Nutrient loading of forest tree seedlings to promote stress resistance and field performance: A Mediterranean perspective

Abstract

The planting environment of Mediterranean areas is highly challenging as summer drought and winter frost jeopardize survival, and soil infertility limits establishment success. We review the potential for seedling nutrient loading to alleviate these post-planting stresses. A growing body of evidence indicates that nitrogen (N) rich seedlings have improved field performance in Mediterranean environments, due to their ability to grow new roots rapidly and out-compete weeds. In addition, frost resistance during hardening is crucial for relatively cold inland nurseries; recent research shows a positive relationship between N and shoot frost resistance though a knowledge gap exists regarding the influence of nutrition on root frost resistance. Some new evidence also implicates phosphorus as an important driver of seedling response in the Mediterranean due to its influence on root growth and physiology. Nutrient status influences other functional attributes critical to survival in Mediterranean areas, such as drought tolerance, root hydraulic conductivity, and mycorrhization. In light of the apparent benefits of high nutrient reserves for seedling performance in Mediterranean areas, we also review techniques for nursery nutrient loading. Exponential fertilization can be applied when species’ growth patterns match this application regime. However, many Mediterranean species exhibit episodic growth indicating that constant or fall fertilization could be more effective in reaching loading. In particular, late-season fertilization has shown good potential to avert nutrient dilution in the fall and increase frost resistance. Several needs for future research are identified, with special emphasis on the necessity to match fertilization regimes to species ecological traits and planting conditions.

Full text

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Nutrient loading of forest tree seedlings to promote stress resistance and field 3
performance: A Mediterranean perspective 4
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Juan A. Oliet1*, Jaime Puértolas2, Rosa Planelles1 and Douglass F. Jacobs3 6
1 Universidad Politécnica de Madrid, Department of Silvopascicultura, Ciudad Universitaria s/n, 28040 7
Madrid, Spain 8
2 Lancaster Environment Centre, Lancaster University, Lancaster, LA1 4YQ, UK. 9
3 Department of Forestry and Natural Resources, Hardwood Tree Improvement and Regeneration Center, 10
Purdue University, West Lafayette, IN 47907-2061, USA 11
12
*Corresponding author (e-mail: juan.oliet@upm.es; phone: 34-91-3367130) 13
14
Submitted to Nutrient dynamics of planted forests Special Issue of New Forests on 28 February 2013 15
Text pages 35, Figures 7 16
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18
NOTE: this s the author’s version of a work that was accepted for publication in New Forests. The final
publication is available at Springer via http://link.springer.com/article/10.1007/s11056-013-9382-8

Abstract 19
The planting environment of Mediterranean areas is highly challenging as summer drought and 20
winter frost jeopardize survival, and soil infertility limits establishment success. We review the 21
potential for seedling nutrient loading to alleviate these post-planting stresses. A growing body of 22
evidence indicates that nitrogen (N) rich seedlings have improved field performance in 23
Mediterranean environments, due to their ability to grow new roots rapidly and out-compete weeds. 24
In addition, frost resistance during hardening is crucial for relatively cold inland nurseries; recent 25
research shows a positive relationship between N and shoot frost resistance though a knowledge gap 26
exists regarding the influence of nutrition on root frost resistance. Some new evidence also 27
implicates phosphorus as an important driver of seedling response in the Mediterranean due to its 28
influence on root growth and physiology. Nutrient status influences other functional attributes 29
critical to survival in Mediterranean areas, such as drought tolerance, root hydraulic conductivity, 30
and mycorrhization. In light of the apparent benefits of high nutrient reserves for seedling 31
performance in Mediterranean areas, we also review techniques for nursery nutrient loading. 32
Exponential fertilization can be applied when species’ growth patterns match this application 33
regime. However, many Mediterranean species exhibit episodic growth indicating that constant or 34
fall fertilization could be more effective in reaching loading. In particular, late-season fertilization 35
has shown good potential to avert nutrient dilution in the fall and increase frost resistance. Several 36
needs for future research are identified, with special emphasis on the necessity to match fertilization 37
regimes to species ecological traits and planting conditions. 38
39
Keywords: plant nutrition; drought avoidance; frost resistance; fertilization; Mediterranean 40
environment 41
42

Introduction 43
A shift in paradigm in forest regeneration science has occurred over the past two decades from 44
emphasis on reforestation following timber harvest to restoration of harsh, degraded sites in order to 45
improve ecosystem function (Oliet and Jacobs 2012). The complex and challenging landscape of 46
restoration ecology requires adaptation and refinement of traditional silvicultural tools to address 47
specific limiting factors to forest establishment in these dynamic and variable systems. In semi-arid 48
Mediterranean environments, where restoration of degraded lands is a major focus of natural 49
resource management activities, new technologies have been identified to improve planting success 50
(Vallejo et al. 2012). Short-term survival is paramount in these systems and various seedling 51
quality attributes collectively help to increase chances of survival (Palacios et al. 2009; Grossnickle 52
2012). 53
Plant mineral nutrition is among the most important seedling quality attributes as it influences plant 54
growth, nutrient storage reserves and resistance to biotic and abiotic stresses (Landis 1985). A 55
growing body of research evidence specific to Mediterranean areas has emphasized the importance 56
of woody plant mineral nutrition to survival and growth in these environments (Oliet et al. 2006). 57
From this collective knowledge, Villar-Salvador et al. (2012) recently presented a conceptual 58
ecophysiological model to explain the relationships between seedling traits and planting response 59
specific to Mediterranean conditions, which emphasizes the importance of both nitrogen (N) 60
concentration and whole plant content in resistance to summer drought due to its role in promoting 61
root growth during the wet season after planting. In this conceptual model, the size of the plant is 62
considered a crucial trait as it integrates two important components of seedling performance after 63
planting: nutrient content (reserves) and competitive ability. Cortina et al. (2013) also focus on the 64
role of mineral nutrition in seedling quality for restoring Mediterranean dry areas, highlighting the 65
necessity of accounting for environmental specificities of drylands to match seedling quality and 66
fertilization to optimize survival. 67

Nutrient loading is a nursery fertilization technique whereby high fertilizer application rates are 68
used to induce luxury uptake in excess of plant growth demand; nutrients are stored for later 69
utilization after planting or allocated to metabolism for stress resistance (Timmer 1997; Salifu and 70
Jacobs 2006). Higher nutrient reserves acquired during nutrient loading have correlated well with 71
improved field performance of seedlings planted under a range of temperate and boreal 72
environments (Malik and Timmer 1998; Salifu et al. 2009). As evidenced below, the potential 73
application of nutrient loading to Mediterranean forest restoration programs has also garnered broad 74
recent interest 75
In this review, we i) present an overview of current knowledge on the importance of nutrient status 76
in high quality seedling production, ii) discuss the relationship between nutrients and functional 77
attributes (i.e., drought and frost resistance) and plant establishment, and iii) analyze the principles 78
and techniques behind the application of nutrient loading of species, with particular emphasis on 79
Mediterranean environments. The recent burst of scientific progress in this area specific to 80
Mediterranean semi-arid regions combined with the need to widen the pool of species used in forest 81
restoration programs in Mediterranean environments (Padilla et al. 2009) provides timely 82
justification for a constructive review of this subject. In this paper, nutrient loading will be 83
considered as a conceptual framework (building of nutrient reserves). But we also present examples 84
of the general benefits of promoting high concentrations of nutrients in tissues (irrespective of 85
reaching optimal [i.e., maximum] nutrient levels via loading), as highly fertilized plants are 86
characterized by many of the same attributes as sensu stricto nutrient loaded seedlings. 87
88

Producing and planting seedlings in the Mediterranean: Implications of nutrient status. 89
Ecological conditions of Mediterranean regions and planting limitations 90
Survival and growth of forest plantations are conditioned by plant-site interactions. Production of 91
high quality seedlings that are likely to thrive on a given outplanting site requires matching of plant 92
attributes to site conditions. The concept of plant quality is strongly related to “fitness for purpose” 93
(Duryea 1985). In the Mediterranean, planting success is limited by some specific ecological 94
circumstances that are clearly distinguished from tropical, temperate or boreal forests. Although a 95
geographical, climatic or floristic definition of the Mediterranean region is ambiguous, all lands 96
from this area are strongly marked by an annual dry season that occurs simultaneously with high 97
temperatures in summer, causing physiological drought for 1-5 months per year (Quezel 1985). In 98
addition, regions such as the Mediterranean Basin are particularly likely to face uncertainties due to 99
climate change, with most models predicting lower precipitation during each season along with 100
increasing temperature (Christensen et al. 2007). This will likely intensify the length and frequency 101
of droughts (Sheffield and Wood 2008). Under these conditions, planting success after summer is 102
highly dependent on ability of seedling roots to reach limited soil moisture that is confined to deep 103
soil layers (Padilla and Pugnaire 2007). The capacity to develop a large, deep root system before the 104
onset of the dry season is not solely a species-specific condition. Phenotypic modifications through 105
nursery culture (e.g., shoot:root ratio, shoot and root biomass; leaf area; nutrient and carbohydrates 106
status) also play an important role, as can optimal timing of planting during the wet season to allow 107
seedlings sufficient time to grow a deep root system before summer (Villar-Salvador et al. 2012). 108
The recommended planting window for Mediterranean zones ranges from early fall to mid-winter 109
(Mollá et al. 2006; Palacios et al. 2009; Andivia et al. 2011). Planting in spring is not 110
recommended, as the inter-annual distribution of spring rainfall is very uneven and summer drought 111
often arrives early in mid-spring. When planting during the wet season, root egress and the 112
overcoming of planting impact is favored by humid soil conditions (Grossnickle 2005). However, 113
frost may also occur during this time, damaging and killing seedlings before the growing period 114

arrives. The range of temporal and spatial variation in temperature can be very wide. Average 115
annual temperature of the Mediterranean climate ranges from 5ºC (inland continental, high 116
elevation regions) to 18ºC (mostly coastal locations of low altitude) (Quezel 1985). In inland 117
locations, early frost can occur at the beginning of the fall (Mollá et al. 2006), and minimum 118
temperature can be as low as -20 ºC in mid-high elevations in January (“Agencia Estatal de 119
Meteorología” and “Instituto de Meteorología de Portugal” 2011). Thus, planting frost resistant 120
(i.e., hardened) seedlings in these locations is crucial to ensure success. However, many 121
Mediterranean forest nurseries are located in sites characterized by mild fall and winter to promote 122
growth to reach adequate plant size in less than one year (Close 2012). Low cold hardiness of 123
seedlings produced in these nurseries is one of the reasons for the poor performance of many 124
plantations in Mediterranean continental areas, which has received little attention (Pardos et al. 125
2003). 126
Mediterranean soils are very diverse in chemical, physical and biological conditions. Large 127
extensions, particularly those from degraded areas, are characterized by shallowness and a lack of 128
fertility (Martínez-Mena et al. 2002; Pigott and Pigott 1993; Sardans et al. 2005; Valdecantos et al. 129
2006). Low soil nutrient availability limits post-transplant growth of woody plants in Mediterranean 130
areas (Silla and Escudero 2003; Querejeta et al. 2008; Pascual et al. 2012). 131
Thus, the ability of seedlings to avoid water stress by growing a large, deep root system from 132
the period ranging from time of planting to onset of drought in summer is the major requirement for 133
seedlings in the Mediterranean (Villar-Salvador et al. 2012). As an example, according to studies 134
from Mediterranean semiarid conditions, roots must reach a depth of at least 30 cm before the 135
beginning of July to ensure access to sufficient supply of water until the end of summer (Oliet et al. 136
2002; Padilla and Pugnaire 2007). The use of hardened seedlings that can both resist frost exposure 137
and cope with low soil nutrient availability are also important attributes when planting in 138
Mediterranean areas. Multiple stress or resource limitations (frost, water, and soil nutrients) require 139
targeting seedling traits that are often impossible to achieve concurrently within a specific nursery 140

cultural regime, especially when a source of stress exerts antagonistic effects on morpho-141
physiological traits. For instance, high nutrient availability reduces the capacity of seedling roots to 142
supply water aboveground (Cortina et al. 2013). Seedling capacity to resist these stresses is linked 143
to physiological processes for which nutrient status is clearly associated. 144
Functional attributes of Mediterranean woody species: Interaction with nutrient status. 145
In general, Mediterranean forest species are adapted to survive in a changing environment 146
where periods of high water availability alternate with long dry and hot seasons. However, species 147
exhibit a wide variety of strategies to survive under these circumstances (Valladares and Sánchez-148
Gómez 2006). Some Mediterranean species evolved in a different climate to the current one but 149
their functional adaptive traits allow survival under Mediterranean conditions (Herrera 1992). 150
Phylogeny of these species is a source of functional variability and plant functional attributes exert 151
a significant influence on the carbon, water and mineral nutrient economy of plants and thus on 152
their fitness. For instance, the well adapted Mediterranean shrub such as Pistacia lentiscus L. 153
originated during the Oligocene under tropical climates and exhibits functional attributes such as 154
high stomatal conductance and specific leaf area that seem to be opposite those of other 155
Mediterranean species (Vilagrosa et al. 2003). However, these features allow rapid root growth 156
which improves establishment rates under dry conditions compared to other sclerophyllous species 157
that are more conservative in their water-use, but has lower root growth such as Quercus coccifera 158
L. (Vilagrosa et al. 2003). Regeneration niche is another important factor explaining functional 159
variability in Mediterranean species (Gómez-Aparicio et al. 2006). Some species, such as Pinus 160
halepensis Mill., can establish faster than others, such as Quercus spp. Attributes like fast root 161
growth (Puértolas et al. 2010), strong stomatal control of water use (Borghetti et al. 1998), and high 162
plasticity in response to resource availability (Baquedano and Castillo 1998) allows Pinus 163
halepensis to survive in unsheltered locations. However, other species that are able to live in semi-164
arid locations like Tetraclinis articulata Masters. has slower root growth rate but higher resistance 165
to embolism than pine species (Oliveras et al. 2003). 166

The abovementioned examples outline the high variability in functional strategies of tree 167
seedlings for species that may co-occur in Mediterranean environments. This functional diversity 168
has a large impact on target seedling features in Mediterranean nurseries. Nowadays, a wide variety 169
of species are currently used in the Mediterranean for restoration purposes, and nursery 170
management must adjust cultural techniques for each functional strategy. One of the most important 171
features to consider is phenotypic plasticity in response to resource availability (Valladares and 172
Gianoli 2007). Some studies suggest that pre-planting traits may have a strong impact on field 173
performance in species with higher morphological and physiological plasticity in response to 174
increased nutrient storage (Cuesta et al. 2010). Species characterized by fast root growth that are 175
also highly responsive in photosynthetic capacity and aboveground biomass accumulation, such as 176
Pinus spp., will benefit more from increasing fertilization or light availability in the nursery than 177
less plastic species like Quercus spp. This is because seedling nutrient concentration and size are 178
positively correlated with survival in Mediterranean plantations through greater capacity to egress 179
new roots and secure access to water (Villar-Salvador et al. 2012). Therefore, nutrient loading 180
would be more effective in some species due to the varying plasticity to availability of resources. 181
Increased internal nutrient storage might be more beneficial for species with stronger responses to 182
nutrient availability. 183
Unlike boreal species, influence of photoperiod on Mediterranean seedling phenology is low 184
(Kostopolou et al. 2011). Growth cessation in autumn is species-specific and mostly temperature-185
specific (Fernández et al. 2008). Therefore, practices like late-season fertilization can have a distinct 186
effect depending on interactions between uptake and growth patterns, which can also be mediated 187
by the environment. This aspect is addressed in more detail in forthcoming sections. 188
Seedling production under Mediterranean conditions 189
Over the past four decades, the demonstrated superior performance of container versus bare-190
root seedlings when planted under stressful conditions has broadened its use in Mediterranean areas 191
(Cortina et al. 2006). Many nurseries produce container seedlings under full sun from sowing in 192

spring to lifting in fall or winter. Container seedlings remain outdoors in autumn prior to planting in 193
fall-winter, and thus cold storage is not practiced in Mediterranean nurseries. However, keeping 194
container seedlings outdoors before planting during fall and early winter may result in root damage 195
from frost. This risk is not present in bare-root seedlings, as root systems are insulated by natural 196
soil. Although cold acclimation of roots during autumn occurs in response to progressively 197
declining temperatures (Stattin and Lindströn 1999), frost induced injury can occur easily, 198
especially at inland container nurseries, where early frosts are frequent (Mollá et al. 2006), leading 199
to ‘hidden death’ of preplanted seedlings (Bigras 1997). Degree of hardening in root tissues is lower 200
than that of shoots (Bigras et al. 2001; Kreyling et al. 2012). Knowledge of root cold hardening 201
dynamics and how N nutrition affects this process is crucial to improve fertilization management. 202
There are not specific studies on this topic in Mediterranean species, although new research on the 203
effect of plant nutrient status on shoot frost resistance has emerged in recent years for 204
Mediterranean forest species, as described below. Irrespective of the occurrence of frost, relatively 205
high temperatures in nurseries characterized by a mild autumn can promote growth of both shoots 206
and roots (Mollá et al. 2006; Trubat et al. 2010; Andivia et al. 2011), due to relatively high activity 207
of plants (Fernández et al. 2008). This results in nutrient dilution during fall that can reduce 208
seedling quality (Oliet et al. 2011). 209
210
Role of nutrients in Mediterranean seedling establishment 211
Nutrients and drought avoidance 212
Nutrients play a crucial role in physiological processes leading to planting success in Mediterranean 213
areas. One major contribution of planting success is the acquisition of traits related to avoidance of 214
drought, like early root growth capacity, stomatal regulation and control of water losses. Studies on 215
plant nutrition and seedling quality usually focus on macro-nutrients as N, P and K. In particular, 216
literature on Mediterranean species emphasizes the strong relationship between N and planting 217

success (Oliet et al. 2006). In Mediterranean plantations, positive relationships between leaf [N] and 218
photosynthesis or stomatal conductance during establishment has been confirmed in several 219
experiments under arid (Fig. 1) and semiarid conditions for Juniperus thurifera L. (Villar-Salvador 220
et al. 2012), and in sub-humid Mediterranean plantations with Pinus halepensis (Cuesta et al. 2010). 221
Under controlled environmental conditions, gas exchange of species like Pinus canariensis C. 222
Smith. (Luis et al. 2010) and Quercus suber L. (Hernández et al. 2009) also positively respond to 223
increments in rate of applied N. Early reestablishment of high assimilation rates guarantees capacity 224
of tree seedling to grow new tissues after planting (Villar-Salvador et al. 2012). 225
Nutrient demand for tree growth can be met either by acquisition from external sources or 226
by remobilization of internal storage (Millard and Grelet 2010). Growing new organs are strong 227
nutrient sinks, and nutrient remobilization from old tissues supplies much of this demand due to 228
lack of uptake capacity of root system immediately after planting (Grossnickle et al. 2005). 229
Nitrogen and P remobilization could be particularly relevant for species that live in nutrient poor 230
soils (Salifu and Timmer 2003; Salifu et al. 2008a and Folk and Grossnickle 2000, respectively) as 231
per much of the Mediterranean (Silla and Escudero 2003; Close et al. 2004). Thus, N remobilization 232
to support new growth after planting is paramount to ensure survival. The proportion of N 233
remobilized in Mediterranean species depends on source strength: seedlings with higher 234
concentration and greater content in old tissues remobilize more N to new roots or shoots (Oliet et 235
al. 2009a; Cuesta et al. 2010). Research under a variety of Mediterranean conditions has shown 236
improved root growth potential (Villar-Salvador et al. 2008; Oliet et al. 2009a; Trubat et al. 2010; 237
Close et al. 2012) and postplanting performance (Cuesta et al. 2010; del Campo et al. 2007; Luis et 238
al. 2009; Oliet et al. 2009b) of largest and N loaded seedlings by promoting assimilation or by 239
supplying reserves to growing tissues. These benefits can be observed even under very arid 240
conditions (Fig. 2). Thus, nutrient loading in the nursery via, for instance, fall fertilization, may 241
enhance seedling performance after transplanting even during the first year after planting (Fig. 3). 242

A growing body of experimental evidence has led to the formulation of a conceptual model 243
providing a physiological basis to explain the superiority of larger and N loaded phenotypes (Villar-244
Salvador et al. 2012). Many of the positive relationships between survival and plant size are 245
supported by nursery fertilization experiments. Some studies have tried to separate the confounding 246
effect of size in the relationship between nutrient status and survival. For instance, Cuesta et al. 247
(2010) demonstrated that this effect clearly depended on species and weed competition, concluding 248
that large Pinus halepensis outperformed small seedlings under strong competition even when both 249
stocktypes were N loaded, while no effect was found for Quercus ilex L. Puértolas et al. (2012) 250
reported a positive effect of better nutrient status on survival of Pinus halepensis planted in a 251
semiarid site only for the smallest stocktype, but larger seedlings showed similar performance 252
irrespective of nutrient status; Oliet et al. (2012) found no changes in survival among seedlings of 253
Zizyphus lotus L. (Lam.) with the same nutrient status and shoot:root ratio but significant 254
differences in size. Variable responses observed in these studies may also be a function of 255
methodological approaches in each experiment. Gradients in morphology have been achieved either 256
by using different sowing dates with the same container size, varying container sizes, choosing 257
specific seed provenances or seedling ages, which affects not only overall development of the 258
stocktypes, but also shoot:root ratio. 259
The general trends outlined above and in Villar-Salvador et al. (2012) conflict with some 260
results indicating that reduction in seedling size and nutrients concentration improved survival and 261
growth of several Mediterranean species under arid conditions (Trubat et al. 2008; 2011; Cortina et 262
al. 2013 and references therein). These results can be important to help to conclude that the relative 263
contribution of seedling size and N status to survival, as well as the point at which increasing N 264
status and plant size relationship turns negative in relation to survival, is probably species and site 265
specific (Puértolas et al. 2012). However, the large body of evidence emphasizing the strong 266
benefits of N loading in many Mediterranean species and ecosystems implicates the benefits of this 267
nursery cultural strategy. 268

The relationship between the nutrient status of other macronutrients like P and K and 269
establishment of Mediterranean species is not well understood. The limited availability of P may 270
affect growth rates and morphology of roots and root hairs (Bucio et al. 2002), altering plant 271
capacity for water transport. Nursery P application has been related to root growth in Quercus ilex 272
(Sardans et al. 2006) and root growth and survival after planting in Acacia salicina Lindl. (Oliet et 273
al. 2005); P concentration in roots is linked to survival or root growth capacity (RGC) in Quercus 274
ilex (Villar-Salvador et al. 2004; Oliet et al. 2011, respectively), to survival in Pinus halepensis 275
(Oliet et al. 2009b) and RGC in Eucalyptus globulus Labill. (Fernández et al. 2007). This element 276
also plays essential roles in metabolic processes driving growth (Landis and van Steenis 2004). For 277
example, strong relationships have been observed between P and maximum rates of photosynthesis 278
(Amax) in Mediterranean conifers, due to the effect of P in the partitioning of N to Rubisco (Warren 279
et al. 2005 and references therein). With regard to K, this element is not part of the structure of the 280
plant and therefore its effect on growth is not as pronounced as that of N and P. To our knowledge, 281
only two studies have shown a significant (but weak) positive relationship between K concentration 282
and Quercus ilex outplanting survival and growth (del Campo et al. 2010, Andivia et al. 2011, 283
respectively). It is probable that physiological functions of this element (stomatal regulation and 284
osmotic roles) are not significantly hindered during establishment unless its content is reduced 285
below critical values (Fernández et al. 2007). 286
Frost resistance and nutrient status 287
Seedling frost resistance has been identified as an important quality attribute for nursery production 288
and subsequent planting success. As in other climatic areas, studies designed to analyze the 289
relationship between nutrient status and frost resistance in Mediterranean species have yielded some 290
contradictory results (Villar-Salvador et al. 2005, 2013). However, a relatively large body of 291
evidence shows significant and positive relationships between cold hardiness and N status for a 292
variety of species commonly used in forest restoration, like Mediterranean Pinus (Puértolas et al. 293
2005), Quercus (Andivia et al. 2011; 2012a) or Eucalyptus (Fernández et al. 2007). In addition, 294

Andivia et al. (2012b) demonstrated that the application of a very small amount of N during the 295
cold hardening phase of Quercus ilex might be sufficient to prevent frost-induce oxidative stress. 296
Primary mechanisms for frost resistance include protection of cell membranes and specific anti-297
freeze proteins that accumulate in the apoplast to inhibit formation of ice crystals or improve 298
membrane fluidity (Lambers et al. 2008). This may explain the reported positive effect of N 299
fertilization on cold hardiness. Nevertheless, the relationship between cold hardiness and the 300
composition of proteins and other organic constituents needs to be defined for many Mediterranean 301
species (Andivia et al. 2011). Along with proteins, accumulation of soluble sugars (SS) can improve 302
cold hardiness through their cryogenic and osmotic properties (Mollá et al. 2006; Fernández et al. 303
2008; Villar-Salvador et al. 2013), and some studies reveal a dilution effect of carbohydrates and SS 304
with increasing N rates in the nursery (Planelles 2004; Andivia et al. 2011; Villar-Salvador et al. 305
2013). However, nutrient deficiency can also impair frost resistance (Bigras et al. 1996). It is also 306
well known that excessive fertilization may affect cold hardiness by delaying growth cessation of 307
boreal and temperate forests species (Repo et al. 2001; Thomas and Ahlers 1999). Studies 308
conducted with Eucalyptus species in Australia concluded that N-deficient seedlings were more 309
tolerant to cold-induced photoinhibition and photodamage resulting from a combination of high 310
radiation levels and low temperature after planting in cold areas (Close 2012 and references 311
therein). Along with the aforementioned functional explanations, the wide range of results obtained 312
from studies examining the relationship between nutrition and cold hardening likely reflects 313
variability in species, differences in tissue nutrient concentrations and fertilization rates, specific 314
growth phase during nutrient application or season in which cold hardiness was assessed (Colombo 315
et al. 2001; Villar-Salvador et al. 2005). Lower frost tolerance in plants fertilized during active 316
growth is probably related to the allocation of N into additional biomass, while N fertilization 317
applied after shoot growth cessation is preferentially incorporated into metabolism, which probably 318
contributes to enhanced cold tolerance (Dehayes et al. 1989). With regard to fertilizer rate it is 319
important to differentiate among optimal, suboptimal and luxury levels of N content and their 320

relationships to cold hardening. For instance, Quercus ilex frost resistance increased with up to 60 321
mg N applied in the fall after which it plateaued (Andivia et al. 2012b). The relationship between 322
frost resistance and nutrient status is mediated by current environmental conditions. Although K is 323
thought to improve the winter survival of trees, the empirical evidence on potential effects of this 324
element on frost resistance of Mediterranean species is scarce (Fernández et al. 2007). To our 325
knowledge, no evidence of the effect of P status on plant frost resistance has been presented in 326
forest nursery experiments. 327
Other functional attributes as affected by nutrient status 328
Along with drought avoidance and frost resistance, other attributes may also play an important role 329
under different planting conditions of the Mediterranean. Those attributes can be affected by 330
nutrient status, although the relationships are not well known for many species. For instance, 331
nutrient availability during growth affects xylem hydraulic conductance, although trends are varied 332
(see citations in Villar-Salvador et al. 2012). Traits associated with drought tolerance (osmotic 333
adjustment, cell wall elasticity) are also an important attribute of seedlings to be planted in the 334
Mediterranean, as it can affect ability to survive during periods of low water potential, particularly 335
immediately after planting. Experiments conducted with Mediterranean oaks and conifers under low 336
and high N availabilities showed no effect of N on drought tolerance measured as osmotic 337
adjustment capacity, except for Juniperus thurifera, whose osmotic potential at turgor loss point 338
was greater for highly fertilized plants (lower tolerance, Villar-Salvador et al. 2005; 2013). 339
On the other hand, studies conducted with Eucalyptus species in Australia concluded that N-340
deficient seedlings were less affected by vertebrate browsing in grazing areas (Close 2012). The 341
author concluded that nutrient loading can have detrimental effects on performance of Eucalypts 342
plantations under certain ecological conditions of the planting areas. But more information about 343
the relationship between nutrient status after planting and degree of browsing damage in 344
Mediterranean systems is needed, as almost all literature on this topic currently originates from 345
temperate and boreal forests (Burney and Jacobs 2013). 346

Finally, fertilization regimes may influence mycorrhizae and nodulation. As per species 347
from other biomes, previous studies have shown that high N concentrations in substrates inhibit 348
ectomycorrhizae development and nodulation in Mediterranean species (Díaz et al. 2010; 349
Valladares et al. 2002, respectively). However, literature indicates that the relationship between 350
ectomycorrhizal colonization and nutrient availability in the growing media is highly dependent on 351
tree and fungi species, as well as fertilization method and regime. For instance, slow release 352
fertilization enhanced percent of ectomycorrhiza colonization in Pinus pinaster Ait. and Pinus 353
pinea but only for one of three fungi species tested (Rincón et al. 2007). In addition, colonization 354
also seems to be nutrient dependent as, for example, frequency of mycorrhizal branching in 355
Eucalyptus globulus was reached at the highest N rates but lowest P rates (Mason et al. 2000). 356
Studies conducted in boreal species concluded that exponential fertilization regimes promoted 357
mycorrhization of seedlings to a much higher extent, probably due to the low nutrient concentration 358
at the beginning of culture when root association takes place (Quoreshi and Timmer 2000). The 359
benefits of root flora symbionts for seedling quality cannot be underestimated. High-dose-nutrient 360
loaded non-inoculated seedlings out-compete low-dose fertilized, inoculated seedlings (Villar-361
Salvador et al. 2008), although the effects may strongly depend on the inoculum status of the area. 362
For instance, most degraded areas have enough inoculum to ensure spontaneous mycorrhizal 363
infection. But this may not be the case in extremely degraded areas such as mine sites and 364
intensively managed agricultural lands (Cortina et al. 2004). Therefore, research in this field should 365
focus on maximizing nutrient status via adequate fertilization strategies that simultaneously do not 366
inhibit mycorrhization/nodulation. 367
368
Nutrient loading of Mediterranean woody species: Principles and procedures 369
All sections above emphasize the importance of seedling mineral nutrition in the nursery for 370
growing Mediterranean target seedlings. Usually, seedling performance in the Mediterranean is 371

improved by increasing nutrient concentration to values where plants accumulate nutrient reserves. 372
We believe that nutrient loading is in the majority of the cases, a good strategy for high quality 373
plant production of Mediterranean seedlings. During the last two decades, several procedures to 374
nutrient loaded seedlings in the nursery have been developed for temperate and boreal forests 375
species (Timmer and Aidelbaum 1996). However, its applicability must be tested for each species 376
and planting environment. 377
Nutrient delivery regime in the nursery and nutrient loading of Mediterranean species 378
The exponential regime of fertilizer application is based on the "steady state" nutrition theory of 379
Ingestad and Lund (1986). According to this theory, maintenance of stable or non-diluted nutrient 380
concentrations in plant tissues over time requires matching relative addition rate of nutrients to 381
relative growth rate of seedlings. This goal can be achieved by employing fertilization schedules 382
that deliver nutrients at exponentially increasing rates that are synchronized with the exponential 383
growth of seedlings. Some studies comparing constant versus exponential regimes show that 384
exponential delivery induces higher nutrient accumulation in the plant at similar or even lower 385
fertilizer rates (Imo and Timmer 1992; Timmer 1997; Hawkins et al. 2005 and references therein; 386
Dumroese et al. 2005). These results led the authors to conclude that nutrient loading can be more 387
effective when combined with exponential fertilization techniques because nutrient delivery to the 388
crop ensures a progressive build up of nutrients in the rooting substrate, thus minimizing potential 389
toxicity (Timmer and Aidelbaum 1996). Unfortunately, many studies examining the effectiveness 390
of exponential delivery regime as a tool for nutrient loading have compared constant regimes at 391
conventional (low) dose with exponential regimes at much higher rates (Malik and Timmer 1998; 392
Salifu and Timmer 2003; Close et al. 2005; Birge et al. 2006; Hawkins et al. 2005 and references 393
therein; Salifu and Jacobs 2006; Salifu et al. 2008a). With this experimental approach it is not 394
possible to elucidate whether nutrient loading is a function of the exponential regime itself or results 395
from the differences in the amount of fertilizer applied (Everett et al. 2007). In fact, other studies 396
comparing effectiveness of nutrient loading using similar rates for both fertilizer delivery regimes 397

are inconclusive (Everett et al. 2007; Hawkins et al. 2005 and references therein; Salifu et al. 2008a; 398
Schott et al. 2013). Along with this methodological issue, the potential of the exponential regime to 399
obtain nutrient loading relies on the existence of a significant period of continuous exponential 400
growth during nursery culture (Burgess 1991). Thus, the effectiveness of exponential delivery on 401
nutrient loading may be species-specific (Everett et al. 2007). Many Mediterranean species exhibit 402
nursery growing patterns at intervals with periods of rapid growth (flush) that alternate with periods 403
of slow or interrupted growth (lags). Shoot and root biomass growth follows a non-continuous 404
(polycyclic) growing model as shown for Quercus ilex (Fig. 4). This pattern implies short periods of 405
high nutrient demand followed by intermediate lag stages. Additionally, ontogeny of each 406
individual is not synchronous and thus nutrient demand varies among individual seedlings. This 407
explains why Quercus ilex fertilized with both constant and exponential regimes at the same rate 408
did not differ in nutrient status, morphology, or in planting response (Oliet et al. 2009a). In addition, 409
growth activation after a lag stage is mediated by endogenous genetic control, but also by 410
environmental conditions and resources availability. In particular, N availability has significant 411
effects on number and length of root and shoot flushes (Schoene and Yeager 2006; Salifu et al. 412
2008b). During the first weeks of culture, the acorn of Q. ilex provides more than two-thirds of N 413
up to the end of the second flush, irrespective of the fertilization rate (Villar-Salvador et al. 2010). 414
Therefore, an efficient strategy to fertilize polycyclic Mediterranean oaks species in the nursery 415
could be to apply high rates of fertilizer after the first or second flush has concluded in order to 416
trigger additional flushes and produce large seedlings. The constant fertilizer regime has shown 417
good results in augmenting size and nutrient concentration of seedlings in Mediterranean 418
sclerophylous species (Planelles 2004; Villar-Salvador et al. 2005). However more information is 419
needed on growth rate and pattern of many of Mediterranean species to better forecast nutrient 420
demand in the nursery before designing the most appropriate fertilization regime to reach nutrient 421
loading (Carrasco et al. 2004). 422

Late-season fertilization as a tool for loading 423
Nutrient application during fall has long been considered a beneficial nursery cultural practice in 424
many areas (Grossnickle 2012 and references therein). Many of these studies were conducted with 425
temperate or boreal species characterized by predetermined growth cessation at the end of summer, 426
and show increments in nutrient concentration without affecting seedling biomass. Many 427
Mediterranean forest species do not have a predetermined fall growth cessation pattern, and 428
continue growing until environmental conditions become unfavorable. For some of these species, 429
late-season fertilization has proven to be an effective way to avert nutrient dilution during fall 430
(Trubat et al. 2008) or even to revert it (Fig. 5), with beneficial consequences after planting for 431
pines (Puértolas et al. 2003; 2012 and Fig. 3). Apart from pines, studies conducted with 432
sclerophylous oaks under mild fall conditions show how nutrient concentration is increased after 433
fertilization during this season despite increased biomass (Trubat et al. 2010; Andivia et al. 2011; 434
2012a;b). In addition, the last three studies reported an increase in shoot frost resistance of fall 435
fertilized seedlings that was attributed mainly to the role of N (incorporated in the proteins 436
metabolism) and the maintenance of high SS values despite greater biomass (absence of strong 437
dilution effects). Other studies with this species reported no effect of late-season fertilization on 438
total non-structural carbohydrate content of seedlings, whereas, it decreased and increased root 439
starch content in leaves and roots, respectively (Cortina et al. 2013). At the same time, the 440
hardening effect of N during fall application depends on the environmental conditions during 441
acclimation. In this sense, Puértolas et al. (2005) showed that while N concentration in needles did 442
not affect frost resistance of Pinus halepensis seedlings grown under mild fall conditions, frost 443
resistance was affected by N status when seedlings of this species hardened under lower 444
temperatures. This may help explain the apparent inconsistencies in the relationship between N 445
status and frost resistance. In addition, fall nutrient delivery must take into account root uptake 446
activity in response to temperature. Oliet et al. (2011) tested early versus late fall applications of 447
fertilizer in a mild season nursery, and concluded that shoot N and K, and root K were unaffected 448

by timing of fall fertilization, but root P concentration was enhanced by applying fertilizer during 449
early fall. This revealed a different nutrient dynamic during fall that was dependent on the specific 450
nutrient and plant component. Along with some evidence concerning positive effects on frost 451
resistance, because both seedling size and nutrient concentration increase root growth capacity, late-452
season fertilization may be an effective tool to improve field performance of many Mediterranean 453
species (Puértolas et al. 2012). 454
Other fertilization methods to accomplish nutrient loading 455
Application of water soluble fertilizers (fertigation) is currently the norm in forest nurseries but 456
application of solid fertilizers to the substrate, particularly controlled-release fertilizer (CRF), 457
represents an alternative to fertigation (Jacobs and Timmer 2005). With improved CRF technology, 458
many nurseries are now routinely incorporating CRF into the growing medium (Haase et al. 2006). 459
In Mediterranean nurseries, similar plant growth rates and nutrient status have been observed in 460
trials in which both types of fertilization were compared (Oliet 1995; Puértolas et al. 2000; Rincón 461
et al. 2007; Trubat et al. 2010). Theoretically, the temperature-dependent nature of nutrient release 462
of CRF (Jacobs and Timmer 2005) results in the largest release during the period of maximum plant 463
growth and the lowest during cold periods or storage. The above mentioned studies were conducted 464
in coastal Mediterranean nurseries with hot summers and mild falls. Under the same environmental 465
conditions, similar experiments showed a potential ability of CRF to reach luxury consumption of 466
some Mediterranean species (Fig. 6). We have detected high values of NO3-N, P and K (up to 305, 467
6 and 275 mg·l-1, respectively) in the growing media saturation extract after 11 months of culture 468
(Oliet et al. 2004) when fertilizing only with CRF; thus, CRF incorporated into the growing 469
medium has the potential to continue releasing following field transplant (Haase et al. 2006). 470
Consequently, CRF may provide an inexpensive and simple way to nutrient load, allowing relative 471
control of nutrient supply during the growing period with higher recovery rates than in fertigation 472
systems (Oliet et al. 2004). However, its application in Mediterranean nurseries requires broadening 473
its experimentation to different species and environmental conditions. 474

Along with root uptake, plants can also absorb nutrients through leaves, and so foliar 475
fertilization is sometimes used to supply nutrients to forest crop species. This can be crucial when 476
nutrients cannot be easily taken up from soil and/or during periods of high nutrient demand, as well 477
as when it is necessary to reduce potential nitrate leaching (Fageria et al. 2009). Nitrogen overload 478
in fall by foliar fertilization can be used without stimulating new growth or delaying dormancy (Bi 479
and Scagel 2008). There is no experience in foliar fertilization for nutrient loading of forests species 480
in general and for Mediterranean species in particular. However, its potential as a tool to nutrient 481
load the plant and/or to correct nutritional deficiencies should be tested for these species. 482
Nutrient ratios in fertilizer and the nutrient loading process 483
Along with nutrient rate, formulation and proportion of N, P and K in the fertilizer can significantly 484
affect nutritional status during culture. A variety of interactive effects occur either in the growing 485
media or within the plant. Such interactions are likely to result from a combination of synergistic 486
and antagonistic soil and plant processes that affect plant response, either directly or indirectly 487
(Zhang et al. 2007). Concerning nutrient loading, these effects must be understood to avoid 488
deficiencies caused by sub-optimal nutrient and source proportions in the fertilizer formulation or to 489
find the most appropriate solution to optimize the supply of all NPK nutrients simultaneously. 490
Along with the well known dilution effect derived from supplying additional increments of a 491
deficient nutrient (mostly N), more complex implications with consequences on nutrient status are 492
observed in nurseries. For example, a study conducted with the Mediterranean species, Ceratonia 493
siliqua, concluded that shoot N and K concentrations are fostered by increasing the rates of K and 494
N, respectively, in the fertigation solution (Fig. 7 A and B), or by interactions with rate of P (Fig. 7 495
C and D, respectively). Similar effects were described by Andivia et al. (2011, 2012a, b) in Quercus 496
ilex. Also with this species, a higher P concentration is promoted by increasing N rates (Villar-497
Salvador et al. 2004). The relatively fixed stoichiometry of these elements in plant matter appears to 498
be a consequence of the requirements of N for proteins and of P for nucleic acids, membranes and 499
metabolism, as well as the K-mediated activation of numerous enzymes and ATPases required for 500

protein synthesis (White and Hamond 2008; Andivia et al. 2011 and references therein). Therefore, 501
we believe that successful nutrient loading of N, P and K requires consideration of the proportions 502
of these macronutrients in the fertilizer, as certain limits to the uptake of a particular nutrient may 503
rely on the availability of other nutrients. 504
505
Synthesis and future research needs 506
Recent research with several key Mediterranean forest tree species suggests that nursery nutrient 507
loading of seedlings is a potential silvicultural tool to improve planting success in Mediterranean 508
areas. However, caution must be taken when broadening this technique to a wider pool of species 509
and nursery/planting site environments with regard to: 510
- The need to match nutrient loading regimes to the high diversity of functional attributes of 511
Mediterranean woody species to ensure a positive response after planting. 512
- Growth patterns of many Mediterranean species are controlled by endogenous rhythms and so 513
nutrient availability via fertilization must match growth and demand patterns. A better 514
knowledge of ontogeny during the seedling stage is needed to match supply and demand. 515
- In this regard, accounting for functional traits and habits is necessary when tailoring late-season 516
or exponential fertilization regimes. For instance, deciduous species may have lower rates of 517
nutrient uptake and storage in fall compared to evergreen species. 518
- The effect of nutrient status on frost tolerance of shoots and (especially) roots in the nursery 519
remains unclear. Despite some inconclusive results, most evidence indicates that N is an 520
important element to improve frost resistance of Mediterranean seedlings when correctly 521
applied in regime, rate and season. New research specifically designed to examine effects of 522
nutrient loading on frost resistance in both the nursery and field is needed. 523
- The importance of achieving a proper balance between nutrient loading regimes and 524
management of symbiots in the nursery that maximizes nutrient status while simultaneously 525

promoting mycorrhization/nodulation, which is a promising research theme to help further 526
improve seedling quality of Mediterranean species. This balance must be species and site-527
specific. 528
- A deeper knowledge of the relative contribution of N reserves to growth of new tissues 529
(especially roots) after planting is needed. We hypothesize that the relative importance is a 530
function of foliar and growth habits of the species, as well as environmental conditions in 531
Mediterranean areas. 532
- In some arid Mediterranean sites, where root growth immediately after planting is restricted 533
either because of scarce rainfall or unfavorable soil conditions, a conservative water use 534
strategy can be more important than an avoidance strategy. Under these circumstances, small 535
seedlings with low shoot:root ratio may be more successful in coping with soil drought 536
immediately after planting. Special attention should be given to designing fertilization regimes 537
for species under this specific ecological context. 538
Among different techniques to achieve nutrient loading, late-season fertilization has emerged as a 539
very important technique for Mediterranean nurseries. Specific research on optimal proportions of 540
macronutrients, as well as the interactions between application time in the fall and frost resistance 541
during the hardening process is needed to develop specific protocols for performing this technique 542
in nursery cultivation. 543
544
Acknowledgements 545
This paper synthesizes results from projects sponsored by multiple funding sources including the 546
Spanish Ministry of Science and Innovation (through Projects OT98-001, AGL2006-12609 Encinut 547
and AGL2011-24296 Ecolpin), the Andalusia Department of Science and Innovation (AGR-6501), 548
Regional Government of Madrid (S2009/AMB-1668), National Institute of Agricultural Research 549
and Polytechnic of Madrid and Cordoba Universities Research Programs. We wish to thank the 550

colleagues with whom we have collaborated on research cited in this review, particularly Francisco 551
Artero, Mari Luz Segura, Pedro Villar-Salvador, and Manuel Fernández. We also thank the 552
organizing committee of the IUFRO Symposium, ‘Nutrient Dynamics of Planted Forests’ for the 553
opportunity to participate. 554
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Nat Resour Can, Canadian Forest Service, Sault Ste Marie, ON 811
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890 814
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seedlinngs in a degraded semi-arid shrubland. Ecol Eng 37:1164-1173 818
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Mediterranean tree seedlings: individual responses versus interspecific trends in eleven 824
species. Plant Biol 8:688–697 825
Valladares F, Villar-Salvador P, Dominguez S, Fernandez-Pascual M, Penuelas JL, Pugnaire FI 826
(2002) Enhancing the early performance of the leguminous shrub Retama sphaerocarpa (L.) 827
Boiss.: fertilisation versus Rhizobium inoculation. Plant Soil 240:253-262 828
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dryland restoration: approaches for climate change adaptation. New For 43:561-579 830

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leaf dieback in seedlings of two co‐occurring Mediterranean shrubs during an intense drought. 832
J Exp Bot 54: 2015-2025 833
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growth in holm oak (Quercus ilex), cultivated with contrasting nutrient availability. Tree 835
Physiol 30:257-263 836
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opposite effects on the stress tolerance of Pinus pinea L. seedlings Tree Physiol 33:221-232 838
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Mediterranean plantations. Insights from an ecophysiological conceptual model of plant 841
survival. New For 43:755-770 842
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nursery on the drought and frost resistance of Mediterranean forest species. Invest Agrar: Sist 844
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Mediterranean leguminous shrub Retama sphaerocarpa: Insights from a provenance, 848
fertilization, and rhizobial inoculation study Env Exp Bot 64:145-154 849
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functional attributes, and field performance relationships in the Mediterranean oak Quercus 851
ilex L. For Ecol Man 196:257-266 852
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N, P and K fertilizers on yield and mineral composition; description and experimental test. 859
Plant Soil 298:81–98 860
861
862

863
Figure captions 864
865
Fig. 1 Photosynthesis rate (±SE, n = 5) of Ceratonia siliqua L. during one season after planting as 866
affected by fertilization rate in the nursery (mg N l-1 of fertigation solution). Rainfall during the 867
entire study period was 157 mm. (Adapted from data in Planelles 2004). 868
Fig. 2 Survival of Zizyphus lotus L. four years after planting in a Mediterranean arid field as 869
affected by leaf N concentration and shoot:root ratio (±SE, n = 30, right axis). Mean and SE, n = 64. 870
Average annual rainfall during the study period was 177 mm. 871
Fig. 3 Height growth (mean ±SE) during the first two years after planting of Pinus halepensis Mill. 872
in an abandoned agricultural land in central Spain. Seedlings were grown under a fall N hardening 873
regime (no N application from September until the planting date in January; solid circles), low N 874
fall fertilization (weekly fertilization with 1 mg l-1 of a N:P:K 4:25:35 soluble fertilizer in the same 875
period; hollow circles), and high N fall fertilization (same application rate as in 4:25:35 but with a 876
20:7:19 formulation). Mean and SE, n = 60. Beside each point, mortality is shown (%). Mortality 877
was identical for both fall fertilization treatments. 878
Fig. 4 Leaf and root biomass accumulation in two provenances of Quercus ilex L. during nursery 879
culture (mean and SE). Acorns were collected from three individuals in each provenance, which 880
were more than 500m apart (4 plants per individual and date were harvested). 881
Fig. 5 Vector diagram for analysis of plant N status in one-year old Pinus halepensis seedlings 882
during fall in a Mediterranean nursery in central Spain. The origin of the three vectors represents 883
whole-plant N status and dry weight on September 21 (n=12). The vector endings represents 884
relative N status and dry weight respect to the values in September measured on November 25 in 885
plants subjected to: N hardening (square); fall fertilization with N:P:K 4:25:35: fall fertilization 886

with 20:7:19. The analysis show that N hardening led to dilution and even loss of N while fall 887
fertilization increased N content and concentration together with dry weight, suggesting that Pinus 888
halepensis has active growth and thus, high demand of N even in the autumn. 889
Fig. 6 Response of seedling dry mass and N concentration (±SE) to increasing N supply (0 to 258 890
mg) as CRF for one growing season. Ceratonia siliqua (n = 30 seedlings per treatment) was 891
cultivated for 36 weeks, and Pinus halepensis (n = 20 seedlings per treatment) for 45 weeks in a 892
costal nursery in southeastern Spain. For C. siliqua species, the luxury consumption zone was 893
reached at 120 mg of N with a plateau thereafter, while P. halepensis luxury consumption occurred 894
at 184 mg of N. No toxicity was observed for across the range of fertilizer rates. 895
Fig. 7 Relationship between N and K concentration (±SE) of Ceratonia siliqua shoots and 896
availability of N, P and K derived from a NPK (3×2×2) factorial experiment. (A) Shoot N 897
concentration as a function of K rate applied or N×P interaction (D). (B) Shoot K concentration as a 898
function of N rate applied or K×P interaction (C). Fertigation was applied during 17 weeks. The 899
experimental unit was a pooled sample of 7 plants, and 4 experimental replicates (in blocks) were 900
analyzed per NPK combination treatment. ANOVA P-value of the effect is presented in the 901
correspondent figure. For main effects presented in (A) and (B), no significant N×K interaction 902
occurred. (Redrawn from data in Planelles 2004). 903
904
905
906

907
Figure 1 908
909
910
911
912
913
b
b
b
b
a
a
ab
b
a
a
a
a
0
2
4
6
8
10
12
January
March
May
August
November
A (μmol m-2s-1)
Measure date
30 mg l-1
150 mg l-1
250 mg l-1

914
Figure 2 915
916
917
918
0,1
0,3
0,5
0,7
0,9
1,1
0
15
30
45
60
75
90
34 36 38 40 42 44 46 48
Shoot:root (g·g-1)
Survival (%)
Leaf [N] mg·g-1
Survival
Shoot to root ratio

Figure 3 919
920
921
922
923
924
Height (cm)
0
20
40
60
80
100
120
140
160
N hardening
4:25:35
20:7:19
0
7
0
7
7
13
31
33
May 1999 Nov. 1999 Nov. 2000 Nov. 2001

Figure 4 925
926
927
928
Leaf biomass (gDW)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4 Alcarria
Alicante
Root biomass (gDW)
0.0
1.0
2.0
3.0
4.0
April 2007 June 2007 Aug. 2007 Oct. 2007 Dec. 2007

Figure 5 929
930
931
932
933
934
40
60
80
100
120
140
160
180
40 60 80 100 120 140 160 180 200 220 240 260 280
N Relative Concentration
N Relative Content
Relative Dry Weight
N hardening
4_25_35
20_7_19
180
80
100
120
140
160

Figure 6 935
936
937
938
939
0
5
10
15
20
25
30
35
40
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
5
050 100 150 200 250 300
Leaves [N] (mg·g-1)
Plant dry mass (mg)
Plant dry mass
Leaves N
0
5
10
15
20
25
30
35
40
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
5
050 100 150 200 250 300
Shoot [N] (mg·g-1)
Plan dry mass (mg)
N supplied as CRF (mg·seedling-1)
Plant dry mass
Shoot N
Ceratonia siliqua
Pinus halepensis

Figure 7 940
941
942
943
944
8
10
12
14
16
18
20
22
24
30
150
Shoot [N] (mg·g-1)
K rate (mg·l-1)
P= 0.041
A
6
8
10
12
14
30
150
Shoot [K] (mg.g-1)
K rate (mg·l-1)
10 mg·l-1 P
70 mg.l-1 P
P= 0.003
C
4
6
8
10
12
14
30
150
250
Shoot [K] (mg·g-1)
N rate (mg·l-1)
P< 0.001
B
8
10
12
14
16
18
20
22
24
26
28
30
30
150
250
Shoot [N] (mg·g-1)
N rate (mg·l-1)
10 mg.l-1 P
70 mg.l-1 P
P< 0.001
D