Available Nitrogen and Responses to Nitrogen Fertilizer in Brazilian Eucalypt Plantations on Soils of Contrasting Texture

Eucalyptus plantations have seldom responded to N fertilization in tropical and subtropical regions of Brazil. This implies that rates of N mineralization have been adequate OPEN ACCESS Forests 2015, 6 974 to supply tree needs. However, subsequent crop rotations with low N fertilization may result in declining concentrations of organic and potentially mineralizable N (N0), and consequent loss of wood productivity. This study investigated (a) in situ N mineralization and N0 in soils of eucalypt plantations in São Paulo state, Brazil; (b) tree growth responses to N fertilizer applied 6–18 months after planting; and (c) the relationships between N0, other soil attributes and tree growth. We established eleven N fertilizer trials (maximum 240 kg ha −1 of N) in E. grandis and E. grandis x urophylla plantations. The soil types at most sites were Oxisols and Quartzipsamments, with a range of organic matter (18 to 55 g kg −1) and clay contents (8% to 67%) in the 0–20 cm layer. Concentrations of N0 were measured using anaerobic incubation on soil samples collected every three months (different seasons). The samples collected in spring and summer had N0 140–400 kg ha −1 (10%–19% total soil N), which were best correlated with soil texture and organic matter content. Rates of in situ net N mineralization (0–20 cm) ranged from 100 to 200 kg ha −1 year −1 and were not correlated with clay, total N, or N0. These high N mineralization rates resulted in a low response to N fertilizer application during the early ages of stand growth, which were highest on sandy soils. At the end of the crop rotation, the response to N fertilizer was negligible and non-significant at all sites.


Introduction
Establishment of a forest plantation using the Eucalyptus genus for pulp and paper production and other products is justified by their high productivity across different soils and climates.However, sustainable wood production might be compromised in the short-or long-term when plantations are established on soils of low fertility, such as Oxisols and Quartzipsamments [1,2].Productive plantations with high capacity for nutrient extraction can greatly impact N pools in the soil.Adequate management practices, including fertilizer application, are required to sustain tree growth rates and ensure soil quality over successive rotations [3].
Although trees require large amounts of N, some researchers found that eucalypts did not respond to N fertilizer application under tropical and subtropical climate conditions [1,4,5].Commonly, N fertilizer application enhances the early growth of a eucalypt stand, but the response is not continued through the entire rotation [3][4][5].The lack of response possibly occurs due to significant mineralization rates of organic N [3,6] and to atmospheric N deposition [7][8][9][10].Mineralization of organic N is the main natural N source for plantations and appears to be sufficient to meet tree demand [3,11], which in southeast Brazil ranges from 20 to 50 kg ha −1 year −1 [3,6].However, intensively managed eucalypt plantations are expected to respond to N fertilizer application following several crop rotations because of high N outputs via harvesting [3,6,12], low N fertilizer rates applied [1,13], and depletion of organic N pools in soil [3].Some studies carried out at sites with low soil organic matter (SOM) and N status showed that eucalypt stands might respond to N application under this condition [14,15].Hence, the need for N fertilization in Brazilian plantations should be examined each rotation across a range of soil types.
The complexity of determining N fertilizer recommendations is attributed to the difficulty in accurately predicting the supply of available N (N mineralization).Gonçalves et al. [2] described a recommendation index for N fertilizer application to eucalypt plantations in Brazil based on SOM content.Although SOM is the main source of N, such an indicator does not consider the effect of SOM quality, climate and forest management [16,17].
The definition of a useful index of N availability is one that is practical, and chemical analyses can be faster than biological assays [18,19].Several laboratory methods have been proposed to estimate potentially mineralizable N (N0) in soils, and the anaerobic incubation method appears promising [11,20,21].
To better understand the interactions of N supply and tree growth at this stage of eucalypt plantation development in southeast Brazil,, this study investigated (a) in situ N mineralization and N0 in soils of eucalypt plantations in São Paulo state, Brazil, (b) tree growth responses to N fertilizer applied 6-18 months after planting, and (c) the relationships between N0, other soil attributes and tree growth.

Site Description
We selected eleven Eucalyptus grandis and Eucalyptus grandis × urophylla sites in São Paulo state, Brazil, ranging from 1 to 11.4 years old.The sites belong to industrial companies and to the Itatinga Experimental Station of Forest Sciences, University of São Paulo and are representative of the majority of soils, climates and plantation management in this state (Figure 1).The climate is Cwa, according to Köppen classification at Altinópolis site; Cfa at Agudos, Angatuba, Capão Bonito, São Miguel Arcanjo and Votorantim sites; and Cfb at Botucatu, Itatinga and Paraibuna sites [22].Cwa is a humid subtropical climate with a dry winter and a hot summer.Cfa is a humid subtropical climate without a dry season and with a hot summer.Cfb is a humid subtropical climate without a dry season and with a temperate summer.C climate types have an average annual temperature below 18 °C.Cw climates have rainfall of the driest month (RDRY) more than 40 mm, while the Cf climates have RDRY less than 40 mm.Average annual rainfall at the studied sites ranges from 1170 to 1517 mm (Table 1).Soils in the municipalities of Itatinga, São Miguel Arcanjo and Paraibuna are Typic Hapludox (Red-Yellow Latosol); at Altinópolis, Angatuba and Botucatu, Typic Quartzipsamment (Quartzarenic Neosol); at Agudos and Capão Bonito 2, Typic Hapludox (Red Latosol); at Capão Bonito 1, Typic Hapludox (Yellow Latosol); at Votorantim, Typic Paleudult (Red-Yellow Argisol); and at Capão Bonito 3, Typic Dystropept (Dystrophic Cambisol) [23].These are the main soils used in forest plantations in São Paulo State [2].Further soil details of Itatinga and Capão Bonito can be found in Gonçalves et al. [24] and Alvares et al. [25], respectively.The contents of SOM ranged from 18 to 55 g kg −1 and clay contents from 8% to 68% in the 0-20 cm layer (Table 2).They are also quite acidic with pH 3.9-4.9.At each site, we used a randomized block experimental design with three replicates.Measured plots were 10 m by 10 m, with 36 trees per plot, surrounded by a two-tree border.The treatments were: (i) control (low N dose of fertilizer application, sufficient to provide good establishment of plants; c. 15 kg ha −1 ); (ii) usual dose (N dose used by the companies; c. 100 kg ha −1 ); and (iii) increased dose (100% higher than commercial doses; c. 200 kg ha −1 ).The N fertilizer application schedule was 25% of total dose at planting, and the rest applied equitably at 6, 12 and 18 months after planting.The fertilizer application after planting was placed in a continuous line under canopy projection.Additional to N fertilization, all treatments received 30 kg ha −1 of P, 130 kg ha −1 of K, 230 kg ha −1 of Ca, 100 kg ha −1 of Mg, 3 kg ha −1 of B, 60 kg ha −1 of S, 1.5 kg ha −1 of Zn and 0.5 kg ha −1 of Cu.Calcium and Mg were supplied through lime application.Throughout the experiment, the stands were kept free of weed competition by herbicide application.

Growth Assessment
In all plots, we measured diameter at breast height (DBH), total height and tree survival, and estimated the mean annual increment (MAI) of solid wood volume (SV) with bark.The SV was estimated by allometric equations using DBH and height developed by companies specifically for the genetic material used in each plantation.To compare eucalypt response to N fertilizer application in all sites, regardless of the growth increment, relative productivity (RP) increase was calculated as follows (Equation ( 1)).
where: "SV" is solid wood volume with bark of a given treatment and "SVmax" is the solid wood volume with bark of the highest dose treatment.

Soil Analysis
Soil chemical and physical properties were determined for the 0-20 cm layer at all sites.Ten soil samples were collected from each plot, in a diagonal transect across the inner part of the plot between planting lines.The samples were used to make one mixed sample per plot, which was air dried and homogenized.Next, the samples were sieved through a 2 mm mesh and mixed up again.
Particle size analysis (pipette method) of soil was performed according to Embrapa [28].We determined the pH in CaCl2 0.01 mol L −1 , available phosphorus and exchangeable calcium, magnesium, potassium extracted by ion-exchange resins and aluminum extracted by KCl 1 M, according to Raij et al. [29].We assessed soil total carbon (Ct) at all sites in the control treatment (0-20 cm layer) using the South Dakota method with modifications by Raij et al. [29].This method consists of organic matter oxidation by dichromate (K2Cr2O7 + H2SO4), and quantification by colorimetry.Total N (Nt) was determined by the micro-Kjeldahl method [30].

Assessment of N Mineralization
At all sites, soil samples from the 0-20 cm layer were collected to evaluate potential N mineralization under anaerobic conditions in the laboratory (N0).The samples were collected in April (fall), July (winter), November (spring), 2007, and in January (summer), 2008.We performed anaerobic incubations (40 °C for 7 days) using chemical methods according to those proposed by Keeney and Bremner [20].Prior to incubation, 30 mL of nutritional solution, consisting of: MgSO4 (0.002 mol L −1 ) and Ca (H2PO4)2 (0.005 mol L −1 ), was added.Jars were manually shaken until soil dispersion, and then covered with polyethylene film to avoid water loss by evaporation and algal growth [11].N was extracted again 7 days after incubation by adding 4.47 g of KCl to each jar, which provide a concentration of 2 mol L −1 of KCl.Jars were then shaken manually for about 60 s, placed to rest 24 h and then filtered and analyzed for NH4 as described above.To calculate potentially mineralizable N we subtracted the initial concentrations of mineral N.
To measure net N mineralization, the in situ method of Raison et al. [31] was used to in the 0-20 cm soil layer of control plots.Five pairs of steel tubes 30 cm long and 5 cm diameter were placed between tree rows in a diagonal transect across the inner part of the plot.The principle of the method is that inserting a sharpened corer severs roots, and adding capping during in situ incubation excludes rainfall.Net N mineralization thereby proceeds without leaching or uptake.By measuring mineral N initially (in additional representative soil samples) and again after incubation, net ammonification and nitrification can be calculated.For this study, one tube of each pair was immediately removed to assess initial mineral N concentrations (t0) for all plots, and the remaining tubes were covered to avoid leaching of N, remaining in soil for 30 days on average.The amount of mineral N at t0 was subtracted from the mineral N content after incubation to calculate in situ net N mineralization for each 30 day period.The same rate was assumed for other periods of the same season.Soil from each set of five tubes per plot was mixed to obtain a composite sample.The in situ method was used at all sites for January-December 2008.
For each collection, samples at all sites were collected within 8 days of each other to minimize time-related climatic variations between sites.The tubes were transported vertically to the lab, to minimize disturbance, and they were kept in thermal boxes (2-5 °C), individually wrapped in plastic bags.Refrigeration was used to minimize microbial activity, reducing mineralization that could occur prior to mineral N extraction [32].Soil samples remained refrigerated until extraction, which was performed within two days of each collection.
For initial N (t0) extraction, 10 g of soil were placed in 110 mL jars and 100 mL of KCl 2 mol L −1 added.Jars were manually shaken for c. 60 seconds for soil dispersion, and placed to rest for 24 h.Afterwards, the suspension was filtered through Whatman No. 42 filter paper, and the filtrate was analyzed to for NH4 + and NO3 − , following the addition of 0.1 mL of microbial inhibitor (mercury acetate phenyl 0.5 mg L −1 ).
For both N0 and in situ N mineralization, NH4 + and NO3 − concentrations were determined using the Analyze System of Injection in Automatic flow-ASIA (Ismatec, Glattbrugg, Switzerland) [33] and detected by colorimetry at 605 nm, which has a detection limit of 0.01 µg mL −1 .

Data Analysis
Data were checked for normality (Shapiro-Wilk) and homoscedasticity (Box-Cox).ANOVA was used to assess the significance of differences between means.Where there was a significant (p < 0.05) F test, a Tukey test (5% probability) was used for contrasting means.The relationship between dependent and independent variables was assessed by Pearson correlation and linear regression.

Results
Net N mineralization in situ rates ranged from 4.8 to 11.5 kg ha −1 month −1 (fall and winter) and from 10.6 to 15.0 kg ha −1 month −1 (spring and summer) for sandy soils, from 7.4 to 15.2 kg ha −1 month −1 (fall and winter) and from 11.0 to 17.6 kg ha −1 month −1 (spring and summer) for loamy soils, and from 3.6 to 19.1 kg ha −1 month −1 (fall and winter) and from 9.4 to 24.3 kg ha −1 month −1 (spring and summer) for clayey soils (Table 3).The average rates of N mineralization were highest in clayey soils, ranging from 110 to 207 kg ha −1 year −1 , and the lowest to sandy soils, ranging from 107 to 140 kg ha −1 year −1 .The average ratio of N-NH4 + /N-NO3 was 1.6 for sand soils, 1.7 for loamy soils and 1.9 for clayey soils.
At all sites, tree survival was higher than 95%.On average, nitrogen fertilizer application resulted in an increase in MAI of 14% at early age, 6% at intermediate age and 0% at the end of the crop rotation (Table 5).Relative production (RP) at early age of control treatment ranged from 74% to 98% (average of 87% ± 2%), and from 90% to 98% (average of 95% ± 1%) at intermediate age.In the treatment that received commercial fertilizer application, RP ranged from 88% to 111% (average of 100% ± 2%) at early age, and from 98% to 107% (average of 100% ± 1%) at intermediate age.After five years of age (approximate harvesting age), RP ranged from 99% to 103% (average of 102% ± 1%).
The relative wood volume response to N application was greater in sandy soils, and, in absolute terms, at soils with higher clay content (Table 5 and Figure 2).During the first two years of age, RP of the control was about 16% lower than in the treatments that received commercial fertilizer application at sandy soils, and 9% to 10% at soils with higher clay content.Greater relative response to N application at early age occurred in soils with lower Nt, N0 and clay contents (Figure 2).At the end of the rotation, regardless of soil texture, no significant responses to N application were found.

N mineralization
Higher rates of N mineralization in clayey soils are attributed to higher stocks of organic N, due to the greater net primary productivity of the ecosystem, and to higher amounts of soil organic-mineral complexes (Tables 3 and 4).Under this condition, microbial activity increases due to higher amounts of substrate [34,35] and water availability.Eaton [36] reported that after two days of intensive rains, clayey soils in subtropical forests showed a significant increase in microbial C and N-NH4 + mineralization rates, compared to sandier soils.The author speculated that part of the active organic matter pool became detached from soil clay particles, and thereby became available to microorganisms.
Low rates of nitrification (Table 3) might be caused by high acidity and low fertility in these soils, and high NH4 + uptake under high growth rate stands could restrict substrate availability for nitrifying bacteria [3,[37][38][39].Under high acidity and low soil fertility, nitrifying bacteria (Nitrobacter and Nitrosomonas) have impaired active.Higher NH4 + than NO3 − availability is usually not a limitation for eucalypt nutrition [40,41].Gonçalves and Carlyle [39] studying N mineralization in Pinus radiata plantation soils under laboratory conditions reported that, despite the rate of nitrification during incubation, it was not proportional to the reduction of NH4 + concentration, showing the possibility of NH4 + immobilization and/or denitrification caused by soil moisture variations.Gonçalves et al. [11] found similar values of N0 in eucalypt stands ranging from 50 to 249 mg kg −1 of soil (average of 111 ± 23 mg kg −1 of soil).Variations in N mineralization rates under laboratory conditions due to season of sampling were also found by Adams and Attiwill [42], Khanna [43], Smethurst and Nambiar [38], and Theodorou and Bowen [44].Those authors also found higher N mineralization rates during temperate summers coinciding with warm, moist soils.For Theodorou and Bowen [44,45], seasonal fluctuations in mineral N availability were related to microbial activities in the soil, which are mostly affected by temperature and soil moisture [39].When incubating a soil sample collected during summer, a higher microbial population is also incubated, leading to a higher mineralization rate.N0 contents were highly correlated with Nt (r = 0.92; p < 0.0001), but less so with SOM (r = 0.69; p = 0,0192) and clay contents (r = 0.83; p = 0,0015) (Figure 3).This confirms that soil total N is a good indicator of potential N mineralization, as also found by Pottker and Tedesco [46] and Noble and Herbert [47].However, N0 was only weakly correlated with annual N mineralization in situ (r = 0.35), and the latter was not correlated with Nt, SOM, or clay content.
The Nt in this study accounted for 3%-5% of SOM.That ratio decreased with increased clay content (Figure 4).Therefore, there may be greater proportional N availability in soils with lower clay content.High N mineralization in sandy soils is related to better soil aeration and less clay protection of SOM.In absolute terms though, soils with higher clay content have more potential availability of N, because Nt stocks are higher.The C/N ratio ranged from 12 to 24 (average of 19 ± 4) (Table 4).Maquere et al. [48], Montero [49] and Lima et al. [50] found higher ratios in eucalypt stands compared to other native vegetation.This explains in part the larger recalcitrance of SOM in eucalypt stands.N release slows as N mineralization proceeds, since reserves of labile N decrease along with microbial activity.This effect is unfavorable to plant nutrition in the short-term, but beneficial for N conservation in the long-term because it decreases N losses by leaching and volatilization [37,51,52].The C/N ratio across sites was inversely correlated with N0 (r = 0.66, p = 0.036), confirming that the more recalcitrant SOM has lower potential N availability.Although not quantified in the current study, temporal changes in C/N ratios at each site can be expected [53], whereby these ratios decrease during the early age of a rotation along with specific rates of mineralization (i.e., rates of N mineralization per unit of carbon).In sandy surface soils (0-15 cm depth) supporting Pinus radiata stands in southeast Australia, the specific rates of N mineralization decreased by more than 50% (from 207 to 93 g N month −1 t −1 C) during the first four years after planting as C/N ratios also decreased in both the <2 mm and >2 mm soil fractions [53].For unsieved soil, the C/N ratio decreased from 38 to 31 during this period.This slow down in specific rate of N mineralization might reflect a decrease in the labile pool of organic N and organic matter quality.Such changes can be expected in any stand prior to replenishment of this labile pool as organic matter and nutrient cycling is restored by above-and below-ground litter production later in the crop rotation.Hence, the relationship C/N ratio and N mineralization depends on poorly understood temporal and spatial factors of SOM quality.

Nitrogen Fertilizer Application Response
The response to N fertilizer application only occurred at an early stage of tree growth, when the canopy was in formation.For eucalypt stands in Brazil, around 70%-80% of N accumulates in aboveground components during the first two or three years of growth [1,54].At this stage, N relative buildup is higher than biomass accumulation, due to formation of N-rich components (mainly, leaf and fine root), and N released from SOM by mineralization might not be enough to meet the N demand of trees [2].After three years of age, competition among trees for water and light intensifies [55] and tree growth rates decline, leading to reduced N demand that is mostly supplied by N released through litter decomposition (biogeochemical cycling) and internal transfers (biochemical cycling) [1,54].Gonçalves et al. [3] found a biochemical cycling of 54 kg N ha −1 year −1 and biogeochemical cycling 42 kg N ha −1 year −1 in E. grandis stands at seven years old, which are higher than the amounts required by trees (50 kg N ha −1 year −1 ).Therefore, during early growth, N fertilizer application may accelerate tree growth rates by increasing N availability when N mineralization rates in soil and litter do not supply enough N to highly demanding trees.However, these responses disappear in subsequent years under the conditions examined here.
The mineral fertilizer requirements of any plantation depend on the nutrient demand required to reach an expected productivity, and the ability of the soil to supply this demand.When plant demand is greater than soil supply, fertilizers must be added.Thus, the criteria for N fertilizer application should involve only situations where response to N fertilizer exists, because fertilizer application aims to fill the deficit of N not released by soils.Fertilizer application practices must be linked with conservative methods of management to minimize N losses from the system, and thereby increasing sustainability [3].

Figure 2 .Table 3 .
Figure 2. Relative productivity (RP) of the control treatments in relation to the usual and increased N rate treatments.(a) at young ages (less than two years) and (b) later ages (more than five years) at all sites, grouped according to soil texture.SeeTable1 for x-axis site codes.

Figure 4 .
Figure 4. Correlation between clay content and N0/Nt ratio across all sites.

Table 4 .
Seasonal values of potentially mineralizable N (N0), total C (Ct), total N (Nt), C/N ratio and N0/Nt ratio at each site grouped by texture class.

Table 5 .
Solid wood volume with bark (SV) and mean annual increment (MAI) in the different treatments and ages at each site.
1Values in the same column and site followed by the same letter do not differ statistically by Tukey mean test (p = 0.05).