Plant Ecol (2017) 218:39–53
DOI 10.1007/s11258-016-0632-7
Improving restoration success through research-driven
initiatives: case studies targeting Pinus pinea reforestation
stock development in Lebanon
Olga A. Kildisheva . Matthew M. Aghai .
Karma Bouazza . Anthony S. Davis
Received: 30 April 2016 / Accepted: 7 July 2016 / Published online: 5 January 2017
Ó Springer Science+Business Media Dordrecht 2016
Abstract Lebanon is located in one of the most
ecologically diverse semi-arid regions of the world,
characterized by endemism and richness of higher
plants, which persist despite centuries of intensive
agro-sylvo-pastoral use and more recent urbanization.
Extensive deforestation, fires, and potential desertifi-
cation have advanced concerns over the sustainability
Communicated by Mrs. Olga Kildisheva, Ms. Lauren Svejcar
and Dr. Erik Hamerlynck.
Electronic supplementary material The online version of
this article (doi:10.1007/s11258-016-0632-7) contains supple-
mentary material, which is available to authorized users.
O. A. Kildisheva (&)
School of Plant Biology, University of Western Australia,
35 Stirling Hwy, Crawley, WA 6009, Australia
e-mail: olga.kildisheva@research.uwa.edu.au
M. M. Aghai
School of Environmental and Forest Sciences, University
of Washington, 4000 15th Ave NE, Seattle, WA 98105,
USA
K. Bouazza
Lebanon Reforestation Initiative, Fourth Floor Hazmieh
Square Building, Hazmieh, Beirut, Lebanon
A. S. Davis
College of Forestry, Oregon State University, Corvallis,
OR 97331, USA
of naturally forested ecosystems in Lebanon, with only
ca. 13 % forest cover remaining. Despite high num-
bers of seedlings planted in recent decades, reforesta-
tion success in Lebanon has been historically low.
Increased success can be achieved through the effec-
tive production of high-quality native plant seedlings,
capable of survival in increasingly arid climatic and
harsh edaphic conditions. Through two trials focused
on Pinus pinea as a model species, we aim to use a
research-based approach to identify techniques
required to produce high-quality seedlings to improve
post-transplant performance in Lebanon. The first trial
examines the suitability of using controlled release
fertilizer for seedling production and evaluates the
effect of fertilizer application rates on seedling
morphological and tissue nutrient parameters. The
second study examines the role of water availability
during the three key periods of seedling development
in the nursery to identify the optimal irrigation regime
needed to achieve functional attributes associated with
seedling drought and cold hardiness. Our findings
serve as the first data-driven recommendations for
improved seedling production in Lebanon and the
greater region as well as demonstrate how locally
integrated planning can result in effective capacity
building.
Keywords Controlled release fertilization 
Irrigation  Container seedling  Cold hardiness 
Stone pine  Target plant concept (TPC)
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Introduction
Regional background
Lebanon is one of the most ecologically diverse
semi-arid regions of the world, characterized by
endemism and richness of higher plants (Médail and
Quézel 1999). Despite millennia of anthropogenic
pressure, the nation’s distinct physiography contin-
ues to shelter over 2600 native species, 12 % of
which are considered regionally endemic (Médail
and Quézel 1999; Talhouk et al. 2001). Extensive
habitat degradation has led to designation of over
170 native herbaceous and 20 woody species as high
conservation priority targets (Khouzami et al. 1996;
Sattout et al. 2007).
The Food and Agriculture Organization’s (FAO)
Global Forest Resources Assessment (FRA) con-
ducted in 2005 classified 13 % (139,376 ha) of
Lebanon as forested; a figure that reflects a loss of
more than 35 % of primary forest cover over the
preceding four decades as a result of fires, ex-urban
development, grazing, agriculture, and an unsta-
ble sociopolitical landscape (Mitri and El Hajj
2007). This decline has negatively impacted many
Lebanese communities that rely on ecosystem
services (Choueiter and Ucenic 2007; Médail and
Quézel 1999) and is expected to increase as a result
of desertification, anthropogenic pressure, and cli-
mate change over the next 20 years (El Hajj et al.
2014).
As a signatory of the United Nations Convention to
Combat Desertification (MoA 2003; UNDP 2008), the
Lebanese government identifies degradation of forests
and woodlands as one of the main factors for
desertification and gives priority for their protection
and restoration. In 2003, a National Action Program
pledged to increase forest cover in Lebanon to 20 %,
but cited the lack of plant propagation infrastructure,
regional scientific knowledge, and cross-institutional
integration as major constraints to the implementation
of this goal (UNDP 2008).
Over the past two decades, a number of non-
governmental groups have been active in the
development of programs that aim to conserve and
restore Lebanon’s native forests. These region- and
nation-wide programs have promoted traditional and
multidisciplinary approaches to building capacity of
local communities and organizations. For one
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Plant Ecol (2017) 218:39–53
program, the Lebanon Reforestation Initiative
(LRI),1 a key factor in developing the forests and
affiliate sectors was improving the capacity of
domestic nurseries to produce plants for reforesta-
tion (USAID 2015). Prior to their involvement in
2012, nursery production followed relatively
underdeveloped and unsystematic practices; while
major overhauls of seedling production efforts were
not easily adopted due to significant barriers for
acquisition of key materials (produced outside the
region), infrastructure limitations, and the lack of
training for local practitioners (UNDP 2008).
Historical nursery practices
Before 2012, typical seedling production was conducted
using an assortment of large ([1000 mL) containers,
ranging from soft-walled polybags to recycled metal
cans filled with locally collected soil (Bouazza, personal
observation). Correspondingly, propagation techniques
(i.e., irrigation quantity and distribution, fertilization,
and handling practices) were inconsistent and uncorre-
lated with container size. Knowledge gaps concerning
nutrient availability and crop development translated
into the use of manure or compost at undefined rates as a
means of nutrient delivery. Moreover, the absence of
record keeping and description of practices used
throughout the growing seasons stifled capacity to
improve propagation techniques and seedling quality
over time. The resultant seedlings were often morpho-
logically unbalanced, root-bound, and nutrient-defi-
cient, which was a likely contributor to plantation failure
(Dominguez-Lerena et al. 2006; Pinto et al. 2011a).
Multiple growing seasons were used to achieve large
seedling sizes, despite higher production costs and low
quality, with in situ seeding survival between 10 and
40 % (Nehme 2014; UNDP 2008).
Seedling quality
Guided by the need for a functional approach, LRI
initiated a capacity-building strategy that focused on
the development of an education and technology
transfer program, which promoted the incorporation of
regionally suitable raw materials and native plant
1
Program funded by the United States Agency for International
Development and implemented by the United States Forest
Service, International Programs.
Plant Ecol (2017) 218:39–53
propagation techniques. This approach used the target
plant concept (TPC) as its basic framework, in which
the biotic and abiotic limitations of the reforestation
site guide the selection of propagation techniques for
seedling production to enhance growth and survival
following outplanting (Dumroese et al. 2016). Fur-
thermore, this strategy emphasized a research-based
approach focused on key elements of seedling pro-
duction (i.e., optimization of container parameters,
plant nutrition, and irrigation) and direct integration
across regional nurseries.
Seedling quality is critical to the success of an
outplanting project (Landis 2003; Pinto et al. 2011b;
Rose et al. 1990), particularly under limiting site
conditions (Palacios et al. 2009). In Lebanon, out-
planting typically occurs in autumn (Villar-Salvador
et al. 2012), prompted by high soil moisture avail-
ability, moderate temperatures, and a lack of capacity
to cold-store seedlings until spring. Given the envi-
ronmental limitations to plant establishment in the
Mediterranean (Mendoza et al. 2009; Vallejo et al.
2006), precise crop management during nursery
propagation is imperative to ensure optimal seedling
quality by the outplanting date (Landis 2003). Quality
driven by plant growth capacity, nutrient storage
reserves, and tolerance of the planting environment
can be enhanced through optimized container selec-
tion, fertilization, and irrigation during nursery prop-
agation (Jacobs and Timmer 2005; Landis 1985; Van
den Driessche 1991).
Container selection
As part of LRI’s initiative, nine nurseries adopted
propagation practices that implemented the use of a
standardized range of rigid-walled containers that
promote downward root growth (300–700 mL) for
seedling production. The standardization of the stock-
type created an opportunity to develop and implement
species- and container-specific propagation practices,
which emphasize irrigation and fertilization regimes
to produce seedlings suited for outplanting conditions
that are characterized by extended summer droughts,
significant frost, vigorous competition from grasses,
rocky soils, and high incidence of damage from
grazing.
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Fertilization
Proper seedling nutrition in the course of nursery
propagation can influence post-planting seedling per-
formance (Landis 2003; Puértolas et al. 2003). The
means of nutrient application is often determined by
the irrigation method employed during production. In
Lebanon, typical irrigation infrastructure does not
allow for water-soluble fertilizers to be used, but
controlled release fertilizers (CRF) can be employed
as an alternative. However, nutrient application via
CRF can pose a challenge, since the rate of application
cannot be altered once incorporated into the growing
medium, thus limiting the ability to adaptively manage
the nutrient regime during the growing season.
Several studies have addressed the optimization of
propagation regimes for Mediterranean pines (Cuesta
et al. 2010; Dominguez-Lerena et al. 2006; Oliet et al.
2004, 2009; Palá et al. 1997; Puértolas et al. 2003; Uscola
et al. 2014; Villar-Salvador et al. 2004, 2005,
2010, 2012). However, all the referenced studies target
restoration in Spain and thus may not provide an accurate
benchmark for generating basin-wide species require-
ments because of differences in species provenance and
restoration landscape physiography (Zahran and Gilbert
2010). In addition, the majority of the previously
published trials fail to decouple the influence of moisture
and fertilizer availability on seedling status—an
approach critical to identifying species-specific nursery
propagation practices (Pinto et al. 2011a).
Irrigation
An irrigation regime that is coupled with the phases of
plant growth in the nursery can be used to control
seedling morpho-physiological development (Landis
1989; Lavender 1984). Specifically, controlled
drought stress during the hardening phase of nursery
propagation has been shown to improve survival,
drought, cold, and stress tolerance after outplanting
among numerous northern temperate conifers (Blake
et al. 1979; Duryea 1984; Grossnickle et al. 1991;
Timmis and Tanaka 1976; Van den Driessche 1991).
However, irrigation thresholds can be species-, con-
tainer-, and site-specific (Dominguez-Lerena et al.
2006; Vilagrosa et al. 2003), as evidenced by the
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mixed results following drought hardening of Mediter-
ranean species (Beikircher et al. 2010; Vilagrosa et al.
2003; Villar-Salvador et al. 1999, 2013). Conversely,
improper irrigation can result in salt build-up and toxic
rhizosphere conditions, unbalanced seedling root and
shoot biomass, or sub-optimal nutrient allocation,
leading to a lower likelihood of survival post-
outplanting (Duryea 1984; Landis and Wilkinson
2009).
Moisture availability throughout the nursery grow-
ing season can have a profound effect on seedling
quality and post-outplanting performance (Duryea
1984). However, most studies of Mediterranean
conifer production have focused on the influence of
fertilization (Cuesta et al. 2010; Oliet et al.
2004, 2009; Puértolas et al. 2003, 2005; Villar-
Salvador et al. 2005) and container parameters
(Dominguez-Lerena et al. 2006; Villar-Salvador
et al. 2012). Among the listed studies, irrigation
effects are only considered in concert with fertilization
(Cuesta et al. 2010; Villar-Salvador et al. 1999, 2012),
or in terms of drought hardening (Royo et al. 2001;
Vilagrosa et al. 2003). Therefore, the influence of
moisture availability dynamics during nursery propa-
gation, independent of fertilization, has not yet been
addressed.
Species selection
Lebanon’s complex physiography and widespread
ecological degradation present inherent reforestation
challenges, requiring the production of field-hardy
planting stock for a diversity of native species with
highly divergent ontogenies. According to the 2005
FRA, the nation’s forests can be classified into
broadleaf (78,887 ha), coniferous (44,879 ha), and
mixed (15,610 ha) types (Mitri and El Hajj 2007).
A range of native species across key genera (i.e.,
Acer, Cedrus, Fraxinus, Ostrya, Pinus, Prunus,
Quercus, Juniperus, etc.) are being grown for refor-
estation of these cover types, with higher emphasis
placed on the production of species that contribute to
regional revenue through timber sales or natural goods
and services. For example, stone pine (Pinus pinea L.)
is an ecologically and economically valued native
species that serves as an important component of
forests throughout the region. In addition to its
distribution in natural systems, it is also often culti-
vated in dense monospecific cohorts for basic
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Plant Ecol (2017) 218:39–53
resources, such as nuts, wood products, firewood,
and resins (Abi-Saheh et al. 1996; Baltaxe 1965;
Talhouk et al. 2001). Within Lebanon, P. pinea is
estimated to cover over 28,000 ha (Fig. 1), with
significant portions of its original endemic range
considered to be highly disturbed or fragmented (Abi-
Saheh et al. 1996; Baltaxe 1965; Talhouk et al. 2001;
UNDP 2008). Due to its mixed-use potential and
hardiness across a variety of edaphic and climatic
conditions, there is a significant interest in seedling
production of P. pinea for use in reforestation efforts
(Dominguez-Lerena et al. 2006; Sáez-Laguna et al.
2014).
Currently, significant knowledge gaps exist
between nursery propagation practices and the corre-
sponding ecophysiological performance potential of
plants used in re-vegetation efforts. Using P. pinea as a
model species, we explore fertilization and irrigation,
as integral parts of nursery practices, with the aim of
(1) optimizing production of seedlings capable of
post-outplanting survival on typical restoration sites in
Lebanon and (2) providing sound and regionally
relevant information to reforestation sector
stakeholders.
Materials and methods
Study A: fertilization with CRF
An in situ experiment was established at a nursery in
the American University of Beirut Nature Conserva-
tion Center (Hawch Sned, Lebanon) (Fig. 1) to
examine the suitability of CRF for seedling produc-
tion; seedling morphological parameters and tissue
nutrient concentrations were evaluated to provide
baseline metrics for the development of quality
reforestation stock.
Pinus pinea seeds were wild-collected in Ramlieh,
Lebanon (33°440 39.600 N 35°390 38.900 E) in autumn
2011 and stored in refrigerated conditions until use.
Prior to sowing, seeds were hydroprimed for 24 h in
aerated DI water, sown into 656 mL containers (D40
DeepotTM, Stuewe and Sons, Corvallis, OR, USA) on
14 May 2012, and mist irrigated through an overhead
sprinklers twice daily until germination. The growing
medium consisted of a 1:1 peat (TPS, JiffyÒ Products
International, Moerdijk, The Netherlands) and coco-
peat mixture (Al Mawasim Co., Beirut, Lebanon). The
Plant Ecol (2017) 218:39–53
Fig. 1 Distribution of Pinus pinea forests throughout Lebanon. Dark shading represents dense cover, while light shading represents
sparse cover
experiment followed a completely randomized design
consisting of five fertilizer rates at 100 seedlings per
treatment level. Containers were randomly amended
with one of five rates (0, 3, 6, 9, and 12 g L-1 of
growing medium or 0, 315, 630, 945, and 1260 mg of
N plant-1) of 16–8–12 (N–P–K) CRF pellets
43
(BasacoteÒ Plus 6 M, COMPO GmbH & Co. KG,
Hamburg, Germany). The CRF pellets had a stated
nutrient release period of 6 months at 21 °C.
Irrigation frequency was determined by gravimetric
water content (GWC) (McDonald 1984; see ‘man-
agers technique’ in Dumroese et al. 2015); with water
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applied using a fixed overhead irrigation system.
Irrigation was tailored to seedling growth phases
(Landis 1999); specifically, seedlings were irrigated
when the container GWC declined to 80 % of the
container weight at field capacity during the estab-
lishment and the rapid growth phases (12 weeks) and
to 60 % GWC during the hardening phase (10 weeks).
Electrical conductivity (EC) and pH of growing
media were measured using Extech EC510 ExStik II
Meter (Extech Instruments, Nashua, NH, USA). Mea-
surements were collected from five randomly selected
seedlings per treatment using the pour-through nutrient
extraction procedure (Wright 1986). A randomly
selected subset of 20 seedlings per treatment was used
to evaluate seedling growth through non-destructive
morphological measurements of height and root-collar
diameter (RCD) collected (1) mid-season (August,
2012) and (2) at the end of the growing season
(November, 2012). Due to unforeseen time and
resource constraints, destructive sampling at the end
of the growing season was conducted on a randomly
selected subset of five seedlings per treatment; seedling
roots were separated from growing media, severed
from the shoot at the root collar, and dried at 70 °C for
72 h. Subsequent assessments of root and shoot dry
mass and nutrient analysis were made according to the
standard methodology by A&L Great Lakes Labora-
tories (Fort Wayne, IN, USA).
Study B: irrigation regime
The second ex situ study was established at the Center
for Forest Nursery and Seedling Research (CFNSR;
Moscow, ID, USA). This location was chosen because
of the capacity to more precisely evaluate the impact
of irrigation on the morphology and hardiness of
container grown P. pinea seedlings following
23 weeks of nursery propagation. The study investi-
gated the influence of irrigation frequency during the
three plant growth phases to better understand the
optimal irrigation regime needed to achieve seedling
functional attributes associated with drought and cold
hardiness.
Pinus pinea seeds were collected from wild stands
(33°440 42.300 N 35°390 57.100 E) in autumn 2011 and
stored (seed treatment and handling described in
Experiment A). Seeds were sown (2 seeds cell-1) into
40 in3 (656 cm3) D40 Deepot containers on May 30,
2012. Containers were filled with sphagnum media
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Plant Ecol (2017) 218:39–53
(SunshineÒ Mix #4) and covered with a layer of perlite
and germination fabric (DeWitt 1oz Deluxe Plus Frost
& Germination Blanket, Agriculture Solutions LLC,
Strong, ME, USA) and irrigated three times daily until
germination.
The study followed a randomized complete block
design, with each of the ten moisture treatments
containing five, 20-seedling blocks. The propagation
regime was broken up into establishment, rapid
growth, and hardening phases (Online Resource 1),
which were determined by plant morphological status
and delineated the experimental changes in moisture
availability throughout the season. Irrigation treat-
ments were based on GWC of entire container trays
(Online Resource 2) and created to represent a
spectrum of water use (see ‘managers technique’ in
Dumroese et al. 2015). Water-soluble fertilizers were
applied through an overhead boom system, and
nutrient rates tailored to each of the irrigation treat-
ments to receive the same cumulative amount of all
macro- and micro-nutrients over the duration of the
growing season. At the time of irrigation, seedlings
were moved to a separate location on the nursery
bench to ensure even application within treatments
and blocks (seedling trays) were re-randomized after
each irrigation event.
Ten seedlings from each irrigation treatment were
randomly selected for morphological assessment of
height, RCD, root and shoot volume (RV and SV) and
dry mass (RDM and SDM), as well as root:shoot
volume (Rv:Sv) and mass (Rm:Sm) during five sam-
pling points throughout the growing season [at
approximately monthly intervals starting in mid-July
(Online Resource 1)]. Seedling RV and SV were
measured using a water displacement method (Burdett
1979). For the evaluation of dry mass, seedling root
and shoot tissue were separated at the root collar and
dried at 70 °C for 72 h and weighed.
The freeze-induced electrolyte leakage (FIEL) test
of foliar tissue was used to evaluate seedling cold
hardiness (Burr et al. 1990). Five seedlings from each
of the ten irrigation treatments were randomly selected
at the end of the nursery propagation in November for
the FIEL analysis (Islam et al. 2009).
Statistical analysis
For both studies, morphological response variables,
EC, and pH were analyzed (9.3, SAS Institute Inc.,
Plant Ecol (2017) 218:39–53 45

Cary, NC) using a one-way ANOVA (PROC

ANOVA); pairwise comparisons were made using
Tukey’s HSD (a = 0.05). When values failed to meet
the assumptions for a general linear model (normality
and homogeneity of variances), a non-parametric
approach was used, where data were ranked (PROC
RANK) and the ranked values were then analyzed
using a one-way ANOVA (PROC ANOVA). All post
hoc comparisons of ranked data were performed using
Tukey’s HSD test (a = 0.05).
For FIEL, adjusted injury percentage values (Lim
et al. 1998), a metric that accounts for both the ion
leakage from the unfrozen control (-2 °C) samples
and the fully frost-injured (-40 °C) samples were
calculated from the FIEL data using a Gompertz
function. The calculations are described in Eqs. (1.1),
(1.2), and (2) (Online Resource 3).

Results
Study A: fertilization with CRF
Mid-season measurements (August, 2012) revealed no
significant treatment differences for height and RCD,
but differed significantly by the end of the growing
season (November, 2012; Fig. 2). Compared to the
control (0 g L-1), all rates of fertilizer amendment
produced taller seedlings, with height increases rang-
ing between 66 and 90 %. The uppermost rate of
fertilizer application (12 g L-1) resulted in seedlings
with larger RCD compared to B6 g L-1, while
seedlings in the 9 g L-1 treatment exhibited an
intermediate RCD response.
Mid-season measurements of media leachate EC
and pH were significantly influenced (p \ 0.0001 and
p = 0.0045, respectively) by fertilizer application rate
(Fig. 3). EC values ranged between 0.3 and
1.0 mS cm-1 among all treatments and increased
with fertilizer rate, while the pH values decreased,
ranging between 6.9 and 4.8.
Fertilizer rate influenced seedling root and shoot
mass as well as R:S (Table 1). Root mass was highest
among seedlings produced with 12 g L-1 rate, which
was the only treatment different from the control.
Seedlings receiving B3 g L-1 produced less shoot
mass than those in the 6–12 g L-1 treatments. Unfer-
tilized seedlings exhibited higher R:S compared to all
other treatments. The 9 and 12 g L-1 fertilizer
Fig. 2 Mean (l ± se) seedling height and root-collar diameter
(RCD) collected mid-season (black bars) in August, 2012 and at
the end of the growing season (gray bars) in November, 2012
from Pinus pinea seedlings subject to five different controlled
release fertilizer rates over a 6-month growing period. Different
letters indicate significant differences (a = 0.05, n = 20)
application rates resulted in the lowest R:S, while
the 6 g L-1 group produced an intermediate response
between the 3 and 9–12 g L-1 treatments. Fertiliza-
tion rate significantly influenced foliar (N, S, P, Mg,
Zn, Mn, Fe, Cu, and Al) and root tissue (N, S, P, K, and
Mn) concentrations (Table 2).
Study B: irrigation regime
Analysis of variance and pairwise comparisons of
treatments revealed treatment differences that were
mostly representative of water availability during the
establishment and rapid growth phases of the study
(Table 3). Treatment differences became more pro-
nounced in the rapid growth phase, with greater water
availability attributed to larger seedling morphologi-
cal features (Online Resource 4) during the first four
sampling periods, which captured establishment and
rapid growth phases.
At the end of the growing season, all measured
morphological parameters differed across irrigation
treatments (Table 3). Seedlings receiving the least

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to the highest irrigation treatment. Neither of the R:S

parameters showed a meaningful difference between
the highest and lowest moisture treatments. Above-
ground growth was higher for seedlings receiving
85 % of GWC through the hardening phase.
Seedlings across all irrigation treatments exhibited
a high degree of cold tolerance, and although the FIEL
test revealed statistically significant treatment differ-
ences (p = 0.0005; Online Resource 5), the specific
LT50 value estimates were between -26.7 and
-29.9 °C—a range well beyond temperatures
encountered in situ and thus not biologically
significant.

Discussion

One of the most important aspects of seedling produc-

Fig. 3 Electrical conductivity (EC) and pH measurements
(l ± se) collected using the pour-through nutrient extraction
procedure from Pinus pinea seedlings subject to five different
controlled release fertilizer rates; black circles indicated EC and
black triangles indicate pH measurements collected in August,
2012. Different letters indicate significant differences
(a = 0.05, n = 5)
moisture (55 % GWC) throughout the growing season
were markedly smaller, exhibiting lower height, RV,
SV, RCD, RDM, and SDM values than those subject
tion is irrigation management. Proper irrigation
depends on the quality and uniformity of water
application and is mediated by container shape and
the permeability of the growth medium used (Landis
1999). Failure to optimize irrigation can result in crop
irregularity and lower quality reforestation stock.
These factors are generally controlled in nations
with developed nursery systems, but often overlooked
in emerging restoration programs. Furthermore,
despite the similarity in production needs with a
number of European species, the materials available
for seedling production differ dramatically between
Europe and the Middle East, highlighting a need to
explore regionally suitable production options. Thus,
through the assessment of seedling growth with the use

Table 1 Mean above- and belowground biomass, R:S, and total plant biomass values for Pinus pinea seedlings subject to five
different rates of controlled release fertilizer assessed at the end of a 6-month growing period
Fertilizer rate Root mass (g), p = 0.0270 Shoot mass (g), p \ 0.0001 R:Sa, p \ 0.0001 Total biomass (g), p \ 0.0001
(g L-1)

0 2.3bc 1.9b 1.2a 4.1b

3 2.0c 3.5b 0.6b 5.5b
6 3.5b 6.9a 0.5bc 10.4a
9 3.5b 8.2a 0.4c 11.7a
12 3.9a 8.0a 0.5c 12.0a
Means followed by a different letter within a column differed significantly (a = 0.05, n = 5)
a
Comparisons were performed on ranked data

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Table 2 Mean tissue Fertilizer rate (g L-1)

nutrient concentration of
Pinus pinea seedlings 0 3 6 9 12
grown with five different
rates of controlled release Shoot nutrient concentration
fertilizer assessed at the end N (%) p < 0.0001 1.4c 1.8b 2.2ba 2.3a 2.3a
of a 6-month growing S (%) p < 0.0001 0.1b 0.1b 0.2a 0.2a 0.2a
period
P (%) p < 0.0001 0.1c 0.2b 0.3a 0.3a 0.3a
K (%) p = 0.7581 1.1 1.2 1.1 1.1 1.2
Mg (%) p < 0.0001 0.3a 0.3a 0.2b 0.2b 0.2b
B (ppm) p = 0.0785 13.2 18.1 17.9 19.3 18
Zn (ppm) p < 0.0001 18.9b 20.5b 35.5a 36.5a 35.5a
Mn (ppm) p < 0.0001 34.0c 81.6b 138.5a 121.2ab 145.7a
Fe (ppm) p < 0.0001 25.2b 22.4b 62.8a 39.2b 32.3b
Cu (ppm) p < 0.0001 1.2b 2.3b 5.1a 4.7a 6.1a
Al (ppm) p = 0.0124 3.4b 27.2a 13.2ab 5.8b 22.4ab
Root nutrient concentration
N (%) p = 0.0003 0.1b 0.2ab 0.2a 0.2a 0.2a
S (%) p < 0.0001 0.7b 1.1b 1.3ab 1.4a 1.4a
P (%) p < 0.0001 0.3ab 0.4a 0.2c 0.2bc 0.2c
K (%) p < 0.0001 1.3a 1.2a 0.8b 0.8b 0.8b
Mg (%) p = 0.1287 0.1 0.1 0.1 0.1 0.1
B (ppm) p = 0.1626 21.4 30.2 38.6 39.8 33.2
Mean values followed by a Zn (ppm) p = 0.4829 225.8 219.8 426.8 224 259
different letter within a row Mn (ppm) p < 0.0001 5.0c 9.4bc 16.2a 14.4ba 19.2a
differed significantly Fe (ppm) p = 0.6948 194 158.8 261.8 115.6 142.6
(a = 0.05, n = 5)

Table 3 Final assessment of the morphological responses of were obtained from ranked values. (Note: RV = root volume;
Pinus pinea seedlings subject to ten different irrigation SV = shoot volume; HT = height; RCD = root collar diameter;
treatments made at the end of a 23-week nursery propaga- Rv:Sv = root:shoot volume; RDM = root dry mass; SDM =
tion period. Reported p-values accompanied by an asterisk shoot dry mass; and Rm:Sm = root:shoot mass)
Treatment RV (cm3) SV (cm3) HT (cm) RCD (mm) Rv:Sv RDM (g) SDM (g) Rm:Sm
Sampling 5 p \ 0.0001 p \ 0.0001 p \ 0.0001 p \ 0.0001 p = 0.0024 p \ 0.0001 p \ 0.0001 p \ 0.0001

85–85–85 12.58ab 27.12a 31.45a 5.66a 0.47b 2.39a 6.85a 0.35c

85–85–70 13.39ab 26.82ab 30.95a 5.30ab 0.50b 2.34a 6.68a 0.35c
85–85–55 11.84abc 26.16ab 32.40a 5.54a 0.46b 2.24abc 6.96a 0.32c
85–70–70 12.81ab 22.76abc 27.17b 4.86bcd 0.56ab 2.18abcd 4.92cb 0.44a
85–70–55 11.12bc 21.29dc 27.80b 4.95bcd 0.54ab 2.02abcd 5.29b 0.38abc
85–55–55 9.60c 20.16dc 26.05bc 4.64cd 0.49b 1.82de 4.82cb 0.38abc
70–70–70 13.93a 22.11bcd 26.20bc 5.05bc 0.66a 2.28ab 5.40b 0.43ab
70–70–55 11.55abc 22.30abc 25.91bc 4.78cd 0.52ab 1.93bcd 5.25b 0.37bc
70–55–55 11.01bc 20.08dc 24.20 cd 4.76cd 0.56ab 1.85cde 5.01cb 0.37bc
55-55-55 9.47c 17.31d 22.05d 4.57d 0.56ab 1.52e 4.16c 0.37bc
Mean values followed by a different letter within a column differed significantly (a = 0.05, n = 10)

of locally available materials and irrigation practices and locally integrated approach aimed at improv-
appropriate for a range of conditions and capacities ing seedling quality and subsequent outplanting
within Lebanon, we have provided a research-based success.

123
48
Study A: fertilization with CRF
Within the 6-month duration of this study, the mean
rhizosphere temperature was 27 °C. Although not
explicitly measured, high temperatures likely
decreased the projected fertilizer release period from
6 to 4–5 months, due to the temperature-mediated
release rates characteristic of CRF (Jacobs and Tim-
mer 2005). Although we observed an increasing trend
in EC and a decreasing trend in pH values with
incremental increases in fertilizer rate across treat-
ments, the EC values were found to be significantly
below values that are considered toxic to plant growth
(Jacobs and Timmer 2005) and coincided with those
observed in the production of P. halapensis, a conifer
native to the Mediterranean Basin (Oliet et al. 2004).
In addition, despite relatively large differences in pH
between the control and the highest fertilizer rate (6.9
and 4.8, respectively) mid-season height and RCD
measurements revealed no differences, suggesting
distinctions in rhizosphere conditions among the
treatments were not large enough to influence seedling
morphological development.
Although seedling height was relatively unaffected
by fertilizer rate, with differences observed only
between the control (0 g L-1) and all other application
rates, fertilization caused seedlings to invest more
resources into aboveground growth as evidenced by
the significant increase in shoot biomass (more than
fourfold) and a smaller change (less than twofold) in
the root biomass. The observed resource allocation to
shoot over root growth at low-to-moderate nutrient
availability in P. pinea may emphasize a potential
strategy for light competition in the absence of water
limitations. Significant gains in shoot mass were
observed with the application of at least 6 g L-1;
thus, increasing the fertilizer rate to C6 g L-1 is the
most cost-effective option for enhancing seedling
shoot mass, a metric which may be important towards
achieving better outplanting performance among
Mediterranean species (Dominguez-Lerena et al.
2006; Villar-Salvador et al. 2010).
Seedling R:S can be used to predict performance
and tolerance to planting stress; with higher values
believed to increase a seedling’s ability to meet the
transpiration demand immediately following outplant-
ing (Grossnickle 2000, 2012). However, this relation-
ship may not be as strong for Mediterranean woody
plants (Navarro et al. 2006); thus, the use of several
123
Plant Ecol (2017) 218:39–53
species-specific morphological standards may be
pertinent. Morphological parameter targets proposed
by the European Union (Navarro et al. 2006; Villar-
Salvador et al. 2010) for P. pinea recommend seedling
height (20–30 cm), RCD (3.5–4.5 mm), and R:S
(0.4–0.5). Based on these recommended targets,
sufficient seedling height and R:S was achieved by
all fertilizer application rates, although additional
in situ testing over multiple growing seasons is needed
to bolster these findings. Seedling RCD values
exceeded recommendations among all fertilizer rates
(Fig. 2; Table 1), which may be of benefit to seedling
performance following outplanting (South et al. 2005).
Typically, increased fertilizer rates contribute to
nutrient assimilation, with increases in foliar N
concentration linked to improved plant growth and
higher post-outplanting survival (Grossnickle 2012;
Palá et al. 1997; Planelles 2004; Villar-Salvador et al.
2004), while excessive N fertilization can lead to
diminished physiological function, reduced cold and
drought hardiness, and decreased outplanting perfor-
mance (Dumroese 2003; Puértolas et al. 2005; Rikala
and Repo 1997; Villar-Salvador et al. 2005). In
general, mean whole plant N concentrations observed
in our study were comparable to other container grown
Mediterranean conifers (Cuesta et al. 2010; Domin-
guez-Lerena et al. 2006; Oliet et al. 2004; Villar-
Salvador et al. 2005), although we detected a higher N
concentrations in the aboveground relative to below-
ground tissue. A possible explanation for this finding
is that seedlings may have been actively growing
during the November measurement period, and thus
did not translocate nutrients to belowground tissue.
Overall, the optimum foliar concentration range
reported for conifers is 1.5–2.5 % (Landis 1989; Oliet
et al. 2009; Puértolas et al. 2012), which corresponded
to the concentrations observed in our study under all
fertilization scenarios except the control.
Study B: irrigation regime
In the Mediterranean, larger seedlings exhibit better
performance following establishment (Cuesta et al.
2010; del Campo et al. 2007; Guehl et al. 1993; Luis
et al. 2007; Oliet et al. 2009; Tsakaldimi et al. 2005;
Villar-Salvador et al. 2004), with a strong positive
correlation between height (20–30 cm) and survival
(Dominguez-Lerena et al. 2006; Villar-Salvador et al.
2010); however, it is unclear if this relationship is
Plant Ecol (2017) 218:39–53
maintained once seedlings exceed the 30 cm height
limit, achieved by the three highest total water
availability regimes in our study. We also found that
target RCD values surpassed recommendations among
all treatments including seedlings subject to the lowest
level of irrigation. In addition, mass-based R:S ratios
were lower than recommended, with Rm:Sm values
ranging between 0.32 and 0.43 and the highest ratio
achieved by the well-to-moderately watered (85–70–
70 % GWC) treatment. There are no listed recom-
mendations for volume-based R:S ratios; however,
Rv:Sv values ranged from 0.46 to 0.66, with all
treatments regardless of irrigation achieving a ratio
above 0.5. Although higher root biomass has been
generally considered beneficial to enhancing estab-
lishment success in arid environments (Grossnickle
2005; Padilla and Pugnaire 2007), with regard to
Mediterranean species higher susceptibility to drought
stress among larger seedlings with lower R:S relative
to smaller seedlings may disappear shortly following
outplanting (Navarro et al. 2006; Villar-Salvador et al.
2005).
The reduction and the eventual cessation of height
growth but continued accumulation of stem diameter
and belowground biomass combined with seedling’s
ability to tolerate subzero air and soil temperatures are
major goals of drought and cold hardening (Landis
2013). During the hardening phase of this study, all
seedlings demonstrated substantial reductions in
height growth; with seedlings subject to moderate
(70–70–70 % GWC), moderate-to-low (70–70–55 %
GWC), and low (55–55–55 % GWC) irrigation
schemes exhibiting no appreciable height difference.
Measurements of RCD growth decreased, but did not
cease completely among all treatments from October
to November. This corresponds to findings of Villar-
Salvador et al. (2013) who observed no differences in
P. pinea seedlings stress tolerance between moderate
and severe drought hardening, although the two levels
of stress were generally lower and less distinct (34–45
and 45–50 %, respectively) than those tested in our
study.
The results of the FIEL revealed that seedlings in all
treatments exhibited LT50 temperatures below
-27 °C. Pardos et al. (2014) reported rapid hardening
on P. pinea seedlings and saplings during November,
ranging between -18 and -20 °C depending on tree
age. These temperatures fall well below the range of
frost damage that seedlings may incur following
49
outplanting (Mitri and El Hajj 2007), and thus indicate
a lower likelihood of frost damage post-outplanting.
Despite statistical significance, we observed no bio-
logically relevant interaction between LT50 tempera-
tures and irrigation. Conversely, Villar-Salvador et al.
(2013) found that well-watered seedlings of P. pinea
(irrigation to field capacity every 1–3 days) exhibited
a higher electrolyte leakage after exposure to -8 °C
compared to seedlings subject to moderate drought
hardening for 3 months (irrigated when containers
reached 43–45 % of their original weight at satura-
tion). This difference between our findings may be
explained by the fact that the well-watered treatments
in our study received less water than those described
by Villar-Salvador et al. (2013). Furthermore, prove-
nance and diurnal light and temperature regimes can
affect seedling hardiness (Mollá et al. 2006; Pardos
et al. 2003; Puértolas et al. 2005; Villar-Salvador et al.
2013) and may have exerted a stronger influence on
the cold tolerance of P. pinea seedlings than irrigation
regime.
Conclusions
In regions, such as Lebanon, where extensive or
scientifically rigorous monitoring of plant perfor-
mance following transplant into field conditions can
be difficult to accomplish, a feedback loop between
growers and land managers can serve as a proxy and
help enhance propagation practices to better suit
restoration objectives (Dumroese et al. 2016). The
cooperative of Native Tree Producers of Lebanon has
adopted standards that build upon the approach
demonstrated in this paper (USAID 2015). Strong
consideration was given to both nursery and local field
conditions that could be tested ex situ and applied to
subsequent seedling production guidelines. While no
refereed data are yet available with regard to seedling
post-outplanting survival, field evaluations provide
insight into the positive survival trends (with 70–90 %
post-outplanting survival) partially attributed to
research-driven plant production guidelines at the
nursery level (Dumroese et al. 2016; Nehme 2014).
The practical outcomes of our results suggest that:
1. High-quality seedlings can be grown using readily
available CRF and an appropriate application rate
can be determined by considering the temperature
123
50
range at the nursery location, enabling seedlings
to receive balanced nutrition while avoiding
toxicity.
2. Irrigation scheduling using the gravimetric method
allows for effective delivery of water when needed
to achieve, or arrest, seedling growth. This can be
achieved using simple, locally adaptable, and
adjustable approaches that overcome some of the
historic challenges found in the region.
3. Engaged seedling growers facilitate the use of the
target plant concept, which requires the incorpo-
ration of monitoring activities into seedling prop-
agation protocol refinement.
Lebanon shares physiographic and climatic features as
well as the similarities in the challenges facing its
ecosystems (e.g., climate limitations, extensive native
vegetation clearing, excessive grazing, agricultural
expansion, soil degradation, etc.) with a number of
regional countries, including Jordan, Syria, Turkey, and
Armenia (Haktanir et al. 2004). In addition, Lebanon is
among a number of countries spanning North Africa, the
Mediterranean, and the Middle East that are experiencing
increased frequency of drought and desertification which
is exacerbated by a shifting climate (Abahussain et al.
2002). It is, therefore, likely that opportunities will arise
for expanding similar efforts or transferring regionally
adapted methods and technologies across borders and
into areas where initiatives for reforestation and conser-
vation may be of much lower funding priority.
Acknowledgments We thank the American University of
Beirut Nature Conservation Center and the Lebanon
Reforestation Initiative for facilitating this work and allowing
for the use of research space. We are grateful to Majd Kashan,
Khaled Slim, Sakra Abou Eid, Sarita Bassil, Raymond Farhat,
Jeremiah Pinto, Kea Woodruff, Don Regan, Brendan Baughn,
and Edward Flathers for assistance.
Compliance with ethical standards
Funding Financial support was provided by the USDA Forest
Service and the University of Idaho, Center for Forest Nursery
and Seedling Research.
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