BIOMASS OF TWO Eucalyptus CLONES (E. grandis × E. urophylla) IRRIGATED WITH SALINE WATER

Using saline water for irrigation relies on strategies that include selecting salt-tolerant cultivars and leaching salts away from zones close to the roots. A greenhouse experiment was carried out to assess early growth and biomass production of two Eucalyptus clones (E. grandis × E. urophylla), CO 865 and CO 1407, irrigated with saline water and under different leaching fractions. Treatments were laid out in a randomized block design and arranged in a 4 × 2 + 2 factorial scheme: four leaching fractions (3, 10, 20, and 30 % of crop water demand for plants irrigated with saline water), two Eucalyptus clones (VCC 865 and CO 1407), and two additional treatments, one for each clone, conventionally irrigated with fresh water. The treatments were replicated four times. Measurements were made at 114 days after transplanting. Soil salinity decreased with increasing leaching fraction where VCC 865 was grown; however, leaf dry weight production was lower in treatments irrigated with saline water. Compared to fresh water-irrigated plants, irrigation with saline water resulted in lower: canopy diameter, leaf number, dry leaf mass, dry root mass, aerial parts dry mass, aerial part/ root ratio, and total plant dry weight. Overall, the VCC 865 Eucalyptus clone performed better under saline irrigation than CO 1407.


INTRODUCTION
Farming faces a major challenge to meet the needs of a growing global population, expected to reach 9.8 billion by 2050 (FAO, 2017;Calicioglu et al., 2019). Major obstacles to accomplishing this goal are water scarcity and salinity (Deinlein et al., 2014;Gorji et al., 2020). High soil salinity affects over 20 % of arable lands around the world and this percentage is rising (Gupta and Huang, 2014). Soil salinity constrains plant production, especially in arid and semi-arid regions (Ashraf and Wu, 1994;Lopes and Klar, 2009;Leksungnoen and Andriyas, 2019;Yang et al., 2020) where evapotranspiration rates always exceed rainfall over a crop year (Hanin et al., 2016). Improper practices, such as overusing fertilizers and irrigation with saline water, may further increase soil salinity.
High salt levels in the soil not only negatively affect its fertility but also its physical properties (Mohamed et al., 2018). As infiltration and storage of water in the soil decrease, runoff and erosion increase (Daliakopoulos et al., 2016), which also has a negative impact on soil biodiversity (Singh, 2016). Therefore, soil salinity is an important abiotic stress that reduce plant growth and yield (Yasar et al., 2016;Afridi et al., 2019). In the soil, sodium chloride (NaCl) is a common salt that adversely affects plant growth through water stress and excessive uptake of Na + and Cl - (Tester and Devenport, 2003;Isayenkov and Maathuis, 2019). These ions also disturb the ionic balance within plant tissues, resulting in lower nutrient uptake; however, plant responses to salinity stress vary with plant genotype, as some species are tolerant to salinity and others are highly sensitive to it (Hanin et al., 2016).
With 5.6 million ha cultivated with Eucalyptus, Brazil is a major producer of pulp and wood (ABAF, 2015). Soil salinity affects many trees stand in Brazil, thus the identification of Eucalyptus species that are tolerant to high-salinity conditions is highly important because the Eucalyptus response to salinity varies across species or even across individuals of the same species (Daas-Ghrib et al., 2011;Bush et al., 2013). E. camaldulensis (Marcar, 1993;Rawat and Banerjee, 1998;Su et al., 2005) and E. tereticornis (Marcar, 1993;Sun and Dickinson, 1995;Tomar et al., 2003) have been reported to be tolerant to salinity, but there is little information on salinity stress in E. pellita (Mendonça et al., 2007). Specific adaptive traits of Eucalyptus species are important in breeding programs aimed at producing hybrids with desirable traits. For example, by crossing E. grandis with E. camaldulensis, breeders could produce a hybrid that grows fast and yields high quality wood, both traits inherited from E. grandis, and is tolerant to low water and nutrient supply, a trait inherited from E. camaldulensis (Pereira et al., 2019).
By reviewing the literature, little information about soil salinity-reducing techniques and/or irrigation management lacks for Eucalyptus species under saline water irrigation, as well as the response of different clones of the same Eucalyptus species under irrigation with saline water. We hypothesized that: (i) the early response of a Eucalyptus species to saline water irrigation depends on the clone; and (ii) leaching fractions alleviates salt-induced stress. The objectives were to evaluate vegetative growth and biomass production of two Eucalyptus clones, VCC 865 and CO 1407 (E. grandis × E. urophylla), irrigated with saline water and to identify the most suitable leaching fraction to offset salinity effects on the plants.

MATERIALS AND METHODS
A greenhouse experiment was carried out at the Universidade Estadual do Sudoeste da Bahia, located in southwestern Bahia State, Brazil (14°51'LS, 40°50'LW, and 876 m altitude). The experiment was conducted using 50 L plastic containers, which served as miniature drainage lysimeters. The drainage system consisted of a hose measuring 40 cm in length and 16 mm in diameter attached to the bottom of the mini lysimeter. The other end of the hose was attached to a 2-L plastic bottle for collecting the drained water.
A eucalypt plant was grown in each mini lysimeter, which represented an experimental unit.
Two Eucalyptus clones, VCC 865 and CO 1407 (E. europhylla × E. grandis), were irrigated with saline water using different leaching fractions (LF). The experimental design was randomized blocks with treatments arranged in a 4 × 2 + 2 factorial scheme with four replicates: four leaching fractions (3, 10, 20, and 30 % of the crop demand with saline water), two clones (VCC 865 and CO 1407), and two additional treatments, one for each clone, conventionally irrigated with fresh water. Saline water had electrical conductivity (EC) of 2.5 dS m -1 and fresh water, 0.31 dS m -1 .
Soil water balance was calculated to determine the crop evapotranspiration (ETc) (Equation 1).
The amount of applied irrigation water for each treatment was calculated using Equation 2.
Electrical conductivity of the drainage water (EC dw ) was measured using a conductivity meter (Micronal, model B330) with which readings were taken from the drainage water. Electrical conductivity of the of saturated soil-paste extract (EC e ) was determined using the standard method, which consisted of separating the extract from the paste using a Büchnere funnel paper filter coupled to a Kitasato flask and a suction pump. Measurements were made in accordance with standards provided by the US Salinity Laboratory Staff (Richards, 1954). The saturated paste was obtained using 300 g of airdried soil to which water was added up to saturation. This was done at both the beginning and the end of the experiment.
To assess the effect of saline water on plant growth, fresh water-irrigated plants were compared to saline water-irrigated ones. Factorial analysis was carried out to determine the responses of saline waterirrigated clones to leaching fractions. Evaluations took place 114 days after transplanting. The collected data were tested by analysis of variance and F-test (p ≤ 0.05) and the means were compared using Tukey's test. Regression analysis was conducted to fit models to the collected data as a function of leaching fractions. The models were chosen considering the significance of the coefficient of determination. The data were further studied by Pearson's correlation (p ≤ 0.05).

Fresh water irrigation: VCC 865 vs CO 1407 and Fresh water irrigated clones vs saline water irrigated clones
Plant height (PH), leaf dry weight (LDW), stem dry weight (SDW), aerial part dry weight (APDW), APDW/ root system dry weight (RSDW), total plant dry weight (TPDW), absolute growth rate (AGR), canopy diameter (CD), leaf number (LN), stem diameter (SD), EC dw , and EC e were statistically similar between the clones irrigated with fresh water. RSDW of CO 1407 was greater than that of VCC 865 by 15.6 % ( Figure 1A). Plant height, SD, AGR, SDW, and APDW/RSDW were not affected by saline water.
The interaction between LF and C affected EC dw and EC e . A quadratic model was fitted to the response of EC dw to LF for both clones. In VCC 865, the increasing curve reaches the maximum value at 13.2 % LF, which corresponded to an EC of 2.4 dS m -1 . In CO 147, however, the curve was decreasing, and the minimum value was at 23.3 % LF, corresponding to 1.6 dS m -1 ( Figure 3C and D). A decreasing straightline function was fitted to EC e values as a function of LF for VCC 865 ( Figure 3E). As for CO 1407, although EC e values decrease as LF increases, no model tested could be fitted to the data. By comparing the EC e values between clones, the difference was significant only at 30 % LF ( Figure 3F).

Correlation analysis of biomass and salinity variables in each clone
Studying how salinity correlates with plant growth biomass parameters revealed a total of 19 linear correlations for clone VCC 865, 14 of which were positive and 5 were negative, and a total of 18 linear correlations for clone CO 1407, all of which were positive (Figures 4A and 4B).
Canopy diameter appears to correlate strongly with biomass parameters in VCC 865 clone. For clone CO 1407, canopy diameter only correlated with the aerial part (p <0.05), having no relationship with the total plant dry weight (p> 0.05).    Stem diameter and LF correlated negatively with EC dw and EC e for VCC 865. However, these variables did not correlate for CO 1407.
EC e correlated positively with EC dw in the mini lysimeters cultivated with VCC 865.
No correlation was observed between EC dw and EC e for clone CO 1407.

DISCUSSION
The greater root system development in CO 1407 might relate to a characteristic intrinsic to the clone. In comparing physiological traits of both clones, Silva et al. (2020) reported that leaf area, photosynthetic pigments, and photosynthesis rates were higher in CO 1407 than in VCC 865.
Osmotic adjustment is the main process by which plants alleviate the salt-induced stress imposed by saline irrigation water (EC 2.5 dS m -1 ) (Lopes and Klar, 2009). Both clones were negatively affected to salinity, which might be associated with the exposure time of plants to saline condition -which lasted 114 days. Conversely, no negative effects were reported for E. urophylla, but the plants were exposed to salinity conditions for only 30 days (Souza et al., 2015).
Elevated soil salinity because of using saline irrigation water negatively affects plant growth by reducing plants' water uptake (decreased osmotic potential) and damaging metabolic and photosynthetic processes due to higher uptake of Na + and Cl - (Mäser et al., 2002;José et al., 2016;Isayenkov and Maathuis, 2019). Particularly, NaCl can negatively impact hormone synthesis and translocation from roots to aerial parts, resulting in decreased leaf area and dry matter (Ferreira et al., 2001). Plant growth may be further affected by the reduction in cell division and elongation due to changes in plant cell wall extensibility (Munns, 2002). In E. urograndis NaCl levels above 4.5 dS m -1 reduced dry matter production in both shoot and roots (Lopes and Klar, 2009). Similarly, Lacerda (2016) reported decreases in dry matter production in leaves and roots of Eucalyptus clones (AEC 144, AEC 1528, VCC 361, and VCC 865). In evaluating responses of Eucalyptus genotypes (E. camaldulensi Dehnh: '169'; E. grandis Hill ex Maiden × E. urophylla S. T. Blake: '5E'; and E. globules Labill: 'Anselmo' and 'Odiel') to salinity validated by the t test at a significance level of 0.05. Squares with an × mean that the correlation was not significant (p > 0.05).

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stress, Sixto et al. (2016) found out that the clones showed a high survival rate even under moderate (50 mM) and severe (125 mM) saline conditions.
The greater plant growth of VCC 865 when irrigated with saline water suggests a better phenotypic adaptation to salinity stress. The wide range of phenotypic responses depends on plant genotype; while some genotypes are tolerant to salinity, others are highly sensitive to it (Hanin et al., 2016). High Na + levels in the soil may impair K + uptake by roots; however, the soil concentration at which Na + has a significant effect on K + uptake depends on crop species (Ferreira et al., 2001) or genotype, as observed in this study. The higher APDW/RSDW in VCC 865 confirms the better adaptation of the genotype to salinity. Lacerda (2016) also reported high APDW/ RSDW ratio for the clone VCC 865.
The decrease in LDW as a result of increasing LF might be due to the fact that some nutrients are more leachable, e.g., K + (Possen et al., 2014). The ratio of APDW to RSDW tends to 1.0 with increasing leaching fraction; values above 1.0 mean that partitioning of nutrients and photoassimilate is imbalanced, leading to reduced plant growth and water uptake (Alfaro et al., 2004;Caldeira et al., 2013). When using a lower leaching fraction, elevated Na + levels in the soil might have induced root damage (Ferreira, 2001), so the growth of aerial parts occurs to the detriment of roots.
In CO 1407, EC dw was lower at 10 and 30 % LF. CO 1407 might have accumulated or taken up higher amounts of salt, which explains the lower growth rate of the clone when irrigated with saline water. When comparing EC dw values of the two clones irrigated with saline water, drainage water collected from mini lysimeters containing VCC 865 clone revealed that less salt was leached (lower EC dw ) at 3 % LF, but more salt was leached at 20 % LF. In average, EC e values increased from 0.2 dS m -1 , before applying saline treatments, to 3.2 dS m -1 , at the end of the experiment. In the mini lysimeters cultivated with VCC 865, the highest EC e values were recorded when using the lowest LF. Similar results were reported for beet and peanut plants (Ferreira et al., 2006;Santos et al., 2012). This shows that using leaching is an efficient way of alleviating the impact of excessive salt in the irrigation water on plant growth. However, this positive response was observed only in the clone VCC 865.
Canopy diameter has a direct relationship with the accumulation of dry weight in the whole plant because the canopy consisted of leaves, where photosynthesis occurs.
Stem diameter and LF correlated negatively with EC dw and EC e for VCC865, i.e., increases in the number of leaves and stem diameter contributed to reductions in the electrical conductivity of the soil and the drained water. However, these variables did not correlate for CO 1407. This might be explained by the influence of saline irrigation on vegetative development of the clone (CO 1407), as shown for the different characteristics in Figure 2. For VCC 865, although salinity affects the accumulation of biomass, this effect is not as pronounced as in CO 1407.
Moreover, EC e correlated positively with EC dw in the mini lysimeters cultivated with VCC 865, that is, despite the increase in soil salinity by saline irrigation (Figure 1B), the largest leaching fractions promoted the reduction of salts in the soil and in the drained water ( Figure 3C and 3E). On the other hand, no correlation was observed between EC dw and EC e for clone CO 1407, probably due to the uptake of salts by the clone.

CONCLUSIONS
Only root dry weight differed between the clones when irrigated with fresh water, with clone CO 1407 outperforming clone VCC 865. However, when irrigating with saline water, both clones had their biomass negatively affected. The negative effects were greater in clone CO 1407, which had lower means for biomass. By using saline irrigation water, soil salinity increases, even when using leaching fraction. Increases in soil salinity are different for each clone. The Eucalyptus clones perform similarly when irrigated with fresh water. However, if saline water is to be used, the results suggest using VCC 865 rather than CO 1407.

AUTHOR CONTRIBUTIONS
All authors made essential contributions to the elaboration of this workThe manuscript is part of the master's thesis of the author OLIVEIRA FS. CASTRO FILHO MN was responsible for and the analysis the writing of the manuscript. OLIVEIRA FS e ALVES RO was responsible for conducting the experiment. Da SILVA BL assisted in the writing and translation of the manuscript. TAGLIAFERRE C was the supervisor teacher professor of the study and contributed to the supervision of the methodology and correction of the work as a whole. De PAULA A and BARROS FM assisted in supervision and review of the work as a whole.