−1 , the seedling height, basal diameter,
f12070922
and dry-matter weight of each organ decreased, as did the length, surface area, and volume of the
Academic Editor: Roger Seco roots in the all diameter classes, indicating that the addition of excessive CaO to fertile soil could
inhibit the growth and root development of cypress. In Ca-deficient low-quality acidic soils, adding
Received: 23 May 2021 CaO fertilizer can promote the development of fine roots and the uptake and utilization of N, P, and
Accepted: 13 July 2021 Ca. The results of this study provide a basis for determining the optimal fertilization strategy when
Published: 15 July 2021 growing cypress in acidic soils in Southern China.
Publisher’s Note: MDPI stays neutral Keywords: Cupressus funebris; root development; nutrient accumulation efficiency; calcium re-
with regard to jurisdictional claims in sponse; clones
published maps and institutional affil-
iations.
1. Introduction
In the subtropics of China, fast-growing plantations are primarily distributed in large
Copyright: © 2021 by the authors.
areas of hills and mountains, and these areas are mostly covered by low-fertility acidic red
Licensee MDPI, Basel, Switzerland.
soils. Due to heavy rainfall and strong weathering in Southern China, soil acidification is
This article is an open access article
serious, and this condition results in soil and water loss, a deficiency of soil nutrients and a
distributed under the terms and
decline in soil fertility [1]. Damaged land is difficult to recover, which results in difficulty
conditions of the Creative Commons
in tree survival and low vegetation coverage [2]. Areas where acid deposition occurs are
Attribution (CC BY) license (https://
continuously expanding along with a serious loss of base ions, especially calcium (Ca2+ ),
creativecommons.org/licenses/by/
4.0/).
in the soil [3]. Ca2+ is an essential nutrient required for plant growth and development
Forests 2021, 12, 922. https://doi.org/10.3390/f12070922 https://www.mdpi.com/journal/forests
Forests 2021, 12, 922 2 of 12
and plays a role in maintaining the stability of plant cell walls, cell membranes, and
membrane-bound proteins; modulating inorganic ion transport; and regulating various
enzyme activities [4–7].
Applying calcium fertilizer and other elements, such as nitrogen (N), phosphorous (P),
and potassium (K), to acidic soil helps regulate the soil pH value and also plays a role in
calcium supplementation, which can significantly increase the content of available calcium
in soil [8,9]. Calcium application can modulate intracellular Ca2+ levels, promote seedling
growth and root development, and enhance plant stress resistance [10,11]. In recent years,
higher yield and quality and higher concentrations of nutrient elements have been obtained
by the application of chelated microfertilizers rather than simple chemical fertilizers [12].
Data have shown that high Ca fertilized Douglas-fir ((Mirb.) Franco) seedlings had greater
new-needle biomass and growth than low Ca fertilized seedlings. High Ca availability
also led to higher foliar membrane-associated Ca, Ca-pectate, and Ca-oxalate than low
Ca seedlings [13]. However, numerous fertilization experiments have been carried out in
low-fertility acidic red soils to determine optimal fertilization regimes for forest trees, and
the issue remains a hot topic [14].
The root is an important organ in plants for resource acquisition, and the spatiotempo-
ral distribution of plant roots determines the quantities of water and nutrients absorbed for
photosynthesis and harvest products [15,16]. Additionally, the root acts as a supporting
organ that allows a plant to be fixed in the ground for a long time, ensuring normal growth
and development [17]. Under different growth conditions, functional attributes such as
root number and morphology show differential responses to changes in underground
resources. For example, fine roots with diameters ≤1.5 mm are key for nutrient uptake,
accounting for over 80% of the total root length and total root surface area [18]. When the
soil environment is altered, the rate at which fine roots lengthen changes, and the mean
root diameter increases or decreases in a short period of time; the duration of such changes
varies [19]. In contrast, roots with a diameter of >1.5 mm mainly play a role in transport
and support, and they constitute relatively low proportions of the total root length and
surface area. Especially for tall arbores, root growth and development status determine
forest tree growth status in subsequent years or even decades.
Cypress (Cupressus funebris Endl.) is highly adaptive, featuring a developed root
system, strong resistance to drought stress, and a strong capacity for self-repair [20]. Thus,
it is also a major tree species for afforestation and forestland restoration under low-fertility
site conditions. However, most subtropical regions have low-fertility acidic soils; only
the residues (5–10%) of dissolved limestone matter can form soil parent material, the soil
layer is shallow, and calcium loss is severe [2]. Previous research has found substantial
variation in the root length of cypress when the soil environment is disturbed; this effect
is long-lasting and especially impactful on the number, morphology, and function of fine
roots [21]. There are few reports on the synergistic relationships between calcium (Ca) and
cypress growth, root development, and nutrient uptake; however, these studies are of great
significance for understanding the development of cypress forests in Ca-deficient acidic
soil [22,23].
The adaptability of plants to the soil Ca environment is related to the absorption,
transport, and accumulation of nutrient elements [8,9]. Considering the differences in the
influence of Ca2+ on root traits and plant growth, adding Ca fertilizer to acidic soil can
increase the soil Ca content [14,24]. We hypothesized that (i) the basal diameter, seedling
height, and dry matter of cypress would respond differently to Ca fertilizer, and soil fertility
may modulate the effect of Ca fertilizer [25]; and (ii) the morphological characteristics
of fine roots differ greatly between fertile and poor soils. The ratio of nutrient uptake in
cypress might differ with Ca addition because fine roots are the most active part of the root
system in nutrient uptake and transport [26,27]. The objectives of this study were to explore
(1) the ground diameter and seedling height growth of cypress clones in response to Ca2+
fertilizer; (2) the effects of Ca2+ addition on the roots of different diameter classes in terms
Forests 2021, 12, 922 3 of 12
of length, surface area, and volume; and (3) the variation in N, P, and Ca accumulation
efficiencies of cypress under different nutrient conditions.
2. Materials and methods
2.1. Study Site and Selection of Materials
The experiment was conducted in a greenhouse at Laoshan Forestry Farm in Zhejiang
Province, China. One-year-old cutting seedlings of cypress were planted as the experi-
mental material. The semi-lignified branches used for cutting came from elite individual
plants of clone 1 (fast height growth) and clone 2 (slow height growth) in the full-sib
progeny. For each clone, robust cutting seedlings aged 1 year were selected. At the time
of planting, seedlings were selected based on plant height (5.15 ± 0.05 cm) and ground
diameter (0.17 ± 0.01 cm). Then, they were planted in containers that were 30 cm in height
and 20 cm in diameter. The potting soil was the local acidic soil, and the soil layer was
0–20 cm thick. This soil is a red acid soil typical of subtropical areas in China. Soil pH value
was determined by Potentiometric method. Total nitrogen was determined by Kjeldahl
method, and available nitrogen was determined by alkaline hydrolysis method. Extraction
of readily available phosphorus was determined by sodium bicarbonate molybdenum anti-
mony colorimetric method. The available potassium was extracted with 1 mol·L−1 neutral
ammonium acetate and measured by flame photometer. Organic matter was determined
by Potassium dichromate external heating method. The exchangeable Ca and Mg were
determined by EDTA volumetric method. The physicochemical properties of the soil are
provided in Table 1.
Table 1. Texture and chemical properties of potted soil.
Hydrolytic Available Organic Exchange Exchange
Nutrient Total N Total P Available K Mg
Texture Soil Type N P Matter Ca pH Value
Elements (g·kg−1 ) (g·kg−1 ) (mg·kg−1 ) (g·kg−1 )
(mg·kg−1 ) (mg·kg−1 ) (mg·kg−1 ) (mg·kg−1 )
Average light red acid 0.75 ± 0.09 0.32 ± 0.05 53.5 ± 4.70 18.50 ± 1.12 0.99 ± 0.14 15.8 ± 1.89 128 ± 12.5 9.24 ± 0.85 4.65 ± 0.21
content loam soil
2.2. Experimental Design
The controlled-release fertilizer used in the experiment was a nursery fertilizer (APEX).
NPK fertilizer was added at 0 and 3 g per kg of soil to simulate low-fertility and fertile
soil, respectively. For Ca2+ fertilization, CaO was added at 0, 3, and 6 g per kg of soil.
Both the NPK fertilizer and CaO were mixed with the soils, stirred uniformly, and placed
into containers. The experiment involved 6 treatments: (1) 0 g·kg−1 soil CaO, (2) 3 g·kg−1
soil CaO, (3) 6 g·kg−1 soil CaO, (4) NPK fertilizer (3 g·kg−1 soil) + 0 g·kg−1 soil CaO,
(5) NPK fertilizer (3 g·kg−1 soil) + 3 g·kg−1 soil CaO, and (6) NPK fertilizer (3 g·kg−1 soil)
+ 6 g·kg−1 soil CaO. The experiment used a completely randomized block design. Twenty
cutting seedlings were planted per treatment per clone, with three replicates each; therefore,
720 potted seedlings were planted. All seedlings were maintained in a greenhouse, under
conventional management.
2.3. Measurement Indices
The experiment started on 2 April 2018. Seedlings were harvested on 23 November,
and height and ground diameter were measured for all plants. Whole plants were collected
and divided into roots, stems, and leaves, with each organ harvested separately. First,
the roots were separated from the soil, washed with deionized water, and stored. Root
diameter was classified as follows: class D1 (root diameter range: 0–0.5 mm), class D2
(0.5–1.0 mm), class D3 (1.0–1.5 mm), class D4 (1.5–2.0 mm), and class D5 (>2.0 mm) [9].
The root length, surface area, and root volume for each diameter class were measured by
using the image analysis software WinRHIZO Pro STD1600 + (Regent Instruments, Quebec
City, QC, Canada). Next, the roots, stems, and leaves were deactivated in an oven at 105 ◦ C
for 30 min and then dried at 80 ◦ C until a constant weight was achieved to obtain the dry
biomass of each part. The N content of each organ was measured by using a FOSS (Foss
Forests 2021, 12, 922 4 of 12
Analytical A/S, Hillerød, Denmark) nitrogen analyzer. The P content was measured by
molybdenum antimony anticolorimetry [28]. The Ca content was measured by atomic
absorption spectrophotometry [29]. The N, P, and Ca contents were multiplied by the
dry biomass of the whole plant to obtain the N, P, and Ca accumulation. N accumulation
efficiency = dry biomass accumulation of whole plant/N uptake of whole plant (g·mg−1 );
P and Ca accumulation efficiencies were calculated by following the same method used for
N accumulation efficiency.
2.4. Data Analysis
One-way analysis of variance (ANOVA) was used to test the significance of differences
in seedling growth, root morphological characteristics, and nutrient accumulation efficiency
in fertile and low-fertility soils. Two-way analysis of variance was used to examine the
differences among clones and calcium treatments under two kinds of soil conditions.
Duncan’s minimum significant difference method was used to assess the significance of
differences among treatments, and the significance level was set at 0.05. All statistical
analyses were performed by using IBM SPSS Statistics 22.0 (IBM Corp, Armonk, NY, USA).
3. Results
3.1. Effects of Soil Fertility and Calcium Fertilizers on Seedling Height, Ground Diameter, and
Dry Biomass
In soil with 0 g·kg−1 NPK fertilizer, there were significant differences in seedling
height, ground diameter, and dry biomass in the roots, stems, and leaves among the CaO
treatments (p < 0.01). Treatment with 3 g·kg−1 CaO significantly promoted seedling height
growth, ground diameter, and dry biomass in the roots, stems, and leaves, resulting in
values 49.9%, 25.6%, 39.4%, 51.2%, and 50.1% higher than those with 0 g·kg−1 soil CaO. In
contrast, the seedling height, ground diameter and dry biomass of roots, stems, and leaves
were lower with 6 g·kg−1 CaO than with 3 g·kg−1 soil CaO (Table 2). As shown in Figure 1,
both clones performed best at 3 g·kg−1 CaO. Moreover, seedling height growth, ground
diameter, and dry biomass in the roots, stems, and leaves were significantly different
between the clones (Table 2). The seedling height, ground diameter, and dry biomass in the
roots, stems, and leaves of clone 1 were significantly higher than those of clone 2, and the
mean values of these parameters were 15.7%, 83.6%, 10.4%, 40.3%, and 60.8% higher for
clone 1 than for clone 2, respectively (Figure 2).
Table 2. Seedling height, ground diameter, and dry matter of cypress clones affected by the CaO level with and without
NPK fertilization. Asterisks * and double asterisks ** indicate significant differences at p < 0.05 and p < 0.01, respectively.
NPK Fertilizer CaO Treatment F Value
Trait
Treatment 0 g·kg−1 3 g·kg−1 6 g·kg−1 CaO Clones Clones × CaO
Seedling height (cm) 29.67 ± 2.56 44.49 ± 3.87 33.17 ± 3.28 8.26 ** 5.57 ** 3.54 *
ground diameter (cm) 4.57 ± 0.35 5.74 ± 0.51 4.89 ± 0.38 15.65 ** 7.21 ** 4.07 **
NPK fertilizer
Root dry matter (g) 2.84 ± 0.21 3.96 ± 0.36 3.43 ± 0.34 7.58 ** 5.53 ** 2.43 *
0 g·kg−1 soil
Stem dry matter (g) 2.09 ± 0.18 3.16 ± 0.34 2.56 ± 0.31 3.59 * 5.32 ** 1.46
Leaf dry matter (g) 4.19 ± 0.36 6.29 ± 0.61 5.33 ± 0.42 6.41 ** 4.34 ** 2.89 *
Seedling height (cm) 48.67 ± 5.17 49.70 ± 4.88 51.36 ± 5.04 0.59 8.65 ** 2.45 *
Ground diameter (mm) 6.02 ± 0.58 6.38 ± 0.54 6.16 ± 0.57 1.57 12.04 ** 4.03 **
NPK fertilizer
Root dry matter (g) 3.91 ± 0.34 5.48 ± 0.52 4.56 ± 0.34 2.74 * 6.81 ** 2.22 *
3 g·kg−1 soil
Stem dry matter (g) 3.76 ± 0.29 4.88 ± 0.39 4.38 ± 0.42 1.20 10.04 ** 2.51 *
Leaf dry matter (g) 7.74 ± 0.59 8.75 ± 0.81 8.59 ± 0.78 0.97 8.12 ** 2.81 *Forests 2021, 12, 922 5 of 12
Figure 1. Differential growth and dry matter of cypress clones in different CaO treatments. CaO and
NPK treatment comparisons for each clone were made separately. Clone 1 represents a fast-growing
clone in terms of tree height, while clone 2 represents a slow-growing clone in terms of tree height.
The error lines in the bar chart are standard errors. Lowercase letters indicate significant differences
at p < 0.05.
In soil with 3 g·kg−1 NPK fertilizer, treatment with 3 g·kg−1 CaO significantly in-
creased the biomass of roots, resulting in a 40.1% increase for the clones relative to the
root biomass under 0 g·kg−1 soil CaO. In contrast, the root biomass was lower than the
control level when the CaO content reached 6 g·kg−1 (Figure 1 and Table 2). The seedling
height, ground diameter, and dry biomass of the roots, stems, and leaves of clone 1 were
significantly higher than those of clone 2, and the mean values of these parameters wereForests 2021, 12, 922 6 of 12
30.1%, 70.3%, 87.2%, 86.1%, and 64.3% higher for clone 1 than for clone 2, respectively
(Figure 2).
Figure 2. Comparison of the mean values of growth traits and dry-matter weight between different
clones. Clone 1 represents a fast-growing clone in terms of the tree height, while clone 2 represents a
slow-growing clone in terms of the tree height. The error lines in the bar chart are standard errors.
Asterisks * indicate significant differences between clones at p < 0.05.
3.2. Effects of Soil Conditions and Calcium Fertilizers on Root Growth and Development
In soil with 0 g·kg−1 NPK fertilizer, the root length, root surface area and root volume
of the diameter classes D1–D4 but not D5 differed significantly among the CaO treatments.
In the 3 g·kg−1 CaO treatment, the root length, root surface area, and root volume sig-
nificantly increased. In the 3 g·kg−1 CaO treatment compared with the 0 g·kg−1 CaO
treatment, for roots in the D1-D4 diameter classes, the root length increased by 32.8%,
24.3%, 35.3%, and 59.5%, respectively; the root surface area increased by 17.9%, 46.8%,
20.2%, and 72.6%, respectively; and the root volume increased by 39.1%, 35.1%, 37.2%, and
53.2%, respectively. With the application of 6 g·kg−1 CaO, the root length, surface area,
and volume in the D1–D4 diameter classes were lower than those in the 3 g·kg−1 CaO
treatment, although the differences were non-significant. Furthermore, the values of these
parameters were significantly higher in the 6 g·kg−1 CaO treatment than in the 0 g·kg−1
CaO treatment, indicating that the fine roots (Forests 2021, 12, 922 7 of 12
Table 3. Root length, surface area, and volume of cypress clones with different diameters affected by CaO fertilization rates
under the treatments with and without NPK fertilization. Asterisks * and double asterisks ** indicate significant differences
at p < 0.05 and p < 0.01, respectively.
NPK Fertilizer Diameter CaO Treatment F Value
Trait
Treatment Class (mm) 0 g·kg−1 3 g·kg−1 6 g·kg−1 CaO Clones Clones × CaO
0–0.5 (D1) 1093.24 ± 89.21 1451.61 ± 100.36 1302.28 ± 98.65 6.11 ** 1.46 1.27
0.5–1.0 (D2) 458.41 ± 31.41 569.99 ± 50.24 566.18 ± 60.04 4.16 ** 2.45 * 1.54
Root length 1.0–1.5 (D3) 92.93 ± 10.02 125.71 ± 10.32 114.12 ± 8.97 5.88 ** 3.90 ** 1.87
(cm) 1.5–2.0 (D4) 21.70 ± 2.14 25.53 ± 2.07 23.21 ± 1.97 7.07 ** 3.18* 1.37
>2.0 (D5) 15.64 ± 1.85 18.52 ± 1.58 17.15 ± 2.04 1.21 2.03 1.17
0–0.5 (D1) 117.38 ± 17.21 138.49 ± 15.47 129.98 ± 16.04 5.32 ** 3.12 * 1.77
NPK fertilizer 0 0.5–1.0 (D2) 87.63 ± 8.36 128.69 ± 13.04 112.37 ± 12.11 4.97 ** 2.43 * 1.54
g·kg−1 soil Root Surface
1.0–1.5 (D3) 38.02 ± 3.21 45.69 ± 4.23 42.97 ± 3.04 6.45 ** 2.76 * 1.52
area (cm2 )
1.5–2.0 (D4) 7.96 ± 0.47 13.74 ± 0.98 10.91 ± 0.75 7.03 ** 0.98 1.04
>2.0 (D5) 15.91 ± 1.02 20.46 ± 1.68 16.46 ± 1.47 1.63 0.34 2.68 *
0–0.5 (D1) 0.90 ± 0.08 1.25 ± 0.11 1.14 ± 0.09 4.93 ** 1.76 7.75 **
0.5–1.0 (D2) 1.64 ± 0.12 2.22 ± 0.09 1.98 ± 0.10 5.08 ** 4.43 ** 0.88
Root volume 0.98 ± 0.06 1.35 ± 0.07 1.22 ± 0.06 7.12 ** 6.75 ** 1.43
(cm3 ) 1.0–1.5 (D3)
1.5–2.0 (D4) 0.40 ± 0.02 0.61 ± 0.03 0.54 ± 0.03 7.54 ** 0.81 1.60
>2.0 (D5) 1.52 ± 0.11 2.31 ± 0.16 1.99 ± 0.16 2.32 0.61 2.20
0–0.5 (D1) 1485.46 ± 105.24 1749.83 ± 135.36 1395.10 ± 101.24 3.27 * 5.67 ** 1.36
Root length 0.5–1.0 (D2) 981.03 ± 85.31 1147.28 ± 91.04 863.67 ± 74.36 7.31 ** 7.22 ** 5.67 **
1.0–1.5 (D3) 205.25 ± 18.36 232.34 ± 17.65 218.88 ± 19.32 6.45 ** 5.16 ** 5.49 **
(cm) 1.5–2.0 (D4) 40.48 ± 3.65 45.28 ± 4.02 37.95 ± 3.21 2.09 9.26 ** 1.24
>2.0 (D5) 22.71 ± 2.11 28.28 ± 2.05 25.02 ± 2.24 1.53 4.89 ** 0.98
0–0.5 (D1) 136.62 ± 9.57 169.39 ± 14.03 149.19 ± 12.32 5.52 ** 7.14 ** 1.65
NPK fertilizer 3 0.5–1.0 (D2) 197.27 ± 15.36 225.99 ± 19.65 191.71 ± 17.65 9.23 ** 5.05 ** 1.88
g·kg−1 soil Root Surface
1.0–1.5 (D3) 79.78 ± 6.32 85.60 ± 7.05 81.28 ± 6.95 6.77 ** 6.28 ** 3.08 *
area (cm2 )
1.5–2.0 (D4) 20.10 ± 1.83 24.09 ± 2.04 20.61 ± 1.76 1.68 8.16 ** 4.73 **
>2.0 (D5) 23.73 ± 1.51 29.72 ± 2.38 25.37 ± 2.01 0.98 4.78 ** 1.69
0–0.5 (D1) 1.18 ± 0.11 1.60 ± 0.13 1.27 ± 0.10 9.32 ** 8.01 ** 4.79 **
0.5–1.0 (D2) 3.14 ± 0.39 4.22 ± 0.32 3.54 ± 0.23 5.11 ** 5.44 ** 2.04
Root volume 2.17 ± 0.19 2.51 ± 0.21 2.40 ± 0.21 7.02 ** 8.26 ** 3.14 *
(cm3 ) 1.0–1.5 (D3)
1.5–2.0 (D4) 0.86 ± 0.07 1.03 ± 0.11 0.90 ± 0.06 1.43 10.31 ** 6.70 **
>2.0 (D5) 2.75 ± 0.21 3.46 ± 0.28 2.83 ± 0.20 1.58 12.21 ** 1.59
Figure 3. Effects of CaO supply levels on root morphology of different cypress clones. Clone 1 represents a fast-growing
clone in terms of the tree height, while clone 2 represents a slow-growing clone in terms of the tree height. CaO0 , CaO3 and
CaO6 represent CaO was added at 0, 3, and 6 g per kg of soil, respectively.Forests 2021, 12, 922 8 of 12
3.3. N, P, and Ca Accumulation Efficiencies
The t-test results showed that the N accumulation efficiency with 3 g·kg−1 NPK
fertilizer was significantly greater than that in low-fertility soil. In the 3 g·kg−1 CaO and
6 g·kg−1 CaO treatment groups, the Ca and P accumulation efficiency was significantly
greater in lower fertility soil than in the soil with 3 g·kg−1 NPK fertilizer. In soil with
0 g·kg−1 NPK fertilizer, the two clones achieved their highest N, P, and Ca accumulation
efficiencies under the 3 g·kg−1 CaO treatment (Figure 4). When the soil was treated with
3 g·kg−1 NPK fertilizer, the P accumulation efficiency in the cypress clones exhibited a
downward trend with increasing CaO concentration, the P accumulation efficiency was
1.3% (3 g·kg−1 CaO treatment group) and 7.5% (6 g·kg−1 CaO treatment group) lower than
that in the 0 g·kg−1 CaO treatment group. However, the differences in P accumulation
efficiency were not significant. The calcium accumulation efficiency was significantly
higher in the 3 g·kg−1 CaO and 6 g·kg−1 CaO treatment groups than in the 0 g·kg−1 CaO
treatment group (Figure 4). As shown in Figure 4, the calcium accumulation efficiency was
25.0% (3 g·kg−1 CaO treatment group) and 34.1% (6 g·kg−1 CaO treatment group) higher
than that in the 0 g·kg−1 CaO treatment group. Moreover, with or without fertilization, the
N and Ca accumulation efficiencies differed significantly between the clones.
Figure 4. Effects of CaO supply levels on uptake and accumulation efficiency in the different clones.
CaO and NPK treatment comparisons for each clone were made separately. Clone 1 represents a
fast-growing clone in terms of the tree height, while clone 2 represents a slow-growing clone in terms
of the tree height. NAE stands for nitrogen accumulation efficiency, PAE stands for phosphorus
accumulation efficiency, and CAE stands for calcium accumulation efficiency. The error lines in the
bar chart are standard errors. Lowercase letters indicate significant differences at p < 0.05.
4. Discussion
The application of NPK fertilizer to soil can significantly increase the growth of
seedlings, but the use of Ca fertilizer in production is often neglected. Moreover, excessive
application of NPK fertilizer can lead to acidic soil; under these conditions, it can cause the
loss of calcium from the soil, which seriously affects the absorption and utilization of Ca by
plants [25]. Compared with the 0 g·kg−1 CaO treatment, regardless of the application ofForests 2021, 12, 922 9 of 12
NPK fertilizer, seedling height, basal diameter, and root development of cypress increased
in the other CaO treatments (Table 2), indicating that the application of CaO fertilizer was
able to promote the growth of cypress. Following treatment with Ca2+ , the soil conditions
were dramatically altered. The cation exchange capacity (CEC) and base saturation (BS)
increased considerably after addition [14], which might promote the migration of N, P, and
Mg plasma in the soil [30]. At the same time, adding an appropriate amount of calcium
fertilizer can improve soil microbial activity [31], enhance the respiration ability of roots in
the soil, and improve the absorption capacity of roots [32].
In this study, in the fertile soils, the seedling height, basal diameter, and stem and
leaf dry biomass of cypress were not significantly different among the Ca2+ treatments,
but the root dry-matter weight, root length, root surface area, and root volume in the
D1–D3 diameter classes (≤1.5 mm) were significantly different among the CaO treatments
(Tables 2 and 3). When CaO application reached 6 g·kg−1 , the root length, surface area,
and volume in the D1–D5 diameter classes decreased, indicating that the growth of the
root system was inhibited, and the seedling height and basal diameter were also reduced.
Although the accumulation efficiency of N and Ca was the highest in the 6 g/kg CaO
treatment, the differences were not significant between CaO treatment. Moreover, the
accumulation efficiency of P was reduced by the application of Ca, and the possible reasons
for this finding are as follows: The orthophosphates in the soil are prone to chemical
precipitation with ions, such as Al3+ , Fe3+ , and Ca2+ , or to adsorption-fixation by soil,
resulting in the poor mobility of P in the soil and difficulty in P uptake by plant roots [23].
The results showed that, in fertile soils, the use of appropriate amounts of Ca can create a
favorable underground environment for the growth of plants, increase the pH of the soil,
and promote the growth and development of fine roots (i.e., ≤1.5 mm in diameter), while
excess Ca can inhibit plant growth.
In the low-fertility soil compared with the fertile soil, cypress seedling growth and
root development show greater responsiveness to CaO. Under low-fertility conditions,
the seedling height of cypress increased with the addition of an appropriate amount of
Ca2+ (3 g·kg−1 ), and the highest N, P, and Ca accumulation efficiencies were all achieved
under the 3 g·kg−1 Ca2+ treatment, with synergy between Ca2+ fertilizer and N and P in
terms of accumulation efficiencies (Figure 4). However, when the Ca2+ concentration was
increased, seedling growth of cypress clones decreased under the 6 g·kg−1 Ca2+ treatment.
These results indicate that the synergy showed a range of adaptation to the level of Ca2+
applied. That is, an appropriate amount of Ca2+ promoted plant N and P uptake, while an
excessively high concentration of Ca2+ fertilizer inhibited uptake [33,34]. In a study con-
ducted on coniferous species such as pine (Pinus massoniana Lamb.), favorable adaptation
was also observed in the soil environment with Ca2+ supplied at 1 to 2 mmol·L−1 , while
the plant height growth of pine seedlings decreased after the Ca2+ supply exceeded this
concentration [35]. Therefore, full consideration should be given to the tolerance of tree
species when applying Ca2+ to promote seedling growth.
The root system determines plant water and nutrient uptake and is closely related
to plant traits, such as height and growth rate [36,37], and a reasonable root configura-
tion can provide the plant with a larger absorption area. As the key parts of plants for
nutrient uptake, fine roots feature small diameters and low lignification levels, with high
sensitivity to changes in soil nutrients [38]. The fine roots in the D1–D3 diameter classes
usually consist of non-lignified components (e.g., cortical tissue); these roots are mainly
involved in the acquisition and absorption of water, nutrients, and other soil resources
and are classified as absorbing roots. The roots in the D4 and D5 diameter classes are
usually composed of lignified components (e.g., secondary xylem) and are mainly used
for transport and storage [26,27,36,39]. Compared with those of coarse roots, the greater
length and surface area of fine roots enable plants to respond to changes in the soil environ-
ment more easily [40]. The quantity of absorbing roots and the root-length density were
significantly correlated with the available nutrients in the soil. Specific root length is the
ratio of root length to biomass. The greater the advantage afforded by higher specific rootForests 2021, 12, 922 10 of 12
length to the plant in obtaining water and nutrients is, the larger the number of fine roots
and the smaller the root diameter [41]. Fine roots of plants in classes D1–D3 (diameter
≤1.5 mm) accounted for more than 96.6% of the total root length and over 88% of the
root surface area (Table 3). In soil treated with 3 g·kg−1 NPK fertilizer, fine root diameters
≤1.0 mm accounted for 89.1%, 90.7%, and 88.9% of the total root length and accounted
for 70.2%, 73.9%, and 71.8% of the total surface area across the three CaO treatments (0, 3,
and 6 g·kg−1 ), respectively (Table 3 and Figure 3). The corresponding proportions of roots
with diameters ≤ 1.0 mm were even higher in the low-fertility soil, reaching 92.1%, 93.4%,
and 92.5% of the total root length and accounting for 75.8%, 77.1%, and 76.1% of the total
surface area across the three CaO treatments (0, 3, and 6 g·kg−1 ), respectively. These results
indicate that cypress can adjust the morphology of its fine roots to adapt to different CaO
environments. In resource-poor locations, increasing the number and longevity of fine roots
may be an optimal option. It can improve fine root turnover efficiency, maximize resource
acquisition efficiency, improve seedling potential adaptability, and balance tolerance with
competitiveness in adversity [15,42]. As expected, when the site conditions were relatively
infertile, cypress formed more roots with diameters ≤ 1.0 mm. Fine, long, fast-growing,
and absorbent roots can improve the root distribution and foraging accuracy, allowing
plants to quickly obtain the nutrients and water needed for growth. Furthermore, fine roots
can spread to fill the soil space, obtaining more soil nutrients and water resources [43].
The main functional unit of a root for nutrient uptake is close to the root-tip re-
gion [44,45]. The process of calcium uptake by roots is active or passive, depending on the
concentration of calcium in the soil. With a low external Ca concentration, the absorption of
Ca by roots has been found to be almost completely determined by the metabolic capacity,
while Ca absorption capacity under a high external Ca concentration was not correlated
with the metabolic capacity of roots but showed a linear relationship with the transpiration
rate [46]. Root metabolism is closely related to root activity, and calcium absorption can be
promoted by improving root activity. In plant roots, Ca2+ can enter epidermal cells and
root hairs directly through Ca2+ channels. Various Ca2+ channels have been found in root
cells, such as voltage-dependent Ca2+ channels, plasma membrane stretching activated
Ca2+ channels, and second-messenger-molecule-activated Ca2+ channels [47]. These find-
ings will provide insight into the study of the Ca2+ dependence, mobility, and absorption
dynamics of cypress in a variety of environments in later stages of development.
5. Conclusions
Cypress roots have a strong ability to adapt to different environments. In Ca-deficient
low-quality acidic soils, fine roots (Forests 2021, 12, 922 11 of 12
Acknowledgments: We acknowledge the help from Zhongcheng Lu with sample collection at the
study site. We thank Jia Du, Yi Zheng, and Chengzhi Yuan for input into the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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