Coconut Coir as a Sustainable Nursery Growing Media for Seedling Production of the Ecologically Diverse Quercus Species

Abstract

Peat, a non-sustainable resource, is still predominately used in forest nurseries. Coconut coir might provide an alternative, renewable, and reliable growing media but few studies have evaluated this media type in forest nurseries. We assessed the influence of pure coir, in combination with various fertilization regimes, on the growth and physiology of three ecologically diverse Quercus species seedlings (Q. robur, Q. pubescens, and Q. ilex) during nursery cultivation. Seedlings were grown using peat and pure coir in combination with three fertilization treatments (standard, K-enriched, and P-enriched). Data were collected for: (1) growth and physiological traits; (2) detailed above- and below-ground morphological traits by destructive analysis; and (3) NPK content in leaves, shoot and roots, and in the growing media, following cultivation. Peat and coir in combination with the various fertilization treatments affected above- and below-ground morphology and, to a lesser extent, the physiological traits of Quercus seedlings. Large effects of the substrate occurred for most morphological variables, with peat being more effective than coir in all studied species. Fertilization also produced significant differences. The effect of K-enriched fertilization on plant growth was clear across the three species and the two growing media. P-enriched fertilization in peat was the only combination that promoted a higher amount of this element in the tissues at the end of cultivation. Despite their smaller size, seedlings produced in coir were compatible with standard Quercus forest stocktype size, and showed a proportionally higher root system development and fibrosity. Our results suggest that coir can be used as an alternative substrate to grow Quercus species seedlings, and that fertilization can offset coir deficiencies in chemical properties. As several functional traits drive planting performance under varying environmental conditions. according to the Target Plant Concept, coir might thus serve as an acceptable material for seedling cultivation in some cases.

Full text

Article
Coconut Coir as a Sustainable Nursery Growing
Media for Seedling Production of the Ecologically
Diverse Quercus Species
Barbara Mariotti 1, * , Sofia Martini 1, Sabrina Raddi 1, Andrea Tani 1, Douglass F. Jacobs 2,
Juan A. Oliet 3and Alberto Maltoni 1
1Dipartimento di Scienze e Tecnologie Agrarie, Alimentari, Ambientali e Forestali–DAGRI, Universitàdi
Firenze, Via San Bonaventura 13, 50145 Firenze, Italy; sofia.martini@unifi.it (S.M.);
sabrina.raddi@unifi.it (S.R.); andrea.tani@unifi.it (A.T.); alberto.maltoni@unifi.it (A.M.)
2Department of Forestry and Natural Resources, Hardwood Tree Improvement and Regeneration Center,
Purdue University, 715 West State Street, West Lafayette, IN 47907, USA; djacobs@purdue.edu
3Departamento de Sistemas y Recursos Naturales, Universidad Politécnica de Madrid,
Ciudad Universitaria s/n, 28040 Madrid, Spain; juan.oliet@upm.es
*Correspondence: barbara.mariotti@unifi.it
Received: 9 April 2020; Accepted: 5 May 2020; Published: 7 May 2020


Abstract:
Peat, a non-sustainable resource, is still predominately used in forest nurseries. Coconut
coir might provide an alternative, renewable, and reliable growing media but few studies have
evaluated this media type in forest nurseries. We assessed the influence of pure coir, in combination
with various fertilization regimes, on the growth and physiology of three ecologically diverse Quercus
species seedlings (Q. robur,Q. pubescens, and Q. ilex) during nursery cultivation. Seedlings were grown
using peat and pure coir in combination with three fertilization treatments (standard, K-enriched,
and P-enriched). Data were collected for: (1) growth and physiological traits; (2) detailed above- and
below-ground morphological traits by destructive analysis; and (3) NPK content in leaves, shoot
and roots, and in the growing media, following cultivation. Peat and coir in combination with
the various fertilization treatments affected above- and below-ground morphology and, to a lesser
extent, the physiological traits of Quercus seedlings. Large effects of the substrate occurred for most
morphological variables, with peat being more effective than coir in all studied species. Fertilization
also produced significant differences. The effect of K-enriched fertilization on plant growth was clear
across the three species and the two growing media. P-enriched fertilization in peat was the only
combination that promoted a higher amount of this element in the tissues at the end of cultivation.
Despite their smaller size, seedlings produced in coir were compatible with standard Quercus forest
stocktype size, and showed a proportionally higher root system development and fibrosity. Our
results suggest that coir can be used as an alternative substrate to grow Quercus species seedlings,
and that fertilization can offset coir deficiencies in chemical properties. As several functional traits
drive planting performance under varying environmental conditions. according to the Target Plant
Concept, coir might thus serve as an acceptable material for seedling cultivation in some cases.
Keywords:
forest nursery stock; coconut fiber; peat; seedling morphology; seedling physiology;
substrate
1. Introduction
Forest seedling field performance is related to plant morphological and physiological
characteristics [
1
–
3
]. Development of morpho-physiological traits, in turn, is strongly influenced by
nursery practices [
1
,
4
–
7
]. In container plant production, the effectiveness of the growing media is
Forests 2020,11, 522; doi:10.3390/f11050522 www.mdpi.com/journal/forests

Forests 2020,11, 522 2 of 19
important to seedling quality [
8
,
9
]. An effective substrate should sustain a favorable balance between
air porosity and water holding capacity, promoting root development and nutrient uptake [
9
,
10
].
Additionally, growing media must have a high cation exchange capacity and be economically viable [
11
].
Sphagnum peat moss (Sphagnum spp.), generally known as peat, is commonly and predominantly
used in Europe and North America in plant propagation [9,12–15]. Peat production globally is about
28 million metric tons [
15
] and peatlands used as growing media cover about 2000 km
2
[
16
]. The
effectiveness of peat is related both to material performance and economic aspects [
17
]. Despite a
low re-wetting capacity [
18
], peat is a light, low bulk density material that contains a low nutrient
content, has a very high cation exchange capacity (CEC) [
8
]), and can easily adsorb fertilizer nutrients;
thus, plant nutrition can be controlled during cultivation [
19
]. Moreover, peat requires relatively
few post-harvest treatments and additives to be used effectively [
9
]. Nevertheless, concerns have
been raised about environmental impacts of peat extraction [
20
–
22
] in relation to—(a) the fragility of
many peatland ecosystems [
16
,
23
–
25
]; (b) their function as a C sink, whose drainage and exploitation
increases C emissions [
22
,
26
]; and (c) the non-sustainable length of the natural processes of peat
production [
24
,
25
,
27
]. Environmental concerns in Europe promoted actions to preserve peatlands as
ecosystem as well as to reduce C emissions [
17
,
28
]. The growing pressure on producers, retailers, and
growers, especially in the horticulture sector, has led to an increasing demand of alternative, renewable,
and reliable growing media [29,30].
Coir (or coconut fiber) has been tested as an alternative to peat in horticulture [
31
], and is the most
used alternative in this sector [
14
,
32
]. Coir is made of husk and short fibers from the nut mesocarp of
Cocos nucifera, which are a waste product of the coconut industry, coming mainly from Philippines,
Indonesia, Sri Lanka, Malesia, and Thailand. Coir is a renewable and largely available resource, and
25% of over 50 million tons of coconut produced annually are waste [
33
]. Coir provides a favorable
balance between air and water, similar to peat [
9
], and a higher re-wetting capacity than peat [
34
].
However, coir has higher pH and lower cation exchange capacity (CEC) than peat [
8
]. In addition, to
be effective as a soilless growing media, coir needs to be further processed, which can lead to a low
standardization of biological, chemical, or physical properties of the material, as compared to peat [
9
].
In particular, when it is produced in coastal marine areas, coir has to be treated to reduce toxic levels
of sodium and potassium [
35
]. Such procedures increase coir production costs, which are generally
favorable to peat [
9
]. According to Schmiliweski [
32
], coir is the third most common growing media
used by nurseries in horticulture after peat (which covers more than 2/3 of the market) and mineral
substrates (such as vermiculite, perlite, rockwool, and others). According to recent reports [
28
,
36
], in
Netherlands and in Italy, the use of coir is increasing.
Seedling physiological and morphological traits can also be strongly altered by fertilization during
nursery cultivation, potentially interacting with substrate chemical properties. This might affect
plenty of attributes, such as biomass accumulation and allocation to shoot- and root-system [
37
,
38
],
leaf morphology and physiology [
7
,
39
], root-system architecture and functionality [
7
,
40
], and xylem
conductance [
41
], with effects on survival and field performance [
42
–
44
]. Hence, by altering fertilization
it might be possible to affect seedling quality [
45
,
46
] and the performance of transplanted seedlings [
47
].
Seedling nutrient availability can influence the amount of reserves available for remobilization after
planting [
44
,
48
]. While many studies have investigated the effects of nitrogen fertilization on forest
tree seedlings, less is known about phosphorous and potassium [
43
]. Phosphorus is an immobile
soil resource whose availability is linked to morpho-functional traits of the root system [
49
–
53
], and,
consequently, it might have indirect effects on seedlings post-planting survival, nutrient uptake and
growth, drought resistance, and more stress resistance, in general [
40
,
54
–
56
]. Less information is
available on forest species about the effect of potassium, which is an element directly related to a
plethora of physiological processes, including, among others, cellular turgor, stomatal conductance,
and photosynthesis (which can influence drought resistance) [57–60].
Although coir was introduced in horti-flori-fructiculture nursery production about two decades
ago, and the extensive literature covers its productive and economic aspects in these systems [
9
], far less

Forests 2020,11, 522 3 of 19
information is available for the forest nursery sector (i.e., [11,61,62]). Moreover, coconut fiber is often
tested as a component at different proportions of a soilless growing media mixture with other materials,
complicating assessment of coir as a single component [
9
], especially in relation to deficiencies in its
chemical properties (i.e., CEC). In this sense, the use of controlled release fertilizer (CRF) with coconut
fiber could help to offset the mentioned low CEC.
In this study, we focused on the Quercus species that is widely used in the Mediterranean region for
reforestation, afforestation, and forest restoration projects [
63
–
66
]. The species were chosen according
to different ecological adaptations, particularly in relation to Mediterranean or arid climates [
67
]—from
the most well adapted to a Mediterranean climate with dry summers, Q. ilex (holm oak), to the
intermediate Q. pubescens (pubescent or downy oak), to the less Mediterranean adapted, Q. robur
(pedunculate oak), which prefers a temperate climate without a dry season, is widespread across
Europe, including the Mediterranean region, and is one of most common species for multi-purpose
hardwood forest plantings in Europe [
68
,
69
]. We aimed to improve the knowledge of the influence of
pure coir on the growth and physiology of these species during nursery cultivation. Coir was combined
with three different fertilization treatments (nursery standard, enriched in K, enriched in P) to assess
the combined effects on seedlings in relation to—height, morphological traits, physiological traits, and
NPK content both in the shoot- and root-system. Specifically, we addressed the following questions:
(1) Is pure coir effective in growing oak seedlings to be used in forest plantation projects? (2) Which
traits are mainly influenced by this renewable and more sustainable growing media? (3) Is it possible
to offset coir deficiencies in chemical properties by fertilization? (4) Do the studied species, which have
different leaf habits despite different ecological adaptations, respond differently to fertilization and
substrate treatments?
2. Materials and Methods
2.1. Nursery Stock Cultivation
The nursery stock was grown in 2017 in a central Italy nursery (43
◦
55
0
31.4
00
N, 10
◦
53
0
09.1
00
E, 85 m
a.s.l.), using multi-pot containers (QuickPot, Herkuplast Kubern, Ering, Germany) with 12 cavities of
650 cm
3
each (frustum of pyramid shape; top width 76 mm, bottom width 17 mm, and depth 180 mm).
Peat substrate (Pe), considered as control, was made of 70% coarse Baltic peat moss (0–40 mm
particles size) and 30% pumice (5–8 mm), which was added to avoid excessive compaction of pure peat.
Main traits of the mixture were—pH 5.7, EC 0.43 dS/m; bulk density 298 kg/m
3
, and porosity 86.9%.
Coir (Co) was composed of 30% coconut fiber (fibrous material that constitutes the thick mesocarp
of the nut) and 70% coconut pith (fine residual material), with pH 5.2, EC 0.38 dS/m; bulk density
122 kg/m
3
, and porosity 92.5%. The week before sowing, the cavities were uniformly filled with
growing media to 0.5 cm beneath the top.
Standard fertilization (St), a Controlled-Release fertilizer (CRF) fertilizer widely used in Italy to
commercially grow oak seedling nursery stock, was considered as control, and it was compared to
two alternative commercial fertilization formulas—enriched in potassium (K-enriched) or phosphorus
(P-enriched). The St formula was Osmocote Exact Standard 12–14 months NPK 15–9–11 plus
micronutrients at 3 kg
·
m
−3
. P-enriched substrate contained an addition of 19% P
2
O
5
fast release (single
superphosphate, SSP, 2 kg
·
m
−3
) to control, while K-enriched substrate was obtained by 4 kg
·
m
−3
of
Osmocote Exact Standard High K 12–14 months NPK 11–11–18 plus micronutrients. N, P, and K
amount per seedling is shown in Table 1.

Forests 2020,11, 522 4 of 19
Table 1. Macro-elements (NPK) content (per volume and per seedling) in the studied fertilizations.
Content per Volume
mg/L
Content per Seedling
(mg per pot)
N P K N P K
Standard 450.0 270.0 330.0 292.5 175.5 214.5
P enriched 450.0 690.0 330.0 292.5 448.5 214.5
K enriched 440.0 440.0 720.0 286.0 286.0 468.0
A total of 6 treatments combinations per species (2 substrates
×
3 fertilizations) were included in
the experimental trial.
Seed was obtained from the National Center for Biodiversity (Italy); seed provenances were
the following for Q. robur,Q. pubescens, and Q. ilex, respectively, Bosco Fontana (43
◦
55
0
31.4
00
N,
10
◦
53
0
09.1
00
E), Ponte di Veja (45
◦
36
0
27.4
00
N, 10
◦
58
0
15.9
00
E), and Torri del Benaco (45
◦
36
0
59.1
00
N,
10
◦
41
0
50.0
00
E). The 1000-seed weight and germination of the three seed lots (according to International
Seed Testing Association procedures [
70
]) were 6.0 kg and 80% for Q. robur, 2.4 kg and 68% for
Q. pubescens, 3.2 kg and 83% for Q. ilex, respectively. From October 2016 to April 2017, the acorns were
stored at 3
±
0.5
◦
C in moist sand to simulate normal overwintering and to prevent acorn germination
before the experiment started. In March 2017, the seeds were moved outside to stimulate germination,
and then the pre-germinated acorns (0.5 cm maximum radicle length) were sown in multi-pots placed
under a tunnel protected by a transparent plastic film that was removed at mid-May. Seedlings were
irrigated daily by sprinklers following seedling evapotranspirative demands (i.e., spring: 5 L
·
m
−2
in
6 min; summer and autumn: 40 L/m
2
and 20 L/m
2
, respectively, in 24 min). Environmental conditions
(air temperature and humidity) were monitored by a weather station (inside the tunnel) and substrate
moisture was assessed once a week (6 pots per stocktype, Soil moisture meter PCESMM1, PCE Instr.
Corp., PCE Holding, Hamburg, Germany).
2.2. Data Collection
Emergence and height were measured weekly from the end of March until September. Here,
final height data are presented. During the season, physiological traits, such as chlorophyll content
(SPAD-502, Konica-Minolta Sensing Europe B.V.) and chlorophyll fluorescence (ChlF, by Handy PEA,
Hansatech, UK) were measured monthly on a sample of three fully expanded leaves on 9 seedlings per
stocktype in dark-adapted leaves (for at least 40 min), during the growing season (June, July, August).
The PSII functionality was described by the F
V
/F
M
(ratio of Variable to Maximum Fluorescence) to
Strasser et al. [
71
]. Results related to physiological traits were shown only for August, the date closer
to plant lifting, in supplementary material.
Macro-element (N, P, K) were assessed in October (before leaf abscission) on 12 seedlings per
stocktype (S
×
F) per species, merging 2 plants of the same multi-pot, by Nutrilab (Universidad Rey Juan
Carlos, Madrid, Spain). Briefly, analysis of leaf, stem, and root N and P concentration of these samples
was done by the standard Kjeldahl method, while K concentration was determined using a perchloric
acid extraction. Substrate analysis at the end of the nursery cultivation was performed by Denetra snc
(Pescia, Italy) on 9 seedlings per stocktype (S
×
F) per species. Concurrently, seedling morphological
traits were assessed, using 20 destructively sampled seedlings per treatment combination (360 in
total). Height (H), number and dry biomass of leaves (Ln and Lb), root-collar diameter (RcD), and dry
biomass of the shoot (stem) were measured. Root-system was assessed by main root (tap-root) dry
biomass (MRb); first-order lateral roots (FOLR) dry biomass (FOLRb) for three diameter classes (<1 mm,
1–5 mm, >5 mm) measured at the junction with the tap root, root-system volume (by immersion), and
dry weight and density (Rv,Rb, and Rd, respectively). Shoot to root ratio (S/R), H/RcD, and specific leaf
area (SLA) were also calculated.

Forests 2020,11, 522 5 of 19
2.3. Statistical Analysis
A randomized complete block design (3 blocks) was used. Each block included the 6 randomized
S
×
Fcombinations per species; each combination comprised 48 sowed cavities (over 4 multi-pots), for
a total of 2592 sowed containers. In a preliminary multifactorial ANOVA (model, Y
ijlmn
=
µ
+Block
i
+Date
j
+Substrate (S)
k
+fertilization(F)
l
+S
×
Finteraction
kl
+error
ijklm
), the block effect was not
significant for any morphological variables, highlighting homogeneous growing conditions. Thus, we
removed block as source of variation and a multifactorial ANOVA was performed, separately for each
species to avoid complex higher-level interactions, considering substrate (S), fertilization (F), and their
interactions (S
×
F) as a source of variation. In case of significant results (p
≤
0.05), the Tukey post-hoc
test was used for multiple comparisons (
α
=0.05) to highlight homogenous groups within species. In
this study, we present results related to substrate (S), fertilization (F), and their significant interactions
(S
×
F). To provide information about the traits of the studied stocktypes (combinations S
×
F), Tukey
test results among combinations are shown in supplementary material. StatSoft Statistica 11 (Tulsa,
OK, USA) was used to process all data.
3. Results
3.1. Emergence, Growth, and Physiological Traits during Nursery Cultivation
Final emergence was not affected by either factor in the three studied oaks. In the Q. ilex, seedlings
emergence started between 3 and 4 weeks later than in the other two species. In all species, substrate
affected height beginning relatively early, from June (from May in Q. robur, data not shown), and
seedlings in Pe were higher than that in Co (Table 2); in all species, fertilization occurred later (September
in Q. robur, July in Q. pubescens, July in Q. ilex), and K-enriched fertilization promoted taller seedlings
(Table 2). As a result, at the end of the growing season, the tallest stocktype was grown in Pe–K in
all species, with differences among the stocktypes in coir, and generally, seedlings grown in Co–St
performed worse (Table S1). Interaction was not significant in any case, excluding Q. robur in August
and Q. ilex in August and September.
In general, in all species, in August (Figure S1), both substrate (Pe >Co) and fertilization (K
generally higher) affected the ChlF content, generally with no interactions (data not shown). In Q. robur
and Q. pubescens, seedlings in peat showed higher values of F
V
/F
M
than in coir (significant in Q. robur,
Figure S1), and K-enriched fertilization generally resulted in higher F
V
/F
M
values (significant in Q. ilex).
3.2. Morphological Traits
In Q. robur, both substrate and fertilization affected most of the analysed variables without any
interaction (Tables 2and 3), and generally, Pe and K-enriched fertilization were more effective in
promoting seedling growth. For total dry biomass, the differences were as follows: Pe >Co by +22.6%,
K>Pand St by +13.4% and +36.6%, respectively (Figure 1). Peat also promoted shoot-system and
leaf biomass, SLA, as well as FOLR1-5 and total biomass of roots. K-enriched fertilization positively
influenced shoot development, SLA,FOLR1-5, root system, and leaf biomass (Figure 1and Figure
S2). Pe stimulated biomass accumulation more in the shoot- than in the root-system with S/Rvalues >
0.5 (Table 3), and K-enriched fertilization promoted higher S/Rthan St. Root volume was affected by
substrate (Pe >Co) and fertilization (K>St); root density was influenced only by fertilization (Co–St >
Pe–K).

Forests 2020,11, 522 6 of 19
Table 2.
Multifactorial ANOVA and Tukey post-hoc test results (p
≤
0.05 in bold) for seedling
morphological traits (mean
±
SD) at the end of the season (N
obs
=20 seedlings per stocktype per
species). Source of variation—substrate (S), fertilization (F), and their interaction (S
×
F). Capital and
lowercase letters indicate homogeneous groups for Sand F, respectively. Variables: H(height); RcD
(root collar diameter in mm); H/RcD (seedling taper); Rv (root-system volume in cm
3
); Rd (root-system
density =Rb/Rv in g cm
−3
); S/R(shoot–root ratio); SLA (specific leaf area, in cm
2
g
−1
); Ln (number of
leaves); and Lb (leaf dry biomass in g).
Q. robur S F S ×F Pe Co St P K
H<0.0001 0.0108 0.8213 57.6 ±10.8 B45.8 ±10.3 A48.3 ±11.4 a
51.5
±
12.2
ab 55.4 ±11.8 b
RcD 0.2265 0.0006 0.6354 11.2 ±1.9 10.8 ±1.7 10.1 ±1.8 a11.2 ±1.8 ab 11.6 ±1.6 b
H/RcD <0.0001 0.4976 0.9521 52.6 ±11.3 B42.9 ±9.1 A48.4 ±12.2 46.1 ±9.8 48.6 ±11.9
Rv 0.0143 0.0001 0.1355 28.0 ±9.2 B24.6 ±7.8 A21.0 ±6.0 a26.9 ±8.0 b31.0 ±8.8 b
Rd 0.3264 0.0040 0.0711 0.43 ±0.1 0.44 ±0.1 0.47 ±0.1 b0.42 ±0.1 ab 0.41 ±0.1 a
S/R<0.0001 0.0419 0.6985 0.65 ±0.14 B0.49 ±0.15 A0.53 ±0.17 a
0.57
±
0.14
ab 0.61 ±0.18 b
SLA 0.0016 0.0120 0.5480 156.1 ±29.2 B142.1 ±18.1 A
147.0
±
20.2
ab
142.2
±
32.0
a
157.9
±
19.3
b
Ln 0.0156 <0.0001 0.7843 56.6 ±26.0 B47.2 ±19.7 A41.6 ±13.2 a
48.0
±
22.0
ab 66.1 ±26.2 b
Lb <0.0001 <0.0001 0.2137 4.5 ±1.5 B3.4 ±1.3 A3.2 ±1.1 a4.0 ±1.3 b4.7 ±1.6 b
Q. pubescens S F S ×F Pe Co St P K
H<0.0001 0.0028 0.4337 36.8 ±11.5 B22.4 ±8.2 A27.9 ±10.7 ab 27.0 ±11.1 a33.9 ±14.0 b
RcD 0.0003 0.0024 0.4202 10.7 ±1.9 B9.4 ±2.2 A9.7 ±1.9 ab 9.4 ±2.3 a10.9 ±2.0 b
H/RcD <0.0001 0.5218 0.5139 34.8 ±10.3 B24.2 ±7.2 A28.9 ±10.6 28.9 ±9.5 30.8 ±11.1
Rv <0.0001 0.3988 0.0951 25.8 ±10.5 B14.9 ±6.1 A20.9 ±12.6 19.1 ±8.6 21.3 ±9.0
Rd 0.0101 0.2732 0.0215 0.41 ±0.1 A0.44 ±0.1 B0.42 ±0.1 0.43 ±0.16 0.42 ±0.1
S/R<0.0001 0.0001 0.0725 0.43 ±0.1 B0.27 ±0.1 A0.33 ±0.1 a0.31 ±0.1 a0.42 ±0.2 b
SLA 0.0600 0.6396 0.7851 104.6 ±13.4 100.6 ±8.7 101.4 ±9.0 103.8 ±13.9 102.7 ±11.2
Ln <0.0001 0.0232 0.4068 60.5 ±27.2 B34.9 ±15.1 A46.5 ±29.7 ab 41.7 ±20.5 a54.9 ±24.0 b
Lb <0.0001 0.0192 0.4390 4.3 ±1.5 B2.1 ±1.1 A3.1 ±1.7 ab 2.9 ±1.4 a3.7 ±1.9 b
Q. ilex S F S ×F Pe Co St P K
H<0.0001 0.0032 0.3332 52.7 ±13.1 B35.0 ±10.3 A40.9 ±13.0 a
41.8
±
13.3
ab 48.9 ±16.7 b
RcD 0.0114 0.1610 0.5929 8.5 ±1.1 B7.8 ±1.6 A8.1 ±1.3 7.9 ±1.5 8.5 ±1.3
H/RcD <0.0001 0.0386 0.2352 62.7 ±15.4 B45.1 ±12.3 A50.1 ±13.4 a
53.6
±
15.7
ab 58.0 ±19.2 b
Rv <0.0001 0.0009 0.4449 14.9 ±5.0 B10.7 ±4.3 A10.8 ±4.2 a12.9 ±5.4 ab 14.6 ±5.1 b
Rd 0.3072 0.1237 0.3875 0.45 ±0.10 0.43 ±0.13 0.45 ±0.13 0.41 ±0.08 0.46 ±0.12
S/R0.0062 0.3922 0.3637 0.71 ±0.3 B0.59 ±0.2 A0.68 ±0.2 0.61 ±0.3 0.67 ±0.2
SLA 0.0104 0.2676 0.0005 73.6 ±8.0 B70.5 ±5.5 A73.2 ±7.6 70.7 ±7.1 72.2 ±6.4
Ln <0.0001 0.0140 0.3323 49.4 ±15.6 B30.0 ±11.1 A38.6 ±16.2 ab 36.0 ±13.6 a44.5 ±19.0 b
Lb <0.0001 0.0040 0.1082 5.2 ±1.5 B2.9 ±1.2 A3.7 ±1.5 a3.9 ±1.8 ab 4.6 ±2.0 b
In Q. pubescens, substrate affected morphological variables more than fertilization, and, generally,
in the case of significant differences, seedlings in peat and K-enriched fertilization were better developed
(Tables 2and 3, Figure 1). Total biomass in Pe was higher than in Co by +78.3%, and the gap between K
and St and Pwas +19.3% and +20.1%, respectively. Pe significantly promoted all biomass variables
excluding FOLR >5. Fertilization with K promoted biomass growth in the shoot-system, leaves, and
in FOLR1-5 over St fertilization (Figure 1and Figure S1). Shoot-to-root ratio was lower than 0.51 in
all stocktypes (Table 2) and was affected by both substrate (Pe >Co) and fertilization (K>Pand St).
Root volume of Q. pubescens seedlings raised with peat was higher than that of coir, while the opposite
occurred for root density.
As per previous species, in the case of Q. ilex, the substrate affected the majority of the analyzed
variables (with a Pe >Co pattern), and generally, when fertilization was significant, seedlings grown in
K-enriched fertilizer had higher values (Tables 2and 3, Figure 1). Total biomass was affected by both
factors (Pe +63.5% than Co;K+36.2% and +50.2% than Pand St, respectively). Similar differences (Pe
>Co) occurred for shoot-system and leaf biomass, main root, FOLR <1,FOLR1-5, and root biomass.
Fertilization affected leaf, main root, FOLR1-5, and root biomass (K>Pand St). S/Rratio was affected
only by substrate (Table 3), both in Co and Pe it resulted higher than 0.50. Root volume was affected by
both substrate (Pe >Co) and fertilization (K>Pand St).

Forests 2020,11, 522 7 of 19
Forests 2020, 11, x FOR PEER REVIEW 7 of 18
(Pe > Co) occurred for shoot-system and leaf biomass, main root, FOLR < 1, FOLR1-5, and root
biomass. Fertilization affected leaf, main root, FOLR1-5, and root biomass (K> P and St). S/R ratio was
affected only by substrate (Table 3), both in Co and Pe it resulted higher than 0.50. Root volume was
affected by both substrate (Pe > Co) and fertilization (K > P and St).
Figure 1. Multifactorial ANOVA and Tukey post-hoc test results (p ≤ 0.05) for seedling dry biomass
(g) allocation at the end of the growing season (Nobs = 20 per stocktype per species). Source of
variation—substrate (S), fertilization (F). Sb—shoot-system dry biomass; MRb—main root dry
Figure 1.
Multifactorial ANOVA and Tukey post-hoc test results (p
≤
0.05) for seedling dry biomass
(g) allocation at the end of the growing season (N
obs
=20 per stocktype per species). Source of
variation—substrate (S), fertilization (F). Sb—shoot-system dry biomass; MRb—main root dry biomass;
FOLRb—dry biomass of first order lateral root split by diameter class (<1 mm, 1–5 mm, >5 mm). Capital
letters indicate homogenous groups for substrate, while lowercase letters indicate homogenous groups
for fertilization; letters in the upper part indicate groups for total biomass. Pe =Peat; Co =Coir; St =
Standard fertilization; K=K-enriched fertilization; and P=P-enriched fertilization.

Forests 2020,11, 522 8 of 19
Table 3.
pvalues from multifactorial ANOVA test results: (p<0.05 in bold) for seedlings biomass
allocation (N
obs
=20 per stocktype per species). Sources of variation—substrate (S), fertilization (F),
and their interaction (S
×
F). Sb: shoot-system biomass; MRb: main root biomass; FOLRb: First Order
Lateral Root biomass split by diameter class (<1 mm, 1–5 mm, >5 mm); Rb: total root-system biomass;
and Sb +Rb: total seedling biomass.
Q. robur S F S ×F
Sb <0.0001 <0.0001 0.6355
MRb 0.9205 0.0610 0.7401
FOLRb <10.0212 0.5548 0.9441
FOLRb 1–5 <0.0001 <0.0001 0.1665
FOLRb >50.8056 0.2704 0.6460
Rb 0.0454 0.0001 0.7101
Sb +Rb 0.0001 <0.0001 0.9119
Q. pubescens
Sb <0.0001 0.0002 0.2060
MRb <0.0001 0.1275 0.2324
FOLRb <10.0081 0.1637 0.0127
FOLRb 1–5 <0.0001 0.0075 0.1767
FOLRb >50.8859 0.3703 0.4351
Rb <0.0001 0.3417 0.4148
Sb +Rb <0.0001 0.0438 0.4563
Q. ilex
Sb <0.0001 <0.0001 0.1204
MRb <0.0001 0.0005 0.0230
FOLRb <10.0059 0.5604 0.0674
FOLRb 1–5 <0.0001 0.0013 0.1812
FOLRb >5---
Rb <0.0001 0.0001 0.0389
Sb +Rb <0.0001 <0.0001 0.0424
3.3. N, P, K Concentration in Seedlings and Substrate at the End of Cultivation
In Q. robur, N concentration was not affected by treatments, excluding fertilization on root-system
(St and P>K), while both substrate (Pe >Co) and fertilization (Pand K>St) affected P concentration in
seedling parts (excluding substrate on shoot; Table 4, Figure 2). Pe–P had higher P concentration than
other stocktypes in leaves and in shoot (Figure S3). Both Sand Fdid not influence K concentration. In
Q. pubescens, N concentration in any tissue was not influenced by either treatment (Table 4, Figure 2);
P concentration was affected by both substrate (Pe >Co) and fertilization (Pand K>St; excluding
substrate on shoot-system Table 4, Figure 2). Pe–P had a generally higher P content than other
stocktypes in leaves, shoot, and roots (Figure S3). Differences in K concentration were found between
substrates in leaves (Pe >Co). In Q. ilex, both substrate (Co >Pe) and fertilization (St >K) influenced
plant N concentration in all tissues (Table 4, Figure 2). Substrate affected leaf P concentration (Co >Pe)
and fertilization affected the root-system concentration (P>St), K concentration was influenced by
substrate in roots (Co >Pe), and by fertilization in the shoot-system (Table 4, Figure 2).

Forests 2020,11, 522 9 of 19
Forests 2020, 11, x FOR PEER REVIEW 9 of 18
Figure 2. Macro-element concentration (mg g−1 for N, P, K) in leaves (Lf), shoot (stem, Ss), and root-
system (Rs) analyzed for substrate and fertilization (mean ± SD). Source of variation substrate (S),
fertilization (F), and their interaction (S × F). Capital letters and lowercase letters indicate homogenous
groups for S and F, respectively. Pe = Peat; Co = Coir; St = Standard fertilization; K = K-enriched
fertilization; and P = P-enriched fertilization.
Nitrate N concentration of the growing media with Q. robur was affected by substrate and
fertilization (Pe > Co; P > K), and only by fertilization for ammonium N (P > K, Table 5). In Q. pubescens,
fertilization affected P concentration (P > K), and both nitric and ammonium N (P > K). In Q. ilex,
nitrate N was affected by fertilization (P > K) and ammonium N by both factors (Co > Pe; P > K), and
the P concentration result was not affected by substrate and fertilization, while both affected K
concentration (Co > Pe; K > P and St). No interaction among factors occurred.
Figure 2.
Macro-element concentration (mg g
−1
for N, P, K) in leaves (Lf), shoot (stem, Ss), and
root-system (Rs) analyzed for substrate and fertilization (mean
±
SD). Source of variation substrate (S),
fertilization (F), and their interaction (S
×
F). Capital letters and lowercase letters indicate homogenous
groups for Sand F, respectively. Pe =Peat; Co =Coir; St =Standard fertilization; K=K-enriched
fertilization; and P=P-enriched fertilization.

Forests 2020,11, 522 10 of 19
Table 4.
pvalues of multifactorial ANOVA (p
≤
0.05 in bold) for macro-element concentration (N,
P, K) in leaves, shoot-system, and root-system, at the end of the growing season (N=6 couples per
stocktype). Source of variation—substrate (S), fertilization (F), and their interaction (S×F).
Q. robur Q. pubescens Q. ilex
S F S ×F S F S ×F S F S ×F
N
Leaves 0.1398 0.8712 0.3971 0.2220 0.2340 0.2340 <0.0001 0.0067 0.3531
Shoot 0.3956 0.1357 0.1760 0.0922 0.0681 0.0681 <0.0001 0.0027 0.1779
Root 0.0711 0.0002 0.9724 0.5779 0.6927 0.6927 <0.0001 0.0017 0.2128
P
Leaves 0.0036 0.0317 0.4204 0.0002 0.0001 0.0001 0.0020 0.3830 0.4956
Shoot 0.4027 0.0013 0.0439 0.3254 0.0001 0.0001 0.1794 0.0923 0.9658
Root 0.0088 <0.0001 0.0208 0.0034 0.0024 0.0024 0.7227 0.0381 0.5865
K
Leaves 0.1448 0.5164 0.1323 0.0469 0.4150 0.4150 0.0751 0.7980 0.2119
Shoot 0.4515 0.7074 0.1073 0.2170 0.5737 0.5737 0.8051 0.0423 0.6514
Root 0.4930 0.6484 0.1861 0.4115 0.3866 0.3866 0.0188 0.3926 0.1645
Nitrate N concentration of the growing media with Q. robur was affected by substrate and
fertilization (Pe >Co;P>K), and only by fertilization for ammonium N (P>K, Table 5). In Q. pubescens,
fertilization affected P concentration (P>K), and both nitric and ammonium N (P>K). In Q. ilex,
nitrate N was affected by fertilization (P>K) and ammonium N by both factors (Co >Pe;P>K),
and the P concentration result was not affected by substrate and fertilization, while both affected K
concentration (Co >Pe;K>Pand St). No interaction among factors occurred.
Table 5.
Multifactorial ANOVA and Tukey post-hoc test results (p
≤
0.05 in bold) for (mean
±
SD, N =6)
macro-element concentration (N, P, K) in meq/L contained in the growing media at the end of the
season (N=12 per combination). Source of variation substrate (S), fertilization (F), and their interaction
(S
×
F). Capital and lowercase letters indicate homogenous groups for Sand F, respectively. Pe =Peat;
Co =Coir; St =Standard fertilization; K=K-enriched fertilization; and P=P-enriched fertilization.
Q. robur S F S ×F Pe Co St P K
Nitrate N 0.0160 0.0004 0.5459 1.32 ±0.49 B0.86 ±0.66 A
1.11
±
0.36
ab
1.65
±
0.47
b
0.52
±
0.39
a
Ammonium N 0.9211 0.0029 0.2031 1.02 ±0.34 1.01 ±0.54
0.92
±
0.26
ab
1.44
±
0.37
b
0.69
±
0.29
a
P 0.2960 0.9878 0.3515 0.26 ±0.08 0.22 ±0.06 0.24 ±0.06 0.23 ±0.05 0.24 ±0.11
K 0.3836 0.0821 0.9781 0.89 ±0.24 0.79 ±0.25 0.66 ±0.13 0.88 ±0.26 0.98 ±0.23
Q. pubescens
Nitrate N 0.0612 0.0020 0.3632 1.22 ±0.70 B0.83 ±0.48 A0.84 ±0.42 a
1.64
±
0.54
b
0.61
±
0.35
a
Ammonium N 0.7522 0.0036 0.6327 0.96 ±0.55 0.91 ±0.37
0.81
±
0.26
ab
1.39
±
0.38
b
0.60
±
0.28
a
P 0.9340 0.0222 0.6351 0.19 ±0.07 0.19 ±0.04
0.18
±
0.04
ab
0.24
±
0.05
b
0.15
±
0.04
a
K 0.5296 0.1437 0.5819 0.73 ±0.25 0.87 ±0.64 0.50 ±0.13 1.07 ±0.70 0.83 ±0.26
Q. ilex
Nitrate N 0.7839 0.0212 0.4642 0.65 ±0.51 0.71 ±0.51
0.77
±
0.35
ab
1.02
±
0.55
b
0.25
±
0.20
a
Ammonium N 0.0299 0.0260 0.3370 0.44 ±0.36 A0.83 ±0.46 B
0.65
±
0.39
ab
0.93
±
0.53
b
0.32
±
0.14
a
P 0.0824 0.3603 0.8507 0.15 ±0.06 0.21 ±0.08 0.17 ±0.05 0.22 ±0.11 0.16 ±0.05
K0.0295 0.0005 0.7127 0.68 ±0.34 A0.90 ±0.28 B0.59 ±0.23 a
0.65
±
0.25
a
1.14
±
0.14
b
4. Discussion
Our study highlighted the early effects of substrate on growth (2 months after sowing in Q. robur
and Q. pubescens; 3 months in Q. ilex, whose seedlings emerged later) and persisted through cultivation.
The absence of any fertilization effect on early growth phases was expected—dependence of Quercus
seedlings on acorn nutrients decreased as the seedlings developed [
7
,
72
]; thus, first growth flush in
many Quercus species was to a higher extent related to acorn size and nutrients [
73
,
74
], rather than to the
quality of the growing media [
7
,
72
], and our results were in line with those of Villar-Salvador et al. [
72
]
in Q. ilex. At the end of cultivation, seedlings of all species grown in peat were taller than those
grown in coir. However, the height of seedlings in coir were at least equal to that of comparable forest

Forests 2020,11, 522 11 of 19
nursery production systems of the Quercus species [
75
,
76
]; in other cases height was higher both in
Q. robur [77,78] and in Q. ilex [27,40,79,80], and were in line with the Italian national regulation [81].
Similar trends were found in other studies with other species using substrate mixtures that
included coir—Rose and Haase [
61
] with Douglas fir, Tsakaldimi and Ganatsas [
11
] with, among others,
Q. ilex, and Radjagukguk et al. [
62
] with two Eucalyptus. Offord et al. [
82
] did not observe differences
in several species, including Eucalyptus melliodora. The reduced growth rate observed in seedlings
grown in coir could be due to the lower CEC than in peat, which reduces nutrients availability for
seedlings [
10
]. This could explain the smaller size of the vast majority of the studied morphological
traits in all coir stocktypes in all species, regardless of fertilization. Additionally, according to previous
studies, such as Handreck [
83
] and Grantzau [
84
], there is a greater immobilization of soluble nitrogen
in coir than peat suggesting the need to add extra N fertilization during cultivation. In our study
we did not find a lower tissue concentration of seedlings raised with coir, suggesting an absence of
nitrogen deficiency. The common mixtures and fertilization protocols used by nursery companies
in horti-flori-fructiculture sector have been optimized [
85
] and plants in coir grow equally as peat
mixtures [29,82,86,87], suggesting that fertilizer can offset substrate deficiencies.
According to our results, morphological traits variability was explained more by substrate than
by fertilization. Fertilization effects on growth occurred later than those of the growing media, in
mid-summer for Q. pubescens and Q. ilex, and near the end of the growing season in Q. robur. In all
species and in both growing media, the K-enriched fertilization improved the growth rate during the
growing season and promoted taller plants. Apart from N fertilization, scarce literature is available
on the effect of the other macro-elements on the growth of forest species in the nursery [
7
]. K is
recognized as a key element of many metabolic processes [
88
], some of which are related to plant stress
responses. Direct connections of K with seedling growth are less investigated, but in a meta-analysis
of forest species (including plants at seedling stage), Tripler et al. [
89
] highlighted that plant growth
responded positively to an increase in K availability. Studies on tropical forest species highlighted a
direct effect of K fertilization in promoting plant growth with a higher shoot/root ratio [
90
,
91
]. Similar
to height responses, seedlings in all species grown in peat and K-enriched fertilization allocated more
resources to above-ground dry biomass (leaves and shoot system). Similar results for substrate effects
were obtained by Rose and Haase [
61
] in Douglas-fir. Coir K content is usually higher than that in
peat [
31
,
61
]. However, in our study, despite the tested rates of K being high compared to the literature
on oaks [
92
–
94
], we observed an outstanding effect of such fertilization on plant growth regardless of
species or substrate, suggesting K deficiency status. K-enriched fertilization included an additional
amount of P, as compared to a standard fertilizer, which could have been helpful in sustaining seedling
growth. However, fertilization with P was not as effective as K in promoting height and biomass in the
studied species, although, generally, it was better than standard fertilization. P plays a key role in plant
metabolism, being a critical element for many physiological reactions [
95
] and root morphological
traits (structure, growth, and articulation) [
49
,
50
,
95
–
97
]. Thus, a non-pronounced effect on the shoot
system growth was expected.
In all studied species and treatments, peat and K-enriched fertilization promoted the greatest
below-ground development. In all cases, shoot/root ratio was <1, and cultivation in coir reduced
this ratio further, particularly for Q. pubescens. Other studies on flowering species pointed out that
cultivation in coir can promote a proportionally higher root system component [
86
,
98
]. The same effect
occurred on the Douglas fir, with a higher root biomass for seedlings in coir and a decreasing trend in
the S/R ratio of peat to a mixture of coir and peat, followed by that of peat to coir [
61
]. In contrast,
Tsakaldimi and Ganastas [
11
] found a higher S/R ratio in 1-year-old seedlings of Q. macrolepis and
Q. ilex grown in a mixture of peat and coconut fiber than in peat with perlite. On the other hand,
Chulaka et al. [
99
] and Wilson et al. [
100
] reported the effects of coir-based substrates on S/R ratio with
non-significant results versus peat-based substrates, and Colla et al. [
98
] found a higher S/R ratio in
horticultural species grown in peat. A lower shoot-to-root ratio has been linked to survival under
drought condition in holm oak [
80
], in Mediterranean shrubs [
101
], and savanna species [
102
]. On the

Forests 2020,11, 522 12 of 19
contrary, Villar-Salvador et al. [
40
] highlighted that Q. ilex seedlings with a higher S/R had a lower
mortality and a larger growth than those with the opposite attributes; however, we found quite similar
shoot-to-root values for holm oak produced in coir to the highest values observed in this study (0.59 vs.
0.63, respectively).
Peat also promoted main root biomass and favored root fibrosity, especially the two smaller classes
of FOLR (>1 mm and 1–5 mm). The proportion of FOLR on total root biomass was statistically affected
by substrate (data not showed) in Q. robur and Q. ilex; however, the gap between substrates in all species
was slight. This result suggests that plants grown in coir, despite a lower absolute root-system biomass,
maintained comparable root fibrosity to peat. This trait, as well as a lower shoot-to-root ratio is crucial
to resist water stress, and is related to post-planting survival and growth under arid conditions, such as
in Mediterranean environments [
103
]. In seedlings grown with K-enriched fertilizer, the S/R ratio was
higher but the biomass allocated to FOLR was positively influenced by such fertilization. However,
studies on tropical tree species and in alpine and dryland environment [
90
,
91
] reported that K promoted
shoot biomass and less FOLR, resulting in a higher S/R ratio. P-enriched fertilization did not promote
the growth and articulation of the root-system, as expected, even though this study did not compare
different levels of P and K, but rather three different fertilizations. Scarce literature has focused on the
direct effect of P on detailed morphology below ground traits in Quercus. According to Pem
á
n [
7
], the
root systems of the Quercus species can be affected by the available P. In Q. ilex, Sardans et al. [
104
]
found that P fertilization can promote root growth, Villar-Salvador et al. [
40
] linked P concentration to
root regeneration, and Oliet et al. [
47
] highlighted a clear effect of root P concentration on root growth
potential (RGP).
Physiological results are in agreement with morphology, demonstrating an overall better
performance of photosynthetic machinery (considering SPAD and F
V
/F
M
in conjunction) of seedlings in
peat and seedlings fertilized with enriched K, in most species. Chlorophyll fluorescence, even though
measured values show absence of stress, has proven to be a sensitive technique to detect differences
among stocktypes, especially those from different fertilization treatments. Results for Chlorophyll
content were generally consistent with what was observed in morphology, and peat and K-enriched
fertilization promoted higher values in all species. The values of chlorophyll content observed for
deciduous oaks were in line with those in other studies [105,106].
We did not find a marked effect of substrate or fertilization on N concentration in plant tissues; at
the end of cultivation. N concentration of Q. robur and Q. pubescens did not depend on the substrate
and only sporadically depended on fertilization, whereas both factors affected Q. ilex. Despite a higher
N content per seedling than that from other studies on the same species [
92
,
107
], higher than the
sufficiency level and the optimum target in Q. ilex [
92
], N concentration in holm oak roots and the
shoot-system was lower. We supplied 27% less N than that of Berger and Glatzer [
108
], as luxury
consumption in Quercus petraea (a European temperate species), and it did not affect Q. robur tissue N
concentration. However, levels of uptake and consumption can vary not only in relation to experimental
factors, but also in terms of intrinsic ecological differences among provenances and species [
92
,
94
].
This makes the hypotheses on this issue for Q. pubescens uncertain, as it is not well-represented in the
literature for this species.
Peat as well as P-enriched fertilization were effective in increasing the level of P in all plant tissues
in Q. robur and Q. pubescens, while in Q. ilex, such fertilization was effective only for P concentration in
roots, and in contrast to the other species, holm oak seedlings in coir performed better than in peat.
Higher P root tissue concentration is important to promote root growth capacity after planting [
109
–
113
].
Campo et al. [
80
], in a study designed to define the quality standards for Q. ilex nursery stock, indicated
that the values of N and P foliar concentration should be higher than 10 and 0.9 mg g
−1
, respectively,
to improve growth performance after planting. Considering this target, our results for N were higher
and better in peat and in P-enriched and standard fertilization, and for P, these were about 10% lower
and higher in peat. No references are available for Q. robur and Q. pubescens; however, in our study,

Forests 2020,11, 522 13 of 19
foliar N and P concentrations in these deciduous species were higher than the standard values for the
evergreen Q.ilex.
In our study, K concentration in plant tissues was generally not affected by both substrate and
fertilization in all species, suggesting that K-enriched fertilization did not increase K concentration.
Considering the higher performance of seedlings grown in K-enriched fertilization, regardless of
species or substrate, this result strengthens the hypothesis that the Quercus species benefits from the
high K availability and that the amount provided in the standard and in P-enriched fertilization was
deficient. According to [
93
], K is the most responsive nutrient at deficiency status. The vast majority of
literature on fertilization of forest nursery stock has focused on the N effects; thus, more investigations
on the optimal K rates to maximize growth is needed. Del Campo et al. [
80
] concluded that nursery
stock with higher K concentration can exhibit improved establishment success due to K retranslocation.
Andivia et al. [
114
] observed that fertilization with K positively influenced morphological development
and NPK content. K effects on field performance on Pinus halepensis have been observed with conflicting
results [
115
,
116
]. Del Campo et al. [
117
] suggested that these multiple responses could be related to
the interference of K with other nutrients, particularly nitrogen. The interaction between K fertilizers
and different types of substrate requires further investigation.
5. Conclusions
Peat and coir in combination with different fertilization treatments affected above- and
below-ground morphology and, to a lesser extent, physiological traits of Quercus seedlings in forest
nursery production. The three studied species, despite being ecologically diverse, provided similar
results, with peat and K-enriched fertilization resulting in larger seedlings and slightly improved
physiological responses. Even though P- and, mostly, K-enriched fertilization partially offset the
difference, seedlings in coir were smaller, which could be linked to deficiencies in the chemical
properties of this material. Seedlings produced in coir showed a proportionally similar root system
development and fibrosity, and a generally lower shoot-to-root ratio than seedlings grown in peat.
According to the target plant concept [
3
,
5
], functional traits driving planting performance vary
according to environmental and operational conditions; and plants with a lower shoot-to-root ratio
might perform better under water stress or in semiarid/arid environments [
80
,
118
]. Pure coir might
thus serve as an acceptable material for seedling cultivation in such cases, despite the tendency to
produce smaller seedlings than peat. However, a more detailed evaluation of the response of seedlings
produced with coir to water stress is needed. NPK content was slightly influenced by treatments,
although P-enriched fertilization in peat was the only combination that promoted a higher amount
of this element in tissues at the end of cultivation; the strong effect of K-enriched fertilization on
seedling growth and biomass suggested a K deficiency in the other fertilization treatments; however,
macro-element content in relation to varying fertilization should be further investigated through a
comparison of the single element rates.
Supplementary Materials:
The following are available online at http://www.mdpi.com/1999-4907/11/5/522/s1.
Table S1: Multifactorial ANOVA and Tukey post-hoc test results (p
≤
0.05 in bold) for seedling morphological
traits (mean
±
SD) at the end of the season (N
obs
=20 seedlings per stocktype). Figure S1: Multifactorial ANOVA
and Tukey post-hoc test results of FV/FM values (box whisker plot) and SPAD units (mean and SD) analyzed for
substrate and fertilization in August. Figure S2: Multifactorial ANOVA and Tukey post-hoc test results (p
≤
0.05)
for seedling dry biomass (g) allocation at the end of the growing season (N
obs
=20 per stocktype per species).
Figure S3: Macro-element concentration (mg g
−1
for N. P. K) in the leaves, shoot-system, and root-system in
stocktypes (mean ±SD).
Author Contributions:
Conceptualization, A.T., B.M., A.M., S.R., D.F.J., and J.A.O.; Methodology, B.M., A.T.,
A.M., S.R., S.M., D.F.J., and J.A.O.; Software, S.M., B.M., and S.R.; Validation, S.M., B.M., and S.R.; Formal Analysis,
B.M. and S.M.; Investigation, B.M., S.M., A.M., and S.R.; Resources, B.M., S.M., A.M., and S.R.; Data Curation,
S.M., and B.M.; Writing–Original Draft Preparation, B.M. and S.M.; Writing–Review & Editing, B.M., S.M., A.M.,
S.R., D.F.J., and J.A.O.; Visualization, B.M. and S.M.; Supervision, A.M.; Project Administration, B.M., A.T., and
A.M.; Funding Acquisition, A.T., A.M., and B.M. All authors have read and agreed to the published version of
the manuscript.

Forests 2020,11, 522 14 of 19
Funding:
This study was funded in Italy in the framework of Regione Toscana PSR FEASR 2014–2020 Regione
Toscana-PIF Verdi Connessioni–Mis. 16.2 VIAA.
Acknowledgments:
Fabio Bandini and Stefano Teri assisted with study maintenance and lab measurements.
Vannucci Piante nursery company hosted the experimental and provided nursery materials. We particularly
want to thank Emilio Resta of Vannucci Piante for sharing his valuable expertise. We appreciate the constructive
comments of the three anonymous reviewers.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
References
1.
Wilson, B.C.; Jacobs, D.F. Quality assessment of temperate zone deciduous hardwood seedlings. New For.
2006,31, 417–433. [CrossRef]
2.
Pinto, J.R.; Dumroese, R.K.; Davis, A.S.; Landis, T.D. Conducting seedling stocktype trials: A new approach
to an old question. J. For. 2011,109, 293–299.
3.
Dumroese, K.R.; Landis, T.D.; Pinto, J.R.; Haase, D.L.; Wilkinson, K.W.; Davis, A.S. Meeting forest restoration
challenges: Using the target plant concept. Reforesta 2016,1, 37–52. [CrossRef]
4.
Duryea, M.L. Nursery cultural practices: Impacts on seedling quality. In Forestry Nursery Manual: Production
of Bareroot Seedlings; Springer: Berlin, Germany, 1984; pp. 143–164.
5.
Landis, T.D. The target plant concept-a history and brief overview. In National Proceedings: Forest and
Conservation Nursery Associations-2010; Proc. Proc. RMRS-P-65; Riley, L.E., Haase, D.L., Pinto, J.R., Eds.;
USDA Forest Service, Rocky Mountain Research Station: Fort Collins, CO, USA, 2011; Volume 65, pp. 61–66.
6.
Haase, D.L.; Davis, A.S. Developing and supporting quality nursery facilities and staffare necessary to meet
global forest and landscape restoration needs. Reforesta 2017,4, 69–93. [CrossRef]
7.
Pem
á
n, J.; Chirino, E.; Espelta, J.M.; Jacobs, D.F.; Mart
í
n-G
ó
mez, P.; Navarro-Cerrillo, R.; Oliet, J.A.;
Vilagrosa, A.; Villar-Salvador, P.; Gil-Pelegr
í
n, E. Physiological keys for natural and artificial regeneration of
oaks. In Oaks Physiological Ecology. Exploring the Functional Diversity of Genus Quercus L.; Springer: Berlin,
Germany, 2017; pp. 453–511.
8.
Landis, T.D.; Jacobs, D.F.; Wilkinson, K.M.; Luna, T. Growing Media. In Tropical Nursery Manual—A Guide to
Starting and Operating a Nursery for Native and Traditional Plants; Agriculture Handbook; Wilkinson, K.M.,
Landis, T.D., Haase, D.L., Daley, B.F., Dumroese, R.K., Eds.; USDA Forest Service: Washington, DC, USA,
2014; Volume 732, pp. 101–122.
9.
Barrett, G.E.; Alexander, P.D.; Robinson, J.S.; Bragg, N.C. Achieving environmentally sustainable growing
media for soilless plant cultivation systems–A review. Sci. Hortic. 2016,212, 220–234. [CrossRef]
10.
Landis, T.D.; Tinus, R.W.; McDonald, S.E.; Barnett, J.P. Containers and Growing Media. In The Container Tree
Nursery Manual; Agriculture Handbook; Landis, T.D., Nisley, R.G., Eds.; USDA Forest Service: Washington,
DC, USA, 1990; Volume 2, pp. 41–85.
11.
Tsakaldimi, M.; Ganatsas, P. A synthesis of results on wastes as potting media substitutes for the production
of native plant species. Reforesta 2016,1, 147–163. [CrossRef]
12.
Caron, J.; Rochefort, L. Use of peat in growing media: State of the art on industrial and scientific efforts
envisioning sustainability. Acta Hortic. 2013,982, 15–22. [CrossRef]
13. Á
ngeles-Arg
á
iz, R.E.; Flores-Garc
í
a, A.; Ulloa, M.; Garibay-Orijel, R. Commercial Sphagnum peat moss is a
vector for exotic ectomycorrhizal mushrooms. Biol. Invasions 2016,18, 89–101. [CrossRef]
14.
Schmilewski, G. Growing media constituents used in the EU in 2013. Acta Hortic.
2017
,1168, 85–92.
[CrossRef]
15. Apodaca, L.E. Peat in 2015. Min. Eng. 2016,68, 3030.
16.
Clarke, D.; Rieley, J. Strategy for Responsible Peatland Management, 6th ed.; International Peat Society: Jyväskylä,
Finland, 2010; pp. 10–25.
17. Schmilewski, G. The role of peat in assuring the quality of growing media. Mires Peat 2008,3, article 2.
18.
Michel, J.-C. The physical properties of peat: A key factor for modern growing media. Mires Peat
2010
,6,
article 2.
19.
Maher, M.; Prasad, M. Organic soilless media components. In Soilless Culture: Theory and Practice, 1st ed.;
Raviv, M., Lierh, J.H., Eds.; Elsevier Science: Amsterdam, The Netherlands, 2008; pp. 479–481.

Forests 2020,11, 522 15 of 19
20.
Alexander, P.D.; Bragg, N.C.; Meade, R.; Padelopoulos, G.; Watts, O. Peat in horticulture and conservation:
The UK response to a changing world. Mires Peat 2008,3, 10.
21.
Carlile, B.; Coules, A. Towards sustainability in growing media. Acta Hortic.
2013
,1013, 341–349. [CrossRef]
22.
Bonn, A.; Reed, M.S.; Evans, C.D.; Joosten, H.; Bain, C.; Farmer, J.; Emmer, I.; Couwenberg, J.; Moxey, A.;
Artz, R. Investing in nature: Developing ecosystem service markets for peatland restoration. Ecosyst. Serv.
2014,9, 54–65. [CrossRef]
23.
Rochefort, L.; Lode, E. Restoration of degraded boreal peatlands. In Boreal Peatland Ecosystems; Wieder, R.K.,
Vitt, D.H., Eds.; Springer: Berlin/Heidelberg, Germany, 2006; Volume 188, pp. 381–423.
24.
Kimmel, K.; Mander, Ü. Ecosystem services of peatlands: Implications for restoration. Prog. Phys. Geogr.
2010,34, 491–514. [CrossRef]
25.
Parry, L.E.; Holden, J.; Chapman, P.J. Restoration of blanket peatlands. J. Environ. Manag.
2014
,133, 193–205.
[CrossRef]
26.
Dunn, C.; Freeman, C. Peatlands: Our greatest source of carbon credits? Carbon Manag.
2011
,2, 289–301.
[CrossRef]
27.
Ugolini, F.; Mariotti, B.; Maltoni, A.; Tani, A.; Salbitano, F.; Izquierdo, C.G.; Macci, C.; Masciandaro, G.;
Tognetti, R. A tree from waste: Decontaminated dredged sediments for growing forest tree seedlings. J.
Environ. Manag. 2018,211, 269–277. [CrossRef]
28.
Wallace, P.; Holmes, S.; Alexander, R.; England, J.; Gaze, R. Review of Growing Media Use and Dominant
Materials (Peat and Alternatives) for Growing Media in Other Countries (European and International); Final Report;
DEFRA Project SP1206; Department for Environment, Food and Rural Affairs: London, UK, 2010.
29.
Ceglie, F.G.; Bustamante, M.A.; Amara, M.B.; Tittarelli, F. The challenge of peat substitution in organic
seedling production: Optimization of growing media formulation through mixture design and response
surface analysis. PLoS ONE 2015,10, e0128600. [CrossRef]
30.
Gruda, N.S. Increasing sustainability of growing media constituents and stand-alone substrates in soilless
culture systems. Agronomy 2019,9, 298. [CrossRef]
31.
Handreck, K.A. Properties of coir dust, and its use in the formulation of soilless potting media. Commun. Soil
Sci. Plant Anal. 1993,24, 349–363. [CrossRef]
32.
Schmilewski, G. Peat covers 77 percent of the growing media production in the EU. Peatl. Int.
2008
,1, 39–43.
33.
Nichols, M.A. Coir—A XXIst Century sustainable growing medium. Acta Hortic.
2007
,747, 91–95. [CrossRef]
34.
Blok, C.; Wever, G. Experience with selected physical methods to characterize the suitability of growing
media for plant growth. Acta Hortic. 2008,779, 239–250. [CrossRef]
35.
Poulter, R. Quantifying differences between treated and untreated coir substrate. Acta Hortic.
2014
,1018,
557–564. [CrossRef]
36.
Gruda, N. Current and future perspective of growing media in Europe. Acta Hortic.
2012
,960, 37–43.
[CrossRef]
37.
Poorter, H.; Nagel, O. The role of biomass allocation in the growth response of plants to different levels of
light, CO2, nutrients and water: A quantitative review. Funct. Plant Biol. 2000,27, 1191. [CrossRef]
38.
Poorter, H.; Niklas, K.J.; Reich, P.B.; Oleksyn, J.; Poot, P.; Mommer, L. Biomass allocation to leaves, stems
and roots: Meta-analyses of interspecific variation and environmental control. New Phytol.
2012
,193, 30–50.
[CrossRef]
39.
Dom
í
nguez, M.T.; Aponte, C.; P
é
rez-Ramos, I.M.; Garc
í
a, L.V.; Villar, R.; Marañ
ó
n, T. Relationships between
leaf morphological traits, nutrient concentrations and isotopic signatures for Mediterranean woody plant
species and communities. Plant Soil 2012,357, 407–424. [CrossRef]
40.
Villar-Salvador, P.; Planelles, R.; Enrıquez, E.; Rubira, J.P. Nursery cultivation regimes, plant functional
attributes, and field performance relationships in the Mediterranean oak Quercus ilex L. For. Ecol. Manag.
2004,196, 257–266. [CrossRef]
41.
Hern
á
ndez, E.I.; Vilagrosa, A.; Luis, V.C.; Llorca, M.; Chirino, E.; Vallejo, V.R. Root hydraulic conductance,
gas exchange and leaf water potential in seedlings of Pistacia lentiscus L. and Quercus suber L. grown under
different fertilization and light regimes. Environ. Exp. Bot. 2009,67, 269–276. [CrossRef]
42.
Grossnickle, S.C. Why seedlings survive: Influence of plant attributes. New For.
2012
,43, 711–738. [CrossRef]
43.
Cortina, J.; Vilagrosa, A.; Trubat, R. The role of nutrients for improving seedling quality in drylands. New For.
2013,44, 719–732. [CrossRef]

Forests 2020,11, 522 16 of 19
44.
Grossnickle, S.C.; MacDonald, J.E. Seedling Quality: History, application, and plant attributes. Forests
2018
,
9, 283. [CrossRef]
45.
Oliet, J.A.; Planelles, R.; Artero, F.; Jacobs, D.F. Nursery fertilization and tree shelters affect long-term field
response of Acacia salicina Lindl. planted in Mediterranean semiarid conditions. For. Ecol. Manag.
2005
,215,
339–351. [CrossRef]
46.
Ovalle, J.F.; Arellano, E.C.; Oliet, J.A.; Becerra, P.; Ginocchio, R. Linking nursery nutritional status and water
availability post-planting under intense summer drought: The case of a South American Mediterranean tree
species. IForest-Biogeosci. For. 2016,9, 758. [CrossRef]
47.
Oliet, J.A.; Salazar, J.M.; Villar, R.; Robredo, E.; Valladares, F. Fall fertilization of Holm oak affects N and P
dynamics, root growth potential, and post-planting phenology and growth. Ann. For. Sci.
2011
,68, 647–656.
[CrossRef]
48.
Villar-Salvador, P.; Valladares, F.; Dom
í
nguez-Lerena, S.; Ruiz-D
í
ez, B.; Fern
á
ndez-Pascual, M.; Delgado, A.;
Peñuelas, J.L. Functional traits related to seedling performance in the Mediterranean leguminous shrub
Retama sphaerocarpa: Insights from a provenance, fertilization, and rhizobial inoculation study. Environ.
Exp. Bot. 2008,64, 145–154. [CrossRef]
49.
Lambers, H.; Shane, M.W.; Cramer, M.D.; Pearse, S.J.; Veneklaas, E.J. Root structure and functioning for
efficient acquisition of phosphorus: Matching morphological and physiological traits. Ann. Bot.
2006
,98,
693–713. [CrossRef]
50.
Trubat, R.; Cortina, J.; Vilagrosa, A. Plant morphology and root hydraulics are altered by nutrient deficiency
in Pistacia lentiscus (L.). Trees 2006,20, 334. [CrossRef]
51.
Lynch, J.P. Root phenes for enhanced soil exploration and phosphorus acquisition: Tools for future crops.
Plant Physiol. 2011,156, 1041–1049. [CrossRef] [PubMed]
52.
Trubat, R.; Cortina, J.; Vilagrosa, A. Root architecture and hydraulic conductance in nutrient deprived Pistacia
lentiscus L. seedlings. Oecologia 2012,170, 899–908. [CrossRef] [PubMed]
53.
Lugli, L.F.; Anderson, K.M.; Arag
ã
o, L.E.; Cordeiro, A.L.; Cunha,H.F.; Fuchslueger, L.; Meir, P.; Mercado,L.M.;
Oblitas, E.; Quesada, C.A.; et al. Multiple phosphorus acquisition strategies adopted by fine roots in
low-fertility soils in Central Amazonia. Plant Soil 2019, 1–15. [CrossRef]
54.
Fern
á
ndez, M.; Marcos, C.; Tapias, R.; Ruiz, F.; L
ó
pez, G. Nursery fertilisation affects the frost-tolerance and
plant quality of Eucalyptus globulus Labill. cuttings. Ann. For. Sci. 2007,64, 865–873. [CrossRef]
55.
Oliet, J.A.; Planelles, R.; Artero, F.; Valverde, R.; Jacobs, D.F.; Segura, M.L. Field performance of Pinus
halepensis planted in Mediterranean arid conditions: Relative influence of seedling morphology and mineral
nutrition. New For. 2009,37, 313–331. [CrossRef]
56.
Oliet, J.A.; Pu
é
rtolas, J.; Planelles, R.; Jacobs, D.F. Nutrient loading of forest tree seedlings to promote stress
resistance and field performance: A Mediterranean perspective. New For. 2013,44, 649–669. [CrossRef]
57.
Egilla, J.N.; Davies, F.T.; Drew, M.C. Effect of potassium on drought resistance of Hibiscus rosa-sinensis cv.
Leprechaun: Plant growth, leaf macro-and micronutrient content and root longevity. Plant Soil
2001
,229,
213–224. [CrossRef]
58.
Egilla, J.N.; Davies, F.T.; Boutton, T.W. Drought stress influences leaf water content, photosynthesis, and
water-use efficiency of Hibiscus rosa-sinensis at three potassium concentrations. Photosynthetica
2005
,43,
135–140. [CrossRef]
59.
Asgharipour, M.R.; Heidari, M. Effect of potassium supply on drought resistance in sorghum: Plant growth
and macronutrient content. Pak. J. Agric. Sci. 2011,48, 197–204.
60.
Ragel, P.; Raddatz, N.; Leidi, E.O.; Quintero, F.J.; Pardo, J.M. Regulation of K+nutrition in plants. Front.
Plant Sci. 2019,10, 281. [CrossRef] [PubMed]
61.
Rose, R.; Haase, D.L. The use of coir as a containerized growing medium for Douglas-fir seedlings. Native
Plants J. 2000,1, 107–111. [CrossRef]
62.
Radjagukguk, B.; Soeseno, O. A comparative study of peats and other media for containerized forest tree
seedlings. Acta Hortic. 1983,150, 449–458. [CrossRef]
63.
Oliet, J.A.; Jacobs, D.F. Restoring forests: Advances in techniques and theory. New For.
2012
,43, 535–541.
[CrossRef]

Forests 2020,11, 522 17 of 19
64.
Leverkus, A.B.; Castro, J.; Delgado-Capel, M.J.; Molinas-Gonz
á
lez, C.; Pulgar, M.; Marañ
ó
n-Jim
é
nez, S.;
Delgado-Huertas, A.; Querejeta, J.I. Restoring for the present or restoring for the future: Enhanced
performance of two sympatric oaks (Quercus ilex and Quercus pyrenaica) above the current forest limit.
Restor. Ecol. 2015,23, 936–946. [CrossRef]
65.
Madrigal-Gonz
á
lez, J.; Ruiz-Benito, P.; Ratcliffe, S.; Rigling, A.; Wirth, C.; Zimmermann, N.E.; Zweifel, R.;
Zavala, M.A. Competition Drives Oak Species Distribution and Functioning in Europe: Implications
Under Global Change. In Oaks Physiological Ecology. Exploring the Functional Diversity of Genus Quercus L.;
Gil-Pelegr
í
n, E., Peguero-Pina, J.J., Sancho-Knapik, D., Eds.; Springer: New York, NY, USA, 2017; pp. 513–538.
66.
Löf, M.; Castro, J.; Engman, M.; Leverkus, A.B.; Madsen, P.; Reque, J.A.; Villalobos, A.; Gardiner, E.S. Tamm
Review: Direct seeding to restore oak (Quercus spp.) forests and woodlands. For. Ecol. Manag.
2019
,448,
474–489. [CrossRef]
67.
Gil-Pelegr
í
n, E.; Saz, M.
Á
.; Cuadrat, J.M.; Peguero-Pina, J.J.; Sancho-Knapik, D. Oaks under
Mediterranean-type climates: Functional response to summer aridity. In Oaks Physiological Ecology. Exploring
the Functional Diversity of Genus Quercus L.; Gil-Pelegr
í
n, E., Peguero-Pina, J.J., Sancho-Knapik, D., Eds.;
Springer: New York, NY, USA, 2017; pp. 137–193.
68.
Ducousso, A.; Bordacs, S. EUFORGEN Technical Guidelines for Genetic Conservation and Use for Pedunculate
and Sessile Oaks (Quercus robur) and (Quercus petraea); International Plant Genetic Resources Institute: Rome,
Italy, 2004.
69.
Löf, M.; Bolte, A.; Jacobs, D.F.; Jensen, A.M. Nurse trees as a forest restoration tool for mixed plantations:
Effects on competing vegetation and performance in target tree species. Restor. Ecol.
2014
,22, 758–765.
[CrossRef]
70.
ISTA. International Rules for Seed Testing; International Seed Testing Association: Bassersdorf, Switzerland, 2020.
71.
Strasser, R.J.; Tsimilli-Michael, M.; Srivastava, A. Analysis of the Chlorophyll a Fluorescence Transient.
In Chlorophyll a Fluorescence: A Signature of Photosynthesis; Advances in Photosynthesis and Respiration;
Papageorgiou, G.C., Ed.; Springer: Dordrecht, The Netherlands, 2004; pp. 321–362. ISBN 978-1-4020-3218-9.
72.
Villar-Salvador, P.; Heredia, N.; Millard, P. Remobilization of acorn nitrogen for seedling growth in holm oak
(Quercus ilex), cultivated with contrasting nutrient availability. Tree Physiol. 2010,30, 257–263. [CrossRef]
73.
Garc
í
a-Cebri
á
n, F.; Esteso-Mart
í
nez, J.; Gil-Pelegr
í
n, E. Influence of cotyledon removal on early seedling
growth in Quercus robur L. Ann. For. Sci. 2003,60, 69–73. [CrossRef]
74.
Quero, J.L.; Villar, R.; Marañ
ó
n, T.; Zamora, R.; Poorter, L. Seed-mass effects in four Mediterranean Quercus
species (Fagaceae) growing in contrasting light environments. Am. J. Bot. 2007,94, 1795–1803. [CrossRef]
75.
Chirino, E.; Vilagrosa, A.; Hern
á
ndez, E.I.; Matos, A.; Vallejo, V.R. Effects of a deep container on
morpho-functional characteristics and root colonization in Quercus suber L. seedlings for reforestation in
Mediterranean climate. For. Ecol. Manag. 2008,256, 779–785. [CrossRef]
76.
Mariotti, B.; Maltoni, A.; Jacobs, D.F.; Tani, A. Container effects on growth and biomass allocation in Quercus
robur and Juglans regia seedlings. Scand. J. For. Res. 2015,30, 401–415.
77.
Mariotti, B.; Maltoni, A.; Jacobs, D.F.; Tani, A. Tree shelters affect shoot and root system growth and structure
in Quercus robur during regeneration establishment. Eur. J. For. Res. 2015,134, 641–652. [CrossRef]
78.
Cabral, R.; O’Reilly, C. Physiological and field growth responses of oak seedlings to warm storage. New For.
2008,36, 159–170. [CrossRef]
79.
Tsakaldimi, M.; Zagas, T.; Tsitsoni, T.; Ganatsas, P. Root Morphology, Stem Growth and Field Performance of
Seedlings of Two Mediterranean Evergreen Oak Species Raised in Different Container Types. Plant Soil
2005
,
278, 85–93. [CrossRef]
80.
Del Campo, A.D.; Navarro, R.M.; Ceacero, C.J. Seedling quality and field performance of commercial
stocklots of containerized holm oak (Quercus ilex) in Mediterranean Spain: An approach for establishing a
quality standard. New For. 2010,39, 19. [CrossRef]
81.
Dlgs 386/03. Available online: https://www.camera.it/parlam/leggi/deleghe/03386dl.htm (accessed on
1 May 2020).
82.
Offord, C.A.; Muir, S.; Tyler, J.L. Growth of selected Australian plants in soilless media using coir as a
substitute for peat. Aust. J. Exp. Agric. 1998,38, 879–887. [CrossRef]
83. Handreck, K.A. Immobilization of nitrogen in potting media. Acta Hortic. 1993,342, 121–126. [CrossRef]
84. Grantzau, E.; Gennrich, J.; DP, D. Mit Kokos Substrate verbessern. Gb Gw 1993,11, 538–541.

Forests 2020,11, 522 18 of 19
85.
Noguera, P.; Abad, M.; Noguera, V.; Puchades, R.; Maquieira, A. Coconut coir waste, a new and viable
ecologically-friendly peat substitute. Acta Hortic. 2000,517, 279–286. [CrossRef]
86.
Meerow, A.W. Growth of two subtropical ornamentals using coir (coconut mesocarp pith) as a peat substitute.
HortScience 1994,29, 1484–1486. [CrossRef]
87.
Stamps, R.H.; Evans, M.R. Growth of Dracaena marginata and Spathiphyllum ‘Petite’in sphagnum peat-and
coconut coir dust-based growing media. J. Environ. Hortic. 1999,17, 49–52.
88.
Wang, M.; Zheng, Q.; Shen, Q.; Guo, S. The critical role of potassium in plant stress response. Int. J. Mol. Sci.
2013,14, 7370–7390. [CrossRef] [PubMed]
89.
Tripler, C.E.; Kaushal, S.S.; Likens, G.E.; Todd Walter, M. Patterns in potassium dynamics in forest ecosystems.
Ecol. Lett. 2006,9, 451–466. [CrossRef] [PubMed]
90.
Santiago, L.S.; Wright, S.J.; Harms, K.E.; Yavitt, J.B.; Korine, C.; Garcia, M.N.; Turner, B.L. Tropical tree
seedling growth responses to nitrogen, phosphorus and potassium addition. J. Ecol.
2012
,100, 309–316.
[CrossRef]
91.
Soliveres, S.; Maestre, F.T. Plant–plant interactions, environmental gradients and plant diversity: A global
synthesis of community-level studies. Perspect. Plant Ecol. Evol. Syst. 2014,16, 154–163. [CrossRef]
92.
Uscola, M.; Salifu, K.F.; Oliet, J.A.; Jacobs, D.F. An exponential fertilization dose–response model to promote
restoration of the Mediterranean oak Quercus ilex. New For. 2015,46, 795–812. [CrossRef]
93.
Salifu, K.F.; Jacobs, D.F. Characterizing fertility targets and multi-element interactions in nursery culture of
Quercus rubra seedlings. Ann. For. Sci. 2006,63, 231–237. [CrossRef]
94.
Villar-Salvador, P.; Peñuelas, J.L.; Nicol
á
s-Perag
ó
n, J.L.; Benito, L.F.; Dom
í
nguez-Lerena, S. Is nitrogen
fertilization in the nursery a suitable tool for enhancing the performance of Mediterranean oak plantations?
New For. 2013,44, 733–751. [CrossRef]
95.
Niu, Y.F.; Chai, R.S.; Jin, G.L.; Wang, H.; Tang, C.X.; Zhang, Y.S. Responses of root architecture development
to low phosphorus availability: A review. Ann. Bot. 2013,112, 391–408. [CrossRef]
96.
Postma, J.A.; Dathe, A.; Lynch, J.P. The optimal lateral root branching density for maize depends on nitrogen
and phosphorus availability. Plant Physiol. 2014,166, 590–602. [CrossRef] [PubMed]
97.
P
é
ret, B.; Desnos, T.; Jost, R.; Kanno, S.; Berkowitz, O.; Nussaume, L. Root architecture responses: In search
of phosphate. Plant Physiol. 2014,166, 1713–1723. [CrossRef] [PubMed]
98.
Colla, G.; Rouphael, Y.; Possanzini, G.; Cardarelli, M.; Temperini, O.; Saccardo, F.; Pierandrei, F.; Rea, E.
Coconut coir as a potting media for organic lettuce transplant production. In VIII International Symposium on
Protected Cultivation in Mild Winter Climates: Advances in Soil and Soilless Cultivation under Protected Environment;
International Society for Horticultural Science: Leuven, Belgium, 2006; pp. 293–296.
99.
Chulaka, P.; Maruo, T.; Takagaki, M.; Shinohara, Y. Organic substrates of tropical origin as an alternative to
growing media for chili and cucumber transplant production. Jpn. J. Trop. Agric. 2004,48, 79–87.
100.
Wilson, S.B.; Muller, K.L.; Wilson, P.C.; Incer, M.R.; Stoffella, P.J.; Graetz, D.A. Evaluation of new container
media for Aglaonema production. Commun. Soil Sci. Plant Anal. 2009,40, 2673–2687. [CrossRef]
101.
Lloret, F.; Casanovas, C.; Penuelas, J. Seedling survival of Mediterranean shrubland species in relation to
root: Shoot ratio, seed size and water and nitrogen use. Funct. Ecol. 1999,13, 210–216. [CrossRef]
102.
Zida, D.; Tigabu, M.; Sawadogo, L.; Od
é
n, P.C. Initial seedling morphological characteristics and field
performance of two Sudanian savanna species in relation to nursery production period and watering regimes.
For. Ecol. Manag. 2008,255, 2151–2162. [CrossRef]
103.
Villar-Salvador, P.; Pu
é
rtolas, J.; Cuesta, B.; Peñuelas, J.L.; Uscola, M.; Heredia-Guerrero, N.; Rey Benayas, J.M.
Increase in size and nitrogen concentration enhances seedling survival in Mediterranean plantations. Insights
from an ecophysiological conceptual model of plant survival. New For. 2012,43, 755–770. [CrossRef]
104.
Sardans, J.; Peñuelas, J.; Rod
à
, F. Plasticity of leaf morphological traits, leaf nutrient content, and water
capture in the Mediterranean evergreen oak Quercus ilex subsp. ballota in response to fertilization and
changes in competitive conditions. Ecoscience 2006,13, 258–270. [CrossRef]
105.
Vollmar, A.; Gunderson, C. Physiological adjustments of leaf respiration to atmospheric warming in Betula
alleghaniensis and Quercus rubra. J. Undergrad. Res. 2006,6, 104–107.
106.
Salifu, K.F.; Apostol, K.G.; Jacobs, D.F.; Islam, M.A. Growth, physiology, and nutrient retranslocation in
nitrogen-15 fertilized Quercus rubra seedlings. Ann. For. Sci. 2008,65, 101. [CrossRef]

Forests 2020,11, 522 19 of 19
107.
Oliet, J.A.; Tejada, M.; Salifu, K.F.; Collazos, A.; Jacobs, D.F. Performance and nutrient dynamics of holm oak
(Quercus ilex L.) seedlings in relation to nursery nutrient loading and post-transplant fertility. Eur. J. For.
Res. 2009,128, 253–263. [CrossRef]
108.
Berger, T.W.; Glatzel, G. Response of Quercus petraea seedlings to nitrogen fertilization. For. Ecol. Manag.
2001,149, 1–14. [CrossRef]
109.
Bigg, W.L.; Schalau, J.W. Mineral nutrition and the target seedling. In Target Seedling Symposium: Proceedings,
Combined Meeting of the Western Forest Nursery Associations; USDA Forest Service: Fort Collins, CO, USA,
1990; pp. 139–160.
110.
Folk, R.S.; Grossnickle, S.C. Stock-type patterns of phosphorus uptake, retranslocation, net photosynthesis
and morphological development in interior spruce seedlings. New For. 2000,19, 27–49. [CrossRef]
111.
Sardans, J.; Peñuelas, J. Increasing drought decreases phosphorus availability in an evergreen Mediterranean
forest. Plant Soil 2004,267, 367–377. [CrossRef]
112.
Sun, Y.; Gu, J.-C.; Zhuang, H.-F.; Wang, Z.-Q. Effects of ectomycorrhizal colonization and nitrogen fertilization
on morphology of root tips in a Larix gmelinii plantation in northeastern China. Ecol. Res.
2010
,25, 295–302.
[CrossRef]
113.
Pascual, S.; Olarieta, J.R.; Rodr
í
guez-Ochoa, R. Development of Quercus ilex plantations is related to soil
phosphorus availability on shallow calcareous soils. New For. 2012,43, 805–814. [CrossRef]
114.
Andivia, E.; Fern
á
ndez, M.; V
á
zquez-Piqu
é
, J. Autumn fertilization of Quercus ilex ssp. ballota (Desf.)
Samp. nursery seedlings: Effects on morpho-physiology and field performance. Ann. For. Sci.
2011
,68, 543.
[CrossRef]
115.
Oliet, J.A.; Planelles, R.; L
ó
pez, M.; Artero, F. Efecto de la fertilizaci
ó
n en vivero sobre la supervivencia en
plantación de Pinus halepensis Mill. Investig. Agrar. Sist. Recur. For. 1997,8, 207–228.
116.
Pu
é
rtolas, J.; Gil, L.; Pardos, J.A. Effects of nutritional status and seedling size on field performance of Pinus
halepensis planted on former arable land in the Mediterranean basin. Forestry
2003
,76, 159–168. [CrossRef]
117.
Del Campo, A.D.; Hermoso, J.; Flors, J.; Lid
ó
n, A.; Navarro-Cerrillo, R.M. Nursery location and potassium
enrichment in Aleppo pine stock 2. Performance under real and hydrogel-mediated drought conditions.
Forestry 2011,84, 235–245. [CrossRef]
118.
Grossnickle, S.C. Importance of root growth in overcoming planting stress. New For.
2005
,30, 273–294.
[CrossRef]
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).