RETRACTED ARTICLE: The effect of soil nutrients and moisture during ontogeny on apparent wood density of Eucalyptus grandis

Knowledge of the effect of soil nutrients, such as K and Na and their interaction with water availability, on the growth and wood properties of the eucalypts, is needed to increase the productivity of commercial plantation forests in Brazil that generate employment and taxes. The present study evaluates the apparent wood density (at 12% wood moisture) of Eucalyptus grandis trees at 12, 24, 36 and 48 months old under ambient and lower than ambient (66%) rainfall conditions and K and Na nutrient enrichment. The treatments were two water availability (100 and 66% of the rainfall) and the three nutrient treatments were: K (4.5 kmol/ha), Na (4.5 kmol/ha) and a control (natural conditions). The apparent wood density of samples at breast height (1.3 m) was determined by X-ray densitometry and digital images. Increased nutrients at all four ages and water availability at 36 and 48 months reduced apparent wood density in E. grandis trees, however, effects of nutrients are lower under water availability reduction. The radial profile of wood density was higher in four-year-old trees, but there was radial variation in apparent wood density at all ages. These findings predict that, under water stress, apparent wood density will not decline in commercial E. grandis plantations fertilized with potassium. The use of sodium, as a substitute of potassium, should consider their negative impacts on wood density of Eucalyptus grandis trees.

In Brazil, eucalyptus species are usually cultivated in soils with low fertility, potassium and other minerals scarcity and water deficit 1 . The expansion of forest plantations in the country depends on the understanding of the interaction between minerals, such as sodium and potassium, and water availability, and their effect on the seasonality of tree growth and wood quality.
The total or partial replacement of potassium by sodium in forest plantations has practical and scientific interest 2,3 . Potassium sources, with sodium in their composition, require less energy in the fertilizer production and, therefore, reducing its final prices. These minerals affect the tree water balance and increase water use efficiency under water stress and improve drought resistance 1,4 .
Because wood is a product of tree growth, factors that affects the growth rate can also affect wood anatomy and therefore wood density and other physical properties. Thus, the application of silvicultural traits in a changing environmental conditions have consider the potential impacts on wood quality.
As well as volume productivity, the wood properties are important factors in forest management, since its affects the transformation process and quality of wood products. In pulp and paper industry wood density is an important attribute for yield and quality of pulp and paper products 5,6 .
However, the effects of mineral fertilizers in wood density of young eucalyptus trees is poorly studied and with controversial results, indicating an increase 7 , decrease or no effect on apparent wood density with moisture content between 12 and 15% 8 . The mineral fertilization effect on the anatomical, physical and mechanical properties of the wood of eucalyptus trees with high growth rates needs further study 9  Climate change and increased eucalyptus stands have reduced water availability. Water stress can be alleviated with silvicultural practices such as proper fertilization. The tree growth under water stress is usually studied in controlled environment 10,11 to evaluate the wood quality and, specifically, its apparent wood density ratio between mass and volume, at 12% wood moisture 12 . Destructive methods do not allow a precise evaluation of small specimens, such as the density between growth rings. On the other hand, non-destructive methods, such as radiographic X-ray, can determine the apparent wood density quickly, with greater accuracy and efficiency in data processing [13][14][15] . X-ray densitometry characterizes wood quality in terms of its apparent density and has been used to evaluate the effect of eucalyptus tree deterioration in response to white-rot fungi, detecting core-sapwood limits and effect of forest management on the wood properties, annual biomass production and relation with wood anatomical structure 16,17 . The apparent wood density of E. grandis, from two to 20 years-old, was 0.46 to 0.80 g/cm³ in the pith and bark, respectively 16,18,19 .
Digital radiography uses X-ray densitometry to obtain the image of the internal wood structure in relation to its chemical composition, density and moisture content. This methodology accurately detects intra and inter-growth ring variations and can be used to interpret tree growth response to change in climatic conditions, fertilization and other silvicultural practices, and carbon fixation on wood quality 13,[20][21][22] .
The wood density is related to mechanical properties 23,24 , dimensional stability 25 , water flow 26 , carbon fixation 27 , and climate change 28 . Hence, precise methods for its determination, such as X-ray densitometry, are becoming more important.
Given the widespread practice of incorporating high amounts of K during the fertilization of Eucalyptus plantations in water-deficit regions 29 and the potential use of Na as a substitute for K 1 , a split plot experiment of 34% rainfall exclusion and fertilization levels of K and Na, was initiated in 2010 in Itatinga, Brazil, to evaluate the interactive effects between K/Na fertilization, water availability, and stand age, on tree growth and wood properties. Here, the objective was to evaluate the effects of K and Na fertilization under water reduction and non-water reduction conditions on the apparent wood density (at 12% wood moisture) using radiographic method, in E. grandis trees from 1 to 4 years after planting.
We intend, therefore, to answer the following questions: How do additions of K and Na to tree fertilization affect apparent wood density? How do effects of K and Na supply on wood properties change under rainfall reduction? The effects of fertilization and water regimes change according tree age?

Results
The apparent wood density (at 12% wood moisture) in the first two years of evaluation was similar between treatments (nutrition and water availability). In the first year, Na nutrition decreased significantly the apparent wood density only under normal water availability (Na/+R). At 24 month old, differences were not significant. K and Na nutrition decreased significantly mean apparent wood density at 36 and 48 months only under normal water availability. Under conditions of rainfall reduction, effects of K and Na on apparent density were no significant over time. The reductions of wood density were higher in Na than K treatments. A reduction of water availability increase significantly wood density only under K or Na nutrition ( Table 1).
The minimum and maximum apparent wood density with 12, 24 and 36 months old was similar between treatments and ranged from 0.19-0.39 to 0.61-0.82 g/cm³, respectively. At 48 months old, minimum and maximum apparent density reached lowest values under K and Na nutrition, respectively, regardless of water availability (Table 1).
In absence of significant interaction between nutrition and water availability in each age, in general, the results indicated that K and Na nutrition decreased significantly apparent wood density from 24 to 48 months old ( Table 2). These effects were stronger in Na nurtured trees. Likewise, reduction of water availability increased significantly apparent wood density.
The first distinct growth ring, formed in the 24th month and representative of the cambial age of eucalyptus trees, was identified in the wood cross section (Fig. 1). The apparent wood density, formed at 24, 36 and 48 months varied in this cross section under rain reduction, between 85 and 100% of its radius.
The radial variation of the apparent wood density showed a same pattern in all ages and treatments lower density (0.35-0.50 g/cm³) in the region near the pith with stabilization in the intermediate region (0.50-0.55 g/cm³) and increase in the external region wood, near to the bark (0.55-0.70 g/cm³). However, radial profiles showed a more homogenous wood density along radial direction in non-water stressed trees.
The digital images of the wood cross section of the E. grandis trees, with 24 and 36 months, showed distinct fibrous zones formation (growth bands), mainly in the treatments with rain reduction and Na/−A and K/−A The (Fig. 2). The formation of such fibrous zones indicates the presence of false growth rings, that confound dendrochronology studies.

Discussion
Variations in the minimum and maximum apparent wood density are due to the alternation of cell regions with high wall fraction and smaller pore area with those with low wall fraction and large pore area 30,31 . Anatomical and wood formation characteristics varied with the rain exclusion, with changes in fiber and vessel growth due to the lower cambial activity and, consequently, the trunk diameter growth of the trees 3,9,32 mechanical and physiological requirements of the tree development process, represented by increased fiber wall thickness and reduced vessel frequency and diameter, as the mature wood is formed in the trunk of E. grandis and E. grandis × urophylla trees 19,34 . The density increase over time is due to the xylem production with thin cell exchange, called juvenile wood, during the beginning of the secondary growth with more robust cells produced, forming the mature wood 35 .
The desired apparent wood density varies according to its use, therefore, the increase of this parameter in wood due to environmental stress 3,36 and the application of potassium and sodium may be positive or not. The increase in the apparent wood density is due to changes in fiber morphology, such as an increase in the cell wall fraction 8 .
The most homogeneous wood formation with K and Na application was also reported with the potassium application in Eucalyptus sp. stands without water stress 37 . This increases the osmotic potential and cellular expansion and, consequently, the cambium activity, altering the wood anatomical characteristics 7,38 .
The highest density of 48-month-old eucalyptus trees in the control treatment and with rainfall reduction may be related to genetic, environmental and tree age factors. Increased density in regimes with lower rainfall  Table 1. Mean, maximum and minimum apparent wood density (mean ± standard error) of E. grandis trees at four ages in treatments (a) C/+R, without fertilization at 100% rainfall; (b) Na/+R, sodium fertilization (4.5 kmol/ha) at 100% rainfall; (c) K/+R, potassium fertilization (4.5 kmol/ha) at 100% rainfall; (d) C/−R, without fertilization at 66% rainfall; (e) Na/−R, sodium fertilization (4.5 kmol/ha) at 66% rainfall; (f) K/−R, potassium fertilization (4.5 kmol/ha) at 66% rainfall. Means followed by the same letter, per column and age, do not differ by the Tukey test (α = 0.05).  www.nature.com/scientificreports www.nature.com/scientificreports/ was also reported for Eucalyptus sp. trees, being explained by the fiber wall thickness and associated to the plants nutritional state and the water availability 39,40 . The trees synthesize more simple sugars by photosynthesis with increasing cambial activity and cellulose biosynthesis and these molecules are incorporated as microfibrils, thickening the secondary wall 37,41 .

Figure 1.
Radial profile of the apparent wood density at breast height diameter of the E. grandis tree trunks at 48 month in the treatments (a) C/+R, without fertilization at 100% rainfall; (b) Na/+R, sodium fertilization (4.5 kmol/ha) at 100% rainfall; (c) K/+R, potassium fertilization (4.5 kmol/ha) at 100% rainfall; (d) C/−R, without fertilization at 66% rainfall; (e) Na/−R, sodium fertilization (4.5 kmol/ha) at 66% rainfall; (f) K/−R, potassium fertilization (4.5 kmol/ha) at 66% rainfall. www.nature.com/scientificreports www.nature.com/scientificreports/ The more variable apparent wood density of eucalyptus trees in water stress treatments, regardless of nutrition, is due to the wall fiber thickening near the bark due to the smaller cambium activity with osmotic potentials and, consequently, lower cell expansion.

R E T R
The cambial activity variation rate in response to climatic conditions explains the radial profile of the apparent wood density due to the increase in this parameter in the pith to bark direction and juvenile wood formation with intra-and inter-annual density variation 16,42,43 . The densitometric profile of eucalyptus trees at four ages differed www.nature.com/scientificreports www.nature.com/scientificreports/ from that obtained for adult trees (five to seven years) and can be explained by mechanical and physiological requirements resulting from the tree development process with increased fiber wall thickness and reduction of the pore frequency and diameter, comparing juvenile and mature wood in eucalyptus trees 16,18,34 .
The different contrasts in the E. grandis wood at four ages in the treatments are due to the transverse dimensions (width, lumen diameter and wall thickness) of the wood tissue cells and the attenuation intensity of X-ray bundles that run longitudinally in wood sample every 50 μm 44 . The high precision of the images shows a darker coloration due to the smaller attenuation of the X-ray bundles in the longitudinal and radial parenchyma and the lumen cells of the vessels (mainly) 20 . The characteristic white spots in the cells of the longitudinal parenchyma indicate the presence of tilose at higher density, filling its lighter color with limits defined by the high X-rays attenuation in the reading process. The light tissue coloring (fibers) indicates narrow fibers with thick walls and reduced lumen. The vessels obstruction by tilose, gums, etc. reduces the treatability, permeability and capillary movement of water in the wood and the drying processes 45 .
In the treatments without rain reduction, the digital wood images by X-ray irradiation make it possible to demarcate and measure the width of the growth rings, being an important tool for dendrochronology 11 . In treatments with rainfall reduction, the false ring formation does not allow to count the rings with precision, resulting from the water stress to the plants with fibrous zones formation in more than one period per year, making dendrochronology studies difficult 37,46 .
The mean apparent wood density at 12% wood moisture of E. grandis trees was lower with mineral nutrition in all ages and 100% rainfall at 36 and 48 months. The wood apparent tree density at 12 and 24 months was similar in treatments with water availability and nutrition. The apparent wood density of trees in the 48 th month was greater with reduced water availability. The eucalyptus wood trees should be continuously evaluated to detect changes in their anatomical structure, density and other properties in K and Na fertilization situations, and water restriction to characterize wood for different uses. The effect of silvicultural practices in forest plantations with water stress and submitted to nutrition with K and Na can be evaluated with digital X-ray images.
In normal conditions of water availability our results indicate an detrimental effects of K and Na nutrition in wood density, mainly in older trees. In the context, which the increase in planted area and climate change will increase water scarcity in many water-limited regions, the effects of fertilizers on wood density of E. grandis could be less severe. Effects of K nutrition are negligible and Na nutrition can slightly decrease wood density. Nevertheless, the beneficial effects of K and Na increasing tree growth, even under a water availability reduction 1,4,26,47 , largely compensate for the loss in wood quality for pulp and paper production. These findings predict that, under water stress, apparent wood density will not decline in commercial E. grandis plantations fertilized with potassium. The use of sodium, as a substitute of potassium, should consider their negative impacts on wood density of Eucalyptus grandis trees.

Methods
Characterization of the area, selection and cutting of trees. The experiment was installed in the municipality of Itatinga, São Paulo state, Brazil (23°00′40.51′′S and 48°44′10.92′′). The climate of the region is humid mesotherm (Cwa), according to Köeppen, with rainfall and average annual temperature of 1,635 mm and 16.2 °C and 28.6 °C, in the colder and hotter months, respectively. The soil is of the type dystrophic yellow red latosol with medium texture and lithology composed by sandstone, Marília formation, of the Bauru group. The soil chemical attributes, in the implantation of the experiment, were characterized up to six meters deep 26 . The cation exchange capacities in the soil were 0.02 cmolc.kg −1 to 5 cm depth and 0.01 cmolc.kg −1 between 0.05 and 6 meters depth, demonstrating severe K and Na deficiencies throughout the profile.
The experiment was implemented in May 2010 with E. grandis clone spaced 3 × 2 m. The basic fertilization was done at the planting, with 2,000 kg of dolomitic limestone/ha and fertilized with 75 kg of P 2 O 5 , 80 kg of N (NH 4 (SO 4 ) 2 ) and 20 kg of FTE (BR-12) per hectare in all treatments.
The experiment was developed in subdivided blocks, split-plot type. First, each block contained a different age, next, these blocks were divided by the fertilization type, then, there was a division into two types of water availability, totaling 24 plots. The treatments were: Four different ages (i): 12; 24; 36 and 48 months-old; Three fertilizer doses (ii): 0 (control); 4.5 kmol/ha and 4.5 kmol/ha in KCl and NaCl form, respectively, applied three months after planting and two water availability (ii): 100% and 63% of rainfall (artificial exclusion by soil cover with 1,700 m² of clear polyethylene tarpaulins). The six treatments were identified as follows: (a) C/+R, control and 100% rainfall; (b) Na/+R, sodium nutrition and 100% rainfall; (c) K/+R, potassium nutrition and 100% rainfall; (d) C/−R, control and 63% rainfall; (e) Na/−R, sodium nutrition and 100% rainfall; (f) K/−R, potassium nutrition and 66% rainfall.
The change in water availability was conducted to reduces 37% of rainfall, installing gutters with plastic sheeting (width 40 cm) supported by wires and covering 37% of the total soil surface (Fig. 3). These gutters were supported on wood cuttings (height from 1.5 to 0.30 m) in slope to withdrawal the rainwater, preventing the arrival of water in the soil. Leaves and branches, retained in the plastic canvas, were removed periodically.
Four eucalyptus trees were selected per treatment (nutrition and water availability) at 12 th , 24 th , 36 th and 48 th months old, totaling 96 trees to determine their apparent wood density (at 12% wood moisture). The basal area of the trees selected was similar to that of the plantation.
Radial profiles of apparent wood density. Four wood discs were sectioned at 1.3 m (DBH) of the trees selected at the four ages analyzed. Samples (20 × 10 mm, width x thickness) were cut from these disks, glued on a wooden support and a 2.0 mm thick sample sectioned radially in parallel double circular sawing equipment. The radial sections were conditioned in an air-conditioning chamber (20 °C, 60% relative humidity and 12% wood moisture) for twenty four hours 13 .