Article Growth and Physiology of senegal (L.) Britton Seedlings as Influenced by Seed Origin and Salinity and Fertility Treatments

Mame Sokhna Sarr 1,2,*, John R. Seiler 1 and Jay Sullivan 1

1 Department of Forest Resources and Environmental Conservation, Virginia Polytechnic and State University, Blacksburg, VA 24061, USA; [email protected] (J.R.S.); [email protected] (J.S.) 2 Centre National de Recherche Forestière of Institut Sénégalais de Recherche Agricole, Dakar 2312, Senegal * Correspondence: [email protected]; Tel.: +221-775-335-483

Received: 2 August 2017; Accepted: 3 October 2017; Published: 11 October 2017

Abstract: Multipurpose trees such as are widespread in arid and semi-arid lands that have natural or induced saline soils and poor soil fertility. Such environmental problems impact growth and have the potential to influence physiological adaptations. Identifying superior genotypes better adapted to these environmental stresses will be of great importance for tree selection for reclamation of degraded drylands. The main objective of this study was to examine the growth performance, and physiological and morphological adaptations to salinity, and fertility treatments of different Senegalia senegal families. We used five families (DB16, DB14, K4B19, K17B19, NB1) selected from 60 families of a Senegalia senegal progeny trial in Dahra, Senegal. Seedlings were grown under greenhouse conditions by watering all for three weeks and then stopping all watering for three more weeks. In a randomized complete block design, a two-level factorial combination was used for salinity (zero and 183.1 mM NaCl added) and fertility (zero and 100 kg/ha N-P-K added) treatments. A significant family × salt × fertilizer interaction was found for all biomass parameters (leaf dry matter, stem dry matter, dry matter, and leaf area). The fertilizer application resulted in a significant increase of total biomass of all families, ranging from 63% to 237% for NB1 and K17B19, respectively. In contrast, salt only decreased total biomass of NB1 and K17B19 increased growth. Despite similar net photosynthetic rates before treatment started, fertilizer and salinity induced different effects between families. Prior to drought stress, fertilizer did not affect photosynthesis of DB16, while salt significantly decreased stomatal conductance of all families. DB16 and N1B1, despite significant differences of stomata size and density, significantly decreased transpiration, and thereby increased their intrinsic water use efficiency. Under drought, relative growth rate was significantly decreased. Given that genotype differences were found, these families and salinity and fertilizer treatments need to be explored in field trials.

Keywords: Senegalia senegal; salt tolerance; fertilizer; drought

1. Introduction In recent years, frequent drought occurrences and anthropogenic pressures on forest resources in arid and semi-arid lands have heavily impacted species composition [1]. The natural vegetation becomes sparse and progressive soil degradation develops, which accelerates desertification and induces salinization [2–4]. Drought and salt tolerant species are seeking for restoration of such affected lands. Senegalia senegal is a drought tolerant species, widely used to control desertification in arid and semi-arid lands. It is particularly exploited because it is the source of many products, including gum arabic which is traded on both local and international markets. Recent studies have revealed its potential tolerance to salinity stress [5–7]. However, salt tolerance among local Senegalia senegal

Forests 2017, 8, 388; doi:10.3390/f8100388 www.mdpi.com/journal/forests Forests 2017, 8, 388 2 of 15 provenances has not yet been investigated. Salt-affected lands in Senegal are estimated at about 1.7 million hectares [8]. Due to increasing population, it is likely that, in the future, tree crops and fuel wood plantations will increase on degraded lands in general and particularly on salt affected soils. In arid and semi-arid areas, salt stress is often combined with water limitations in the context of poor soil management. Afforestation/reforestation plans are often proposed solutions for soil improvement of degraded lands. However, plantation failures can occur when inappropriate species are selected. An understanding of plant response to salinity is of great importance for reclamation of salt affected lands. Yet, strategies to improve salt and/or drought tolerance in such ecosystems require an understanding of tree physiological mechanisms that links their growth and adaptation. Growth characteristics and photosynthesis efficiency are traditionally used to select higher quality family or genotypes more adapted to hostile environments. Stomatal response is among one of the most important factors influencing photosynthetic capacity under limiting environmental factors [9]. Different stomatal characteristics may account partially for different stomatal resistance that consequently impact the photosynthetic rate [10]. However, little attention has been given to the leaf physiology of Senegalia senegal, and particularly stomata characteristics. Under water stress, stomatal density is known to impact leaf photosynthesis and water use efficiency [11,12]. Reduced stomatal density can be advantageous under saline stress [13,14]. Drought and salinity stress both induce a reduced photosynthetic rate through stomata and mesophyll restriction [15,16]. Furthermore, selection of adapted genotypes based on leaf traits and water use efficiency to such environments are highly recommended. This study investigates adaptive features in Senegalia senegal from different families which may allow them to grow better in both saline and dry conditions. The main objective is to examine physiological and morphological changes among Senegalia senegal provenances subjected to salinity and fertility treatments. We tested the hypothesis that salinity tolerance and fertilizer effect on the physiology and biomass accumulation of Senegalia senegal seedlings are influenced by seed origin. We expected that leaf gas exchanges, growth and biomass accumulation will decrease under the effect of salinity whereas the fertilizer may increase those potentials. We are interested in seeking genotypes that perform best in terms of dry matter accumulation and intrinsic water use efficiency under well-watered and then drought conditions, and whether stomatal characteristics explain potential changes among families and environmental conditions.

2. Materials and Methods

2.1. Plant Material Senegalia senegal families from five randomly selected mother trees (seed origin) were used in this study. Seeds were harvested in January 2015 from each of the 5 different trees in a 20-year-old Senegalia senegal progeny trial in Dahra, Senegal. Ngane21B1, Kidira4B19, and Diamenar27B14 maintained a relatively high gum production over 4 successive years while Kidira17B19 and Diamenar27B16 displayed low gum yields for the same period (Table1). Each tree label represents a mother tree, and families within the same name and different numbers belong to the same provenance.

Table 1. Families and mean annual gum yields (g) by tree of the mother trees from which seeds in this study were harvested. (-) indicates that the tree died.

2013 Gum 2014 Gum 2015 Gum 2016 Gum Provenance Families Tree Label Yield Yield Yield Yield Diamenar Diamenar27B16 DB16 29.3 9.7 0 - Diamenar Diamenar27B14 DB14 899.5 791.7 386.1 758.3 Kidira Kidira4B19 K4B19 555.3 551.8 583.3 990.3 Kidira Kidira17B19 K17B19 131.3 62.1 22.2 51.7 Ngane Ngane21B1 NB1 522.2 476.4 945.2 1103.5 Forests 2017, 8, 388 3 of 15

2.2. Growth Conditions, Treatment, and Experimental Design The experiment was conducted from June to October 2015 in a greenhouse where the temperature was maintained above 25 ◦C and ventilated with box fans when temperatures went above 32 ◦C. Light was extended using sodium vapor lights to maintain a 16-h photoperiod. Seeds were chemically scarified with concentrated sulfuric acid (98%) in order to perforate the tough seed coat and then rinsed with water to remove the acid before germinating them in shallow flats filled with Promix BX as a growth medium. Once emerged, young germinals were transplanted individually into small tubes (SC10 super cell, 3.8 cm diameter × 21 cm deep, Stuewe and Sons, Tangent, OR, USA) containing Promix and kept well-watered for about six weeks. They were then transplanted into 2.3-liter plastic pots (Treepots, 30.5 cm height and 10.2 cm width, IGC Greenhouse Megastore, Danville, IL, USA) containing either soil substrate (a 2:1 mix of Promix BX and washed sand) or soil substrate with salt added. A factorial combination of salinity and fertilizer treatments was then established. The salinity treatment consisted of mixing 1070 g of sodium chloride with 100 kg of soil substrate. The salt treatment used was based on an early study which revealed a significant decrease of about 50% on biomass of Senegalia senegal seedlings [17]. The average corresponding electrical conductivity of the saline soil was 7.525 dSm−1, whereas the electrical conductivity of a control soil was 0.31 dSm−1. For the measurement of electrical conductivity, a soil suspension was prepared in distilled water at a ratio of 1:2. The suspension was shaken and allowed to stand overnight [17]. Thereafter, electrical conductivity of the supernatant solution was determined with an electrical conductivity meter (2265FS/2265FSTP, Spectrum Technologies, Inc., Aurora, IL, USA). The fertilization treatment consisted of applying 2.3 g of N-P-K (10-10-10) in each pot to give the equivalent fertilizer supply of 100 kg/ha. The experiment was designed as a randomized complete block with five replications of the 2 by 2 factorial treatments (no salt/no fertilizer (control), salt only, fertilizer only, and salt plus fertilizer) making a total of 100 experimental units. When treatments started, all plants were kept well-watered for three weeks, and then the drought stress treatment was started where all watering was stopped in all treatments for three more weeks. This procedure was adopted to mimic field conditions (alternation between a wet season and a dry season) under which Senegalia senegal is naturally subjected. The induction of drought in this experiment is not considered as a treatment factor, but a common condition subjected to all experimental units. Prior to drought stress, shallow trays were placed beneath pots that received the salt treatment (the salted and salt-fertilized) in order to recover any salt discharge which occurred when pots were watered. Any recovered water was then poured back into each tree pot. This step was to maintain the same salt concentration in each pot prior drought stress.

2.3. Stomatal Density and Stomatal Size Measurements Stomatal density and size were assessed at the end of the experiment before seedlings were harvested by using a stereo zoom microscope SMZ 1500 (Nikon Corporation, Chiyoda-ku, Tokyo, Japan) equipped with a digital camera (Nikon digital sight). A colorless nail polish was coated on the abaxial and adaxial leaflet surfaces from the newest fully mature leaf, then leaflets were mounted on glass slides. Images were taken on both leaf faces at 70× magnification. Stomatal density was expressed as the number of stomata per mm2 and stomatal size reflected the length in micrometers between the junctions of the guard cells for each stoma. Two leaflets per face and per plant were sampled and 2 subsamples of 0.01 mm2 were measured on each leaflet face. The number of stomata for each sample was counted under a photomicroscope system with a computer attachment.

2.4. Leaf Gas Exchanges and Chlorophyll Content Measurements

Measurements of net photosynthetic rate (A), stomatal conductance (Gs), and transpiration (E) of a fully expanded leaf per plant were made using a portable infrared gas analyzer (LI-6400, Lincoln, NE, USA). Environmental conditions in the leaf cuvette were set to 2000 µmols m−2 s−1 photosynthetic Forests 2017, 8, 388 4 of 15

−1 ◦ photon flux density (PPFD), 398 µmols mol reference CO2 concentration, 25 C block temperature, and a flow rate of 400 µmols sec−1. Gas exchange was measured weekly from day 0 (when planted into large pots with fertilizer and salt) for the 6 weeks of the experiment. The intrinsic water use efficiency (WUEi) was calculated as the ratio of photosynthetic to transpiration rates. Total chlorophyll content (mg/g) was estimated at the same time in intact leaves using a portable chlorophyll meter CCM-300 (Opti-Sciences Inc., Hudson, NH, USA). Three leaflets per plant were randomly chosen from new fully mature leaves and an average taken for analysis.

2.5. Relative Growth Rate and Biomass The relative growth rates of height (RGH, cm cm−1day−1) and diameter (RGD, mm mm−1day−1) were determined [18], where RGR = (ln X2 − ln X1)/(t2 − t1), t is the number of days, X is either plant height or diameter, ln X is the natural log of height or diameter. All plants were harvested at the end of the experiment. We manually washed soil from and then separated leaf, stem, and root from each other. Root area from each plant was measured by using WinRHIZO Software (version 2005b Pro, Regent Instruments Inc., Québec, Canada) and an EPSON Expression 1680 scanner (Epson America Inc., Long Beach, CA, USA). The area of leaves for each plant was determined with Photoshop software CS6 (version 13.0.1, Adobe Systems Inc., Mountain View, CA, USA). Different plant parts were then dried at 65 ◦C to a constant weight before weighing them to determine dry matter.

2.6. Statistical Analysis Data were analyzed using JMP software (version 12, SAS Institute, Cary, NC, USA). Normality and constant variance were examined across all data. We used a log (natural) transformation of the root/shoot ratio for analysis to meet assumptions of analysis of variance (ANOVA). Also, an outlier identified from specific leaf area dataset was omitted in the analysis. A full factorial model analysis was conducted to test treatments and family effects on all measured variables twice, once under well-watered conditions (prior drought) and then again under water stress conditions (drought). Student’s t-test and Tukey-Kramer Honestly Significance Difference were used for separation of means tests.

3. Results

3.1. Prior Drought Conditions

3.1.1. Leaf Gas Exchanges and Chlorophyll Content

Prior to drought stress, a family × fertilizer, and family × salt interaction was significant for A, Gs, E, and WUEi (Table2). No interaction effect was found to be significant for total chlorophyll content, which was similar among families at the starting treatment ranging from 264.7 mg m−2 (K17B19) to 330.0 mg m−2 (in K4B19). However, main effects of salt (p < 0.0001) and fertilizer (p < 0.0001) (Table2) were found on total chlorophyll content. Salt and fertilizer increased chlorophyll from 275.3 to 314.20 mg m−2 and from 281.2 to 309.1 mg m−2, respectively. With the exception of DB16, fertilizer significantly increased A (Figure1a,b). G s and E were also generally increased with fertilizer, but were only significantly increased in a few families (Figure1a). Since both A and E increased with fertilizer, the WUEi was generally unaffected by fertilizer. However, KB19 displayed higher WUEi. The salt treatment significantly decreased Gs in all families and E was lower in all families, but only significantly decreased in DB16, K4B19 and NB1. A was lower in the salt treatments in all families, but only significantly decreased in K4B19. WUEi was increased in all families, but only significantly in DB16 and NB1 (Figure1b). Forests 2017, 8, 388 5 of 15

Table 2. Analysis of variance (ANOVA) results for the response of different Senegalia senegal families to salinity and fertility for leaf gas exchanges (net photosynthetic rate (A), stomatal conductance (Gs), transpiration (E), and intrinsic water use efficiency (WUEi)), relative growth of height (RGH) and Forests 2017, 8, 388 5 of 15 diameter (RGD), and chlorophyll content.

Forests 2017With, 8 ,the 388 exception of DB16, fertilizer significantly increased A (Figure 1a,b). Gs and E were5 of also 15 p-Value Sourcesgenerally of Variation increased dfwith fertilizer, but were only significantly increased in a few families (Figure 1a). RGH RGD A Gs E WUEi Chlorophyll Content SinceWith both the A exception and E increased of DB16, fertilizerwith fertilizer, significantly the WUE increasedi was Agenerally (Figure 1a,b). unaffected Gs and byE were fertilizer. also Salinity 1 0.5807 0.2407 0.0025 <0.0001 <0.0001 <0.0001 <0.0001 generallyHowever, increased KB19 displayed with fertilizer, higher butWUE werei. The only salt significantly treatment significantly increased in decreaseda few families Gs in (Figure all families 1a). Fertility 1 <0.0001 0.0023 <0.0001 0.0008 0.0133 0.1899 <0.0001 Since both A and E increased with fertilizer, the WUEi was generally unaffected by fertilizer. andFamily E was lower in 4all families, 0.0004 but 0.3738only significantly 0.9632 decreased 0.0375 in 0.3877 DB16, K4B19 0.0054 and NB1. 0.0922A was FamilyHowever,lower× salinity in KB19the salt displayed treatments 4 higher 0.6415 in all WUE families, 0.7013i. The saltbut 0.622 treatmentonly significantly 0.0188 significantly 0.0494decreased decreased 0.079in K4B19. Gs in all WUE families 0.3469i was Familyandincreased ×E fertilitywas inlower all families, in all 4 families, but 0.7818 only but significantly only 0.1669 significantly in 0.0302 DB16 decreasedand 0.1636 NB1 (Figure in 0.0265DB16, 1b). K4B19 0.0246 and NB1. A 0.4621 was Family × salinity × fertility 4 0.1288 0.6976 0.8522 0.5166 0.9227 0.8606 0.5800 lower in the salt treatments in all families, but only significantly decreased in K4B19. WUEi was increased in all families, but only significantly in DB16 and NB1 (Figure 1b).

(a) (b)

Figure 1. The effect of fertilizer (a) and salt (b) treatments on net photosynthetic rate (A), stomatal Figure 1. The effect of fertilizer(a) (a) and salt (b) treatments on net photosynthetic(b) rate (A), stomatal conductance (Gs), transpiration (E), and intrinsic water use efficiency (WUEi) of Senegalia senegal conductanceFigurefamilies. (G1. ThesAsterisk), transpirationeffect marks of fertilizer on bars (E), ( amean) andand significantsalt intrinsic (b) treatments effect water of usefertilizeron net efficiency photosynthetic (a) and salt (WUE (b )rate ati) p of (A),< 0.05.Senegalia stomatal senegal families.conductance Asterisk marks (Gs), transpiration on bars mean (E), significantand intrinsic effect water of use fertilizer efficiency (a )(WUE and salti) of (Senegaliab) at p < senegal 0.05. 3.1.2.families. Relative Asterisk Growth marks Rate on bars mean significant effect of fertilizer (a) and salt (b) at p < 0.05. 3.1.2. Relative Growth Rate 3.1.2. Relative Growthgrowth Raterate of height of Senegalia senegal seedlings was significantly different among Relativefamilies growth (Table 2, rate Figure of height2) varying of betweenSenegalia 1.18% senegal for NB1seedlings and 0.80% was for significantlyK17B19. Significantly, different NB1 among hadRelative the highest growth RGH rate in comparison of height of with Senegalia the rest senegal of the seedlings families, waswhich significantly did not differ different significantly. among familiesfamilies (Table 2(Table, Figure 2, Figure2) varying 2) varying between between 1.18% 1.18% for for NB1NB1 and and 0.80% 0.80% for for K17B19. K17B19. Significantly, Significantly, NB1 NB1 had thehad highest the highest RGH RGH in comparison in comparison with with the the rest rest ofof the families, families, which which did didnot differ not differ significantly. significantly. 1.4 A 1.41.2 B B A 1.21 B B 0.8 B B 1 B B 0.6

RGH (%) RGH 0.8 0.60.4 RGH (%) RGH 0.40.2 0.20 DB14 DB16 K17B19 K4B19 NB1 0 Family DB14 DB16 K17B19 K4B19 NB1 Figure 2. Variation of relative growth rate of heightFamily (RGH) among Senegalia senegal families. Different

letters on bars mean significant differences among families at p < 0.05. Figure 2. Variation of relative growth rate of height (RGH) among Senegalia senegal families. Different Figure 2. Variation of relative growth rate of height (RGH) among Senegalia senegal families. Different letters on bars mean significant differences among families at p < 0.05. letters on bars mean significant differences among families at p < 0.05.

Forests 2017, 8, 388 6 of 15

Forests 2017, 8, 388 6 of 15 Salinity had no significant effect on the relative growth (Table2), whereas the fertilizer significantly Salinity had no significant effect on the relative growth (Table 2), whereas the fertilizer increased RGH and RGD by 43% and 24%, respectively (Figure3). significantly increased RGH and RGD by 43% and 24%, respectively (Figure 3).

1.2 A 1 B 0.8 A 0.6 B

0.4

0.2

0 RGH (%) RGD (%) Fert No Fert

Figure 3. Effect of fertilizer (Fert) on relative growth rate of height (RGH) and diameter (RGD) among Figure 3. Effect of fertilizer (Fert) on relative growth rate of height (RGH) and diameter (RGD) among Senegalia senegal seedlings. Different letters on bars mean significant differences between fertilized Senegalia senegal (Fert) and no seedlings.fertilized (No Different Fert) at p letters < 0.05. on bars mean significant differences between fertilized (Fert) and no fertilized (No Fert) at p < 0.05. 3.2. Under Drought Conditions 3.2. Under Drought Conditions 3.2.1. Stomatal Density and Size 3.2.1. StomatalAt the Densityend of 3 weeks and Size of drought, we found the presence of stomata on both abaxial and adaxial Atleaf the surfaces endof and 3 weeksstomatal of density drought, and we length found did thenot differ presence significantly of stomata between on both leaf surfaces. abaxial andHence, adaxial leaf surfacesstomatal anddensity stomatal was considered density and to lengthbe the didaverag note differof abaxial significantly and adaxial between surfaces. leaf No surfaces. family by Hence, treatment interactions were found to be significant in stomatal density and stomatal size (Table 3). stomatal density was considered to be the average of abaxial and adaxial surfaces. No family by However, salinity significantly increased stomatal density from 82 to 89 stomata per mm2, and a treatment interactions were found to be significant in stomatal density and stomatal size (Table3). significant effect of family was found on both stomatal length and distribution (Figures 4 and 5a,b). 2 However,The salinity Diamenar significantly provenance increased (DB16) had stomatal the highest density stomatal from density 82 to but 89 stomatawas only persignificantly mm , and a significantgreater effect than ofNgane family NB1 was (Figure found 5a). on Ngane both stomatal displayed length significantly and distribution larger stomata (Figures than 4all and other5a,b). Theprovenances Diamenar and provenance significantly (DB16) lower stomatal had the density highest than stomatal the Diamenar density butprovenance was only (DB14 significantly and greaterDB16) than (Figure Ngane 4 and NB1 5). (Figure5a). Ngane displayed significantly larger stomata than all other provenances and significantly lower stomatal density than the Diamenar provenance (DB14 and DB16) (Figures4 and5).

Forests 2017, 8, 388 7 of 15

Table 3. Analysis of variance (ANOVA )results for the response of different Senegalia senegal families to salinity and fertility for stomatal density and size, total biomass, specific leaf area (SLA), root/shoot, leaf gas exchanges (net photosynthetic rate (A), stomatal conductance (Gs), transpiration (E), and intrinsic water use efficiency (WUEi)), relative growth of height (RGH) and diameter (RGD), and chlorophyll content following a drought period.

p-Value Sources of Variation df Stomatal Stomata Total Chlorophyll SLA Root/Shoot RGH RGD A Gs E WUEi Density Size Biomass Content Salinity 1 0.0158 0.2604 <0.0001 0.0059 <0.0001 0.0435 0.6073 0.452 <0.0001 <0.0001 <0.0001 <0.0001 Fertility 1 0.6565 0.91 <0.0001 0.6169 <0.0001 0.623 0.0012 0.2827 0.0142 0.0057 <0.0001 <0.0001 Family 4 0.0091 <0.0001 <0.0001 0.0159 0.023 0.0421 0.0019 0.4944 0.7874 0.3642 <0.0001 0.2614 Family × Salinity 4 0.2459 0.5518 0.0634 0.9215 0.2997 0.2108 0.4326 0.0438 0.1426 0.5021 0.233 0.6956 Family × fertility 4 0.7811 0.0733 0.6723 0.2896 0.1454 0.1914 0.5758 0.9961 0.4089 0.514 0.2803 0.5889 Family × Salinity × fertility 4 0.5933 0.6437 0.0002 0.2343 0.7375 0.0925 0.1375 0.4873 0.1361 0.3306 0.0410 0.7770 Forests 2017, 8, 388 8 of 15 Forests 2017, 8, 388 8 of 15

Forests 2017, 8, 388 8 of 15

Figure 4. Stomata on the abaxial and adaxial leaf surfaces of 12-week-old Senegalia senegal seedlings. FigureFigure 4. Stomata 4. Stomata on onthe the abaxial abaxial and and adaxial adaxial leafleaf surfacessurfaces of of 12-week-old 12-week-old Senegalia Senegalia senegal senegal seedlings. seedlings. NB1 (NB1a), K4B19 (a), K4B19 (b), ( K17B19b), K17B19 (c), (c DB14), DB14 (d (d),), DB16 DB16 ((ee).). NB1 (a), K4B19 (b), K17B19 (c), DB14 (d), DB16 (e).

(a) (b)

Figure 5. Variation of stomatal density (a) and size (b) among Senegalia senegal families. Different Figure 5. Variation of stomatal(a) density (a) and size (b) among Senegalia senegal(families.b) Different letters letters on bars mean significant differences among families at p < 0.05. on bars mean significant differences among families at p < 0.05. Figure 5. Variation of stomatal density (a) and size (b) among Senegalia senegal families. Different 3.2.2.letters Leaf on bars Gas meanExchanges significant differences among families at p < 0.05. 3.2.2. Leaf Gas Exchanges There was a significant salt × family × fertilizer and salt × family interactions for WUEi and A, 3.2.2.Thererespectively Leaf Gas was Exchanges a(Table significant 3). The saltmagnitude× family of the× effectfertilizer varied and with salt family× family (Figure interactions 6). Gas exchange for WUE ratesi and A, respectivelywere lower (Tableafter the3). drought The magnitude stress and ofthe the salt effect treatment varied generally with family still decreased (Figure6 G).s Gasand E, exchange and There was a significant salt × family × fertilizer and salt × family interactions for WUEi and A, increased WUEi. ratesrespectively were lower (Table after 3). the The drought magnitude stress of and the theeffect salt varied treatment with generally family (Figure still decreased 6). Gas exchange Gs and E, rates and increased WUEi. were lower after the drought stress and the salt treatment generally still decreased Gs and E, and

increased WUEi.

ForestsForests2017 2017, 8, ,8 388, 388 99 of of 15 15

salt 5 * * N 4 Y WUEi 3 2 1 0

4 *** 3 2 1 0 * 0.25 * * 0.20 0.15 0.10 0.05 0.00 * 12.5 10.0 7.5 5.0 2.5 0.0 DB14 DB16 K17B19 K4B19 NB1 Fam

Figure 6. Effect of salt on net photosynthetic rate (A), stomatal conductance (Gs), transpiration (E), and Figure 6. Effect of salt on net photosynthetic rate (A), stomatal conductance (Gs), transpiration (E), intrinsic water use efficiency (WUEi) of Senegalia senegal families under drought stress. Asterisk marks and intrinsic water use efficiency (WUEi) of Senegalia senegal families under drought stress. Asterisk on bars mean significant effect of salt at p < 0.05. marks on bars mean significant effect of salt at p < 0.05.

3.2.3.3.2.3. Biomass Biomass and and Relative Relative Growth Growth Rates Rates ThereThere was was a a significant significant family family× ×salt salt× × fertilizer interactioninteraction forfor allall biomassbiomass parametersparameters (Table(Table4 ).4). K17b19K17b19 was was the the only only family family which which increased increased biomass biomass in in response response to to the the salt salt treatment. treatment. TotalTotal weight, weight, leafleaf area, area, and and root root weight weight in in this this source source were were increased increased by by 51%, 51%, 17%, 17%, and and 47%, 47%, respectively. respectively. Ng21B1 Ng21B1 growthgrowth was was significantly significantly decreased decreased by by the the salt salt (Table (Table4). 4). No No other other family family had had growth growth changed changed by by the the saltsalt treatment. treatment. Growth Growth was was generally generally increased increased (more (more than than 100%) 100%) in in all all families families in in response response to to added added fertilizerfertilizer (Table (Table4). 4). Salt Salt generally generally had had a negativea negative impact impact on on this this fertilizer fertilizer growth growth enhancement. enhancement.

TableTable 4. The4. The effect effect of of salt salt and and fertilizer fertilizer treatments treatments on on growth growth parameters parameters of ofSenegalia Senegalia SenegalSenegal families.families.

FamiliesFamilies Variable Treatment Treatment Variable DB14DB14 DB16DB16 K4B19K4B19 K17B19K17B19 NB1NB1 Control 1.4 ± 0.47Aa 1.7 ± 0.28Aab 2.3 ± 0.61ABab 1.5 ± 0.17Aa 3.7 ± 0.40Bb Control 1.4 ± 0.47Aa 1.7 ± 0.28Aab 2.3 ± 0.61ABab 1.5 ± 0.17Aa 3.7 ± 0.40Bb Salt 1.4 ± 0.24Aa 1.6 ± 0.12Aa 1.7 ± 0.18Aa 2.1 ± 0.16Aab 1.6 ± 0.17Aa Leaf (g) Salt 1.4 ± 0.24Aa 1.6 ± 0.12Aa 1.7 ± 0.18Aa 2.1 ± 0.16Aab 1.6 ± 0.17Aa Leaf (g) Fert 4.2 ± 0.11Ab 4.9 ± 0.45Ac 4.2 ± 0.33Abc 4.8 ± 0.41Ac 5.3 ± 0.30Ab Salt × fertFert 2.9 ± 0.09BCab 4.2 ± 0.11Ab 2.3 ± 0.21Aab 4.9 ± 0.45Ac 3.7 ± 0.36ABbc 4.2 ± 0.33Abc 3.3 ± 0.30ABCbc 4.8 ± 0.41Ac 4.2 ± 5.30.32Cb ± 0.30Ab Salt × fert 2.9 ± 0.09BCab 2.3 ± 0.21Aab 3.7 ± 0.36ABbc 3.3 ± 0.30ABCbc 4.2 ± 0.32Cb Control 1.3 ± 0.40Aa 1.9 ± 0.34Aa 1.7 ± 0.47Aa 1.2 ± 0.16Aa 2.7 ± 0.23Aab SaltControl 1.3 ± 0.23Aa 1.3 ± 0.40Aa 2.0 ± 1.90.11Ba ± 0.34Aa 1.8 ± 0.14ABa 1.7 ± 0.47Aa 2.1 ± 1.20.15Bab ± 0.16Aa 1.5 ± 2.70.17ABa ± 0.23Aab Stem (g) FertSalt 3.9 ± 0.24Ab 1.3 ± 0.23Aa 5.3 ± 0.65Ac 2.0 ± 0.11Ba 4.8 ± 1.80.62Ab ± 0.14ABa 5.0 ± 2.10.52Ac ± 0.15Bab 5.2 ± 1.50.25Ac ± 0.17ABa Stem (g) Salt × fertFert 3.0 ± 0.17ABab 3.9 ± 0.24Ab 2.5 ± 0.21Aab 5.3 ± 0.65Ac 4.0 ± 4.80.35Bb ± 0.62Ab 3.3 ± 0.31ABbc 5.0 ± 0.52Ac 4.2 ± 5.20.35Bbc ± 0.25Ac ControlSalt × fert 2.0 ± 0.35ABab 3.0 ± 0.17ABab 2.7 ± 0.17ABab 2.5 ± 0.21Aab 2.9 ± 0.45ABa 4.0 ± 0.35Bb 1.7 ± 3.30.25Aa ± 0.31ABbc 3.3 ± 4.20.29Bab ± 0.35Bbc SaltControl 1.5 ± 2.00.30Aa ± 0.35ABab 2.4 ± 0.22ABab 2.7 ± 0.17ABab 2.6 ± 2.90.38Ba ± 0.45ABa 2.5 ± 0.25ABab 1.7 ± 0.25Aa 1.7 ± 3.30.07ABa ± 0.29Bab Root (g) FertSalt 3.9 ± 0.36Ac 1.5 ± 0.30Aa 5.4 ± 2.40.40Ac ± 0.22ABab 5.2 ± 2.60.34Ac ± 0.38Ba 5.1 ± 2.50.68Ac ± 0.25ABab 5.2 ± 1.70.20Ac ± 0.07ABa Root (g) Salt × fertFert 1.8 ± 0.19Aab 3.9 ± 0.36Ac 1.7 ± 0.15Aa 5.4 ± 0.40Ac 3.3 ± 0.14Bab 5.2 ± 0.34Ac 2.0 ± 5.10.22Aab ± 0.68Ac 3.6 ± 5.20.41Babc ± 0.20Ac ControlSalt × fert 242.2 ± 1.8111.3Aa ± 0.19Aab 279.9 ± 1.757.1Aa ± 0.15Aa 366.9 ± 3.3106.9Aa ± 0.14Bab 295.1 2.0± 52.9Aa ± 0.22Aab 551.5 3.6± 61.6Aab ± 0.41Babc Leaf area SaltControl 263.2 242.2± 58.7Aa ± 111.3Aa 237.7 ± 279.922.4Aa ± 57.1Aa 380.6 366.9± 90.6Aa ± 106.9Aa 346.0 295.1± 72.1Aab ± 52.9Aa 226.2 551.5± 50.5Aa ± 61.6Aab 2 (cm ) FertSalt 891.6 ± 263.280.6Ac ± 58.7Aa 909.9 ± 237.7113.8Ac ± 22.4Aa 686.8 ± 380.6111.3Aab ± 90.6Aa 818.0 346.0± 134.8Ac ± 72.1Aab 756.1 226.2± 106.1Ab ± 50.5Aa Leaf area (cm2)Salt × fert 435.1 ± 74.0Aab 323.9 ± 38.4Aab 560.8 ± 118.0Aab 522.5 ± 49.2Aabc 546.6 ± 92.7Aab Fert 891.6 ± 80.6Ac 909.9 ± 113.8Ac 686.8 ± 111.3Aab 818.0 ± 134.8Ac 756.1 ± 106.1Ab ControlSalt × fert 4.6 ± 435.11.16Aa ± 74.0Aab 6.2 ± 323.90.75ABa ± 38.4Aab 6.8 ± 560.81.41ABab ± 118.0Aab 4.4 522.5± 0.49Aa ± 49.2Aabc 9.7 546.6± 0.76Bb ± 92.7Aab ± ± ± ± ± Total dry SaltControl 4.2 0.71Aa 4.6 ± 1.16Aa 6.0 0.22ABa 6.2 ± 0.75ABa 6.2 6.80.63ABa ± 1.41 ABab 6.7 4.40.53Bab ± 0.49 Aa 4.8 9.70.38Aa ± 0.76Bb matter (g) Fert 12.0 ± 0.57Ab 15.6 ± 1.39Ab 14.1 ± 0.90Acd 14.9 ± 1.51Ac 15.7 ± 0.43Ac Salt 4.2 ± 0.71Aa 6.0 ± 0.22 ABa 6.2 ± 0.63 ABa 6.7 ± 0.53Bab 4.8 ± 0.38 Aa Total dry matter (g)Salt × fert 7.8 ± 0.30ABab 6.5 ± 0.45Aa 11.0 ± 0.77BCbc 8.6 ± 0.80ABb 12.0 ± 0.99Cbc Fert 12.0 ± 0.57 Ab 15.6 ± 1.39Ab 14.1 ± 0.90Acd 14.9 ± 1.51 Ac 15.7 ± 0.43 Ac For each variable, valuesSalt × infert each 7.8column ± 0.30ABab (between treatment) 6.5 ± 0.45Aa with different 11.0 ± 0.77 small BCbc letters 8.6 are ± 0.80 significantly ABb different12.0 ± 0.99 Cbc (p < 0.05 Tukey test). Values in each line (between families) with different capital letters are significantly different (pFor< 0.05 each Tukey variable, test). Values values represent in each the meancolumn of five (betw replicateseen treatment)± standard error.with different small letters are significantly different (p < 0.05 Tukey test). Values in each line (between families) with different capital letters are significantly different (p < 0.05 Tukey test). Values represent the mean of five replicates ± standard error.

Forests 2017, 8, 388 10 of 15

Forests 2017,The8, 388 main effects of family and salt treatments had a significant influence on specific leaf area10 of 15 (SLA) and root/shoot ratio. NB1 had the lowest SLA and root/shoot ratio while the other families did Forests 2017, 8, 388 10 of 15 not differ significantly (Figures 7 and 8). Forests 2017, 8, 388 10 of 15 TheThe main main effects effects of familyof family and and salt salt treatments treatments had had a a significant significant influence influence on specific on specific leaf area leaf area (SLA)(SLA) and root/shootand root/shoot ratio. ratio. NB1 NB1200 had had the Athe lowest lowest SLA and and root/shoot root/shoot ratio ratio while while the theother other families families did did The main effects of family and salt treatmentsA ABhad a significantAB influence on specific leaf area not differnot(SLA) differ significantly and significantly root/shoot (Figures ratio. (Figures NB17 and 7 ha andd8 ).the 8). lowest SLA and root/shoot ratioB while the other families did ) 150 -1 g

not differ significantly (Figures2 7 and 8). 100 200 A A AB AB SLA (cm 20050 A A B ) 150 AB

-1 AB g

2 B

) 1500

-1 100 g

2 DB14 K17B19 K4B19 DB16 NB1

SLA (cm 100 Family 50

SLA (cm 50 Figure 7. Variation of specific0 leaf area (SLA) of Senegalia senegal families. Different letters on bars DB14 K17B19 K4B19 DB16 NB1 mean significant differences among families at p < 0.05. 0 Family DB14 K17B19 K4B19 DB16 NB1 Both SLA and root/shoot ratio were significantlyFamily reduced by the salt, decreasing from 169.7 to Figure 7. Variation of specific leaf area (SLA) of Senegalia senegal families. Different letters on bars Figure152.2 cm 7. 2Variation g−1 and 0.61 of specificto 0.47 leafin controls area (SLA) versus of Senegalia salt treated. senegal Thefamilies. root/shoot Different ratio also letters decreased on bars meanfrom mean significant differences among families at p < 0.05. significant0.66 toFigure 0.43 differences 7.in Variationcontrols among ofversus specific familiesfertilized leaf area at plants.p (SLA)< 0.05. of Senegalia senegal families. Different letters on bars mean significant differences among families at p < 0.05. Both SLA and root/shoot ratio were significantly reduced by the salt, decreasing from 169.7 to 152.2 cm2 g−1 and 0.61 to 0.47 in 0.8controls versus salt treated. The root/shoot ratio also decreased from Both SLA and root/shoot ratio were significantly reducedA by the salt, decreasing from 169.7 to 0.7 0.66152.2 to cm 0.432 g −in1 and controls 0.61 to versus 0.47 in fertilized controlsAB plants. versusA salt treated. The root/shoot ratio also decreased from AB 0.66 to 0.43 in controls versus fertilized0.6 plants. B 0.5 0.8 0.4 A 0.7 0.80.3 AB A AB A Root/shoot 0.6 0.70.2 A B 0.5 AB 0.60.1 AB B 0.4 0.50 0.3 DB14 DB16 K17B19 K4B19 NB1 0.4 Root/shoot 0.2 0.3 Family

Root/shoot 0.1 0.2 Figure 8. Variation of root/shoot0 ratio among families of Senegalia senegal seedlings. Different letters Figure 8. Variation of root/shoot0.1 ratioDB14 among DB16 families K17B19 of Senegalia K4B19 NB1 senegal seedlings. Different letters on barson meanbars mean significant significant differences differences0 among among families families atat p < 0.05. 0.05. DB14 DB16Family K17B19 K4B19 NB1 There was a positive effect of fertilizer applicatFamilyion on RGD prior to and during the drought stress Figure 8. Variation of root/shoot ratio among families of Senegalia senegal seedlings. Different letters Bothperiod SLA (Figures and root/shoot3 and 9). In contrast, ratio were fertilizer significantly did not increase reduced RGH by .during the salt, drought decreasing (Figure from 9). 169.7 to on bars mean significant differences among families at p < 0.05. 152.2 cm2Figureg−1 and 8. Variation 0.61 to of 0.47 root/shoot in controls ratio among versus families salt treated. of Senegalia The senegal root/shoot seedlings. ratio Different also decreased letters from on bars mean significant differences among families at p < 0.05. 0.66 to 0.43 in controls versus fertilized0.7 plants. There was a positive effect of fertilizer application on RGDA prior to and during the drought stress ThereperiodThere was(Figures was a positive a3 positive and 9). effect Ineffect contrast, of0.6 of fertilizer fertilizer fertilizer application applicat did notion increase on on RGD RGD RGHprior prior .duringto toand and during drought during the (Figure drought the drought 9). stress stress A periodperiod (Figures (Figures3 and 3 9and). In 9). contrast, In contrast,0.5 fertilizer fertilizer didA did not not increase increase RGHB .during .during drought drought (Figure (Figure 9).9 ). 0.70.4 A 0.60.70.3 A A 0.50.60.2 A B A 0.40.50.1 A B 0.30.40 RGH (%) RGD (%) 0.20.3 Fert No Fert

0.10.2 Figure 9. The effect of fertilizer0.10 (Fert: fertilized, No fert: no fertilized) on the relative growth rate of diameter (RGD) and relative growthRGH rate (%) of height (RGH) RGD of(%) Senegalia senegal seedlings during 0 Fert No Fert drought stress. Different letters on barsRGH me (%)an significant differences RGD (%) among families at p < 0.05. Fert No Fert Figure 9. The effect of fertilizer (Fert: fertilized, No fert: no fertilized) on the relative growth rate of

diameterFigure 9. The(RGD) effect and of relativefertilizer growth (Fert: fertilized, rate of height No fert: (RGH) no fertilized) of Senegalia on the senegal relative seedlings growth duringrate of Figure 9. The effect of fertilizer (Fert: fertilized, No fert: no fertilized) on the relative growth rate of droughtdiameter stress. (RGD) Different and relative letters growth on bars ratemean of significant height (RGH) differences of Senegalia among senegalfamilies seedlings at p < 0.05. during diameter (RGD) and relative growth rate of height (RGH) of Senegalia senegal seedlings during drought drought stress. Different letters on bars mean significant differences among families at p < 0.05. stress. Different letters on bars mean significant differences among families at p < 0.05.

Forests 2017, 8, 388 11 of 15 Forests 2017, 8, 388 11 of 15

A significantsignificant difference difference among among families families was was found found on RGHon RGH (Figure (Figure 10), whereas 10), whereas RGD was RGD similar was betweensimilar between families. families. Prior to Prior the drought, to the drought, Ng21B1 Ng21B1 had significantly had significantly greater RGHgreater than RGH all otherthan all families. other Duringfamilies. drought, During drought, RGH was RGH greatly was reduced greatly andreduce K17B19d and displayed K17B19 displayed the highest the RGH, highest but RGH, it was but only it significantlywas only significantly higher than higher DB16 than (Figure DB16 10 (Figure). 10).

0.7 A 0.6 AB 0.5 AB AB 0.4 B 0.3 RGH (%) RGH 0.2 0.1 0 DB14 DB16 K17B19 K4B19 NB1 Family Figure 10. Variation of relative growth rate of height (RGH) between Senegalia senegal families) and Figure 10. Variation of relative growth rate of height (RGH) between Senegalia senegal families) and during drought stress. Different lettersletters onon bars mean significantsignificant differencesdifferences amongamong familiesfamilies atatp p << 0.05.

4. Discussion 4. Discussion This study was the first attempt to look for growth performance and physiological adaptation This study was the first attempt to look for growth performance and physiological adaptation under salt, and fertility conditions among different Senegalia senegal families collected in Senegal. under salt, and fertility conditions among different Senegalia senegal families collected in Senegal. Results of this study confirmed that photosynthetic capacities of Senegalia senegal seedlings are similar Results of this study confirmed that photosynthetic capacities of Senegalia senegal seedlings are among families in non-limiting water conditions [19]. However, significant differences in stomatal similar among families in non-limiting water conditions [19]. However, significant differences in density and size, SLA, root/shoot ratio, and RGH may be indicative of different responses to stomatal density and size, SLA, root/shoot ratio, and RGH may be indicative of different responses environmental stress among the tested families. The significant salinity by family interaction on dry to environmental stress among the tested families. The significant salinity by family interaction on matter accumulation found in this study implies that some Senegalia senegal families may perform dry matter accumulation found in this study implies that some Senegalia senegal families may perform better on salt affected drylands than on non-salty drylands [20]. NB1 had significantly reduced better on salt affected drylands than on non-salty drylands [20]. NB1 had significantly reduced growth growth with salt addition, whereas K17B19 grew better in the salt treatment. An earlier study showed with salt addition, whereas K17B19 grew better in the salt treatment. An earlier study showed a a significant decrease of Senegalia senegal shoot and root dry weights in response to the same salinity significant decrease of Senegalia senegal shoot and root dry weights in response to the same salinity level used in our study [21]. However, they used a mixture of seeds collected from one coastal area level used in our study [21]. However, they used a mixture of seeds collected from one coastal area and did not examine families, so it is possible they utilized a susceptible source such as our NB1. and did not examine families, so it is possible they utilized a susceptible source such as our NB1. The significant decrease of total biomass observed in NB1 reveals its higher sensitivity to salt The significant decrease of total biomass observed in NB1 reveals its higher sensitivity to salt stress than the other genotypes tested in this study. Despite originating from more saline areas, NB1 stress than the other genotypes tested in this study. Despite originating from more saline areas, NB1 tended to be more sensitive to salinity, a finding similar to that which was reported in an earlier study tended to be more sensitive to salinity, a finding similar to that which was reported in an earlier study which revealed that topodemes from areas subjected to salinity are more salt tolerant than those from which revealed that topodemes from areas subjected to salinity are more salt tolerant than those from non-salted areas [22]. In contrast, salt tolerance in Senegalia tortilis might be attributed to genetic non-salted areas [22]. In contrast, salt tolerance in Senegalia tortilis might be attributed to genetic factors factors that may be site independent [23]. Senegalia senegal is known as a salt tolerant species [5–7] that may be site independent [23]. Senegalia senegal is known as a salt tolerant species [5–7] and this is and this is confirmed for most of our sources. Further, results on relative growth rate revealed a confirmed for most of our sources. Further, results on relative growth rate revealed a positive effect positive effect of salinity on RGH, which is significantly increased by 21% under drought (P = 0.0435). of salinity on RGH, which is significantly increased by 21% under drought (p = 0.0435). Our results Our results suggest potential salt tolerance improvement of Senegalia senegal through genotype suggest potential salt tolerance improvement of Senegalia senegal through genotype selection. Genetic selection. Genetic variation for salt tolerance has been reported for many other species [23–27]. variation for salt tolerance has been reported for many other species [23–27]. Surprisingly, K17B19 Surprisingly, K17B19 grew better in saline conditions, increasing total biomass by 51%, compared to grew better in saline conditions, increasing total biomass by 51%, compared to non-saline conditions, non-saline conditions, even though the increase was not significant. Increased growth of plants under even though the increase was not significant. Increased growth of plants under saline soils may be saline soils may be due to specific genes that control cell growth and leaf function [26]. K17B19, due to specific genes that control cell growth and leaf function [26]. K17B19, K4B19, DB14, and DB16 K4B19, DB14, and DB16 may have inherent ability to control water loss and improve ionic balance to may have inherent ability to control water loss and improve ionic balance to their favor, therefore their favor, therefore maintaining growth and biomass under salinity [28]. Recently, a sodium maintaining growth and biomass under salinity [28]. Recently, a sodium hydrogen antiporter gene hydrogen antiporter gene (NHX1) has been identified in the Senegalia senegal tonoplast cells, which (NHX1) has been identified in the Senegalia senegal tonoplast cells, which through an overexpression through an overexpression may confer capacity to tolerate salinity stress [5,29]. may confer capacity to tolerate salinity stress [5,29]. Unexpectedly, stomatal density and total chlorophyll content were significantly increased by salinity. Past studies found salinity to negatively affect stomatal density and chlorophyll content [14,23,30–33]. The presence of salt increased WUEi and decreased SLA (Figures 1b and 7). Reduced

Forests 2017, 8, 388 12 of 15

Unexpectedly, stomatal density and total chlorophyll content were significantly increased by salinity. Past studies found salinity to negatively affect stomatal density and chlorophyll content [14,23,30–33]. The presence of salt increased WUEi and decreased SLA (Figures1b and7). Reduced SLA (thicker leaves) usually have more mesophyll cells with a higher density of chlorophyll and proteins per unit leaf area, hence a higher photosynthetic capacity than thinner leaves. Our results confirmed the negative association between WUEi and SLA reported by [34], who suggested lower SLA as a selection criterion for enhanced WUEi. Our study revealed that Senegalia senegal response to salt depended on genotypes and whether or not drought was induced. The increased WUEi in DB16 and NB1 in response to salt when water was not a limiting factor may be attributed to the significant decrease of transpiration relative to the assimilation rate through stomata closure (Figure1b). The same response was observed on K17B19 under drought conditions. The increased WUEi likely is due to high photosynthesis, or low transpiration, or both [35,36]. However, under drought stress, the higher WUEi in response to salt without a significant change in stomata conductance observed in DB16 might be influenced by other traits such as mesophyll conductance. Besides stomatal control, inherent differences in mesophyll CO2 fixation capacity may influence the rate of photosynthesis and accordingly WUEi [37,38]. It is reported that enhancing mesophyll conductance/stomata conductance ratio can improve WUEi [37,39]. Moreover, notable effects of salinity and drought stress on mesophyll conductance have also been reported in Vitis vinifera [40]. Stomatal conductance was decreased in saline conditions by 39% prior to drought and by 37% during drought, averaged across all families. Still, influence of stomatal closure on A inhibition and regulation of stomatal conductance are strongly determined by both mesophyll and stomatal resistance to CO2 diffusion [41]. As expected, the fertilizer significantly increased the total biomass and biomass partition of all families. A positive effect of nutrient supply on Senegalia senegal growth has been revealed in an earlier study [42]. The significant increase in total biomass was likely due, in part, to the significant increase in the net photosynthetic rate under fertilizer conditions in most families (Figure1a). However, the increased biomass partitioning was higher in leaves and stems than in roots, inducing a lower root/shoot ratio (Figure8). It is reported that, in young seedlings, nutrient addition favored shoot growth more than root growth [38,43,44]. Our results confirm that fertilizer supply influences not only plant dry matter, but also biomass partitioning. Trends in growth observed in seedling plants, showing lower RGH for DB16, were similar to what was found in their parent trees grown in the field which displayed lower height for DB16 (396 cm) compared to other genotypes. In other families, height varied from 510 cm for K4B19 to 590 cm for K17B19 [19]. We assume that height and RGH are highly heritable traits among Senegalia senegal genotypes. According to Dvorak et al. [45], heritability of growth traits for some forest trees does not change significantly between early and mature growth stages. With the exception of NB1, all sources (and in particular K17B19) used in this study would be suitable for testing on salt impacted soils. However, it would be important to test these findings in field conditions with more salt and fertilizer levels. Also, gum yield for such potential adapted genotypes would be important to take into account in the future to assess expected profit from growing Senegalia senegal in marginal and harsh dry lands.

5. Conclusions This study testing the physiological and morphological adaptation of Senegalia senegal seedling under salinity and fertility conditions provided valuable information for improving Senegalia senegal tolerance to harsh environments through genotype selection. Our results revealed significant differences among Senegalia senegal genotypes in their leaf morphology. Large stomata size with low density was observed in NB1, which tended to be more sensitive to salinity despite lower specific leaf area. Photosynthetic capacities and growth rate performances showed significant interactions between families and salinity or fertility treatments. Diamenar and Kidira provenances in general and particularly the K17B19 family might be suitable for afforestation of salt affected drylands. All genotypes have notably increased biomass under the fertilizer effect. Our hypothesis that at least one Forests 2017, 8, 388 13 of 15 family would be better adapted to salinity or fertilizer effect could be supported. However, despite the importance of intraspecific variation in changing environmental conditions, more research is needed to test findings in field conditions and to determine salt and fertilizer thresholds for optimized growth of Senegalia senegal in arid lands.

Acknowledgments: This work is made possible, in part, by the generous support of the American people through the United States Agency for International Development (USAID) as part of Feed the Future, the U.S. Government’s global hunger and security initiative, under the terms of Contract No. AID-685-A-00-10-00194-00 (USAID/Education and Research in Agriculture). The contents are the responsibility of the author(s) and do not necessarily reflect the views of USAID or the United States Government. We would like to thank Audrey Zink-Sharp, professor and associate department head of sustainable biomaterials, Virginia Polytechnic Institute and state university, USA for the material provided and assistance on stomata measurements. Our gratitude to Momar Wade, research assistant at Centre National de Recherche Forestière of Institut Sénégalais de Recherche Agricole (CNRF/ISRA) for his help on harvesting Senegalia senegal seeds. We thank John Petersen, a laboratory specialist of the department Forest Resources and Environmental Conservation (FREC), Virginia Tech for his support along the greenhouse experiment. Author Contributions: J.R.S. and M.S.S. conceived and designed the experiments; M.S.S. performed the experiment; J.R.S., J.S. and M.S.S. analyzed data and wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Gonzalez, P. Desertification and a Shift of Forest Species in the West African Sahel. Clim. Res. 2001, 17, 217–228. [CrossRef] 2. Green Economy Sector Study on Water Resources in Senegal. Available online: https://www.unep.org/ greeneconomy/sites/unep.org.greeneconomy/files/publications/ge_water_senegal_final.pdf (accessed on 10 October 2017). 3. Bradley, D.; Grainger, A. Social resilience as a controlling influence on desertification in Senegal. Land Degrad. Dev. 2004, 15, 451–470. [CrossRef] 4. Rapport Exploratoire sur L’Économie Verte au Sénégal. Available online: http://www.cepod.gouv.sn/sites/ default/files/L’%C3%A9conomie%20verte%20%20rapport%20provisoire.pdf (accessed on 10 October 2017). 5. Fall, D.; Bakhoum, N.; Fall, F.; Diouf, F.; Ly, M.O.; Diouf, M.; Gully, D.; Hocher, V.; Diouf, D. Germination, Growth and Physiological Responses of Senegalia senegal (L.) Britton, seyal (Delile) P. Hurter and Prosopis Juliflora (Swartz) DC to Salinity Stress in Greenhouse Conditions. Afr. J. Biotechnol. 2016, 15, 2017–2027. 6. Pandey, S.; Zhang, W.; Assmann, S.M. Roles of ion channels and transporters in guard cell signal transduction. FEBS Lett. 2007, 581, 2325–2336. [CrossRef][PubMed] 7. Sambou, A.; Ndour, B.; Cheng, S.; Senghor, E. Ligneous species tolerance in acid sulphated and saline soils of sine saloum: Case of rural community of Djilass and Loul Secene. J. Sustain. Dev. 2010, 3, 174. [CrossRef] 8. Sonneveld, B.G.J.S.; Keyzer, M.A.; Zikhali, P.; Merbis, M. National Land Degradation Assessment Senegal and Review of Global Socio-Economic Parameters in the LADA Data Base–Land Degradation Assessment (LADA) Project Paper; Centre for World Food Studies: Amsterdam, The Netherlands, 2010. 9. Farooq, M.; Wahid, A.; Kobayashi, N.; Fujita, D.; Basra, S.M.A. Plant drought stress: Effects, mechanisms and management. Agron. Sustain. Dev. 2009, 29, 185–212. [CrossRef] 10. Aroca, R. Plant Responses to Drought Stress; Springer: Berlin/Heidelberg, Germany, 2012. [CrossRef] 11. Masle, J.; Gilmore, A.; Farquhar, G. The ERECTA Gene Regulates Plant Transpiration Efficiency in Arabidopsis. Nature 2005, 436, 866–870. [CrossRef][PubMed] 12. Xu, Z.; Zhou, G. Responses of leaf stomatal density to water status and its relationship with photosynthesis in a grass. J. Exp. Bot. 2008, 59, 3317–3325. [CrossRef][PubMed] 13. Ouyang, S.Q.; Liu, Y.F.; Liu, P.; Lei, G.; He, S.; Ma, B.; Zhang, W.; Zhang, J.; Chen, S. Receptor-like kinase OsSIK1 improves drought and salt stress tolerance in rice (Oryza sativa) plants. Plant J. 2010, 62, 316–329. [CrossRef][PubMed] 14. Reginato, M.A.; Reinoso, H.; Llanes, A.S.; Luna, M.V. Stomatal abundance and distribution in Prosopis strombulifera plants growing under different iso-osmotic salt treatments. Am. J. Plant Sci. 2013, 4, 41195. [CrossRef] Forests 2017, 8, 388 14 of 15

15. Chaves, M.M.; Costa, J.M.; Saibo, N.J.M. Recent Advances in Photosynthesis under Drought and Salinity. Elsevier Sci. Technol. 2011, 57, 49–104. [CrossRef] 16. Chaves, M.M.; Flexas, J.; Pinheiro, C. Photosynthesis under drought and salt stress: Regulation mechanisms from whole plant to cell. Ann. Bot. 2009, 103, 551–560. [CrossRef][PubMed] 17. Hardikar, S.A.; Pandey, A.N. Growth, Water Status and Nutrient Accumulation of Seedlings of senegal (L.) Willd. in Response to Soil Salinity. An. Biol. 2008, 30, 17–28. 18. Blackman, V.H. The compound interest law and plant growth. Ann. Bot. 1919, 33, 353–360. [CrossRef] 19. Sarr, M.S. Morphological and Physiological Responses of Senegalia senegal (L.) Britton Provenances to Drought, Salinity, and Fertility. Ph.D. Thesis, Virginia Tech, Blacksburg, VA, USA, 2017; p. 121. 20. Flowers, T.J.; Yeo, A.R. Ion Relations of Plants Under Drought and Salinity. Aust. J. Plant Physiol. 1986, 13. [CrossRef] 21. Briggs, D. Genecological Studies of Salt Tolerance in Groundsel (Senecio vulgaris L.) with Particular Reference to Roadside Habitats. New Phytol. 1978, 81, 381–389. [CrossRef] 22. Muturi, G.M. Provenance Variation of Salt Tolerance and Seedling Nutrition in Acacia tortilis (Forsk.) Hayne. Ph.D. Thesis, University of Nairobi, Nairobi, Kenya, 1993. 23. Cha-um, S.; Somsueb, S.; Samphumphuang, T.; Kirdmanee, C. Salt tolerant screening in eucalypt genotypes (Eucalyptus spp.) using photosynthetic abilities, proline accumulation, and growth characteristics as effective indices. In Vitro Cell. Dev. Biol. Plant 2013, 49, 611–619. [CrossRef] 24. Fung, L.E.; Wang, S.S.; Altman, A.; Hutterman, A. Effect of NaCl on Growth, Photosynthesis, Ion and Water Relations of Four Poplar Genotypes. For. Ecol. Manag. 1998, 107, 135–146. [CrossRef] 25. Greenway, H.; Munns, R. Mechanisms of Salt Tolerance in Nonhalophytes. Annu. Rev. Plant Physiol. 1980, 31, 149–190. [CrossRef] 26. Munns, R. Genes and Salt Tolerance: Bringing them Together. New Phytol. 2005, 167, 645–663. [CrossRef] [PubMed] 27. Ghoulam, C.A.F.; Fares, K. Effects of Salt Stress on Growth, Inorganic Ions and Proline Accumulation in Relation to Osmotic Adjustment in Five Sugar Beet Cultivars. Environ. Exp. Bot. 2002, 47, 39–50. [CrossRef] 28. Yadav, R.S.; Sehgal, D.; Vadez, V. Using genetic mapping and genomics approaches in understanding and improving drought tolerance in pearl millet. J. Exp. Bot. 2011, 62, 397–408. [CrossRef][PubMed] 29. Taiz, L.; Zeiger, E.; Moller, I.M.; Murphy, A. Plant Physiology and Development; Sinauer Associates, Incorporated: Sunderland, MA, USA, 2015. 30. Barbieri, G.; Vallone, S.; Orsini, F.; Paradiso, R.; De Pascale, S.; Negre-Zakharov, F.; Maggio, A. Stomatal Density and Metabolic Determinants Mediate Salt Stress Adaptation and Water Use Efficiency in Basil (Ocimum basilicum L.). J. Plant Physiol. 2012, 169, 17–37. [CrossRef][PubMed] 31. Larcher, W. Physiological Plant Ecology: Ecophysiology and Stress Physiology of Functional Groups; Springer: Berlin/Heidelberg, Germany; New York, NY, USA, 1995.

32. Yang, H.; Wang, G. Leaf stomatal densities and distribution in Triticum aestivum under drought and CO2 enrichment. Acta Ecol. Sinica 2000, 25, 312–316. 33. Zhang, H.; Li, J.; Yoo, J.H.; Yoo, C.C.; Cho, S.H.; Koh, H.J.; Seo, H.S.; Paek, N.C. Rice Chlorina-1 and Chlorina-9 Encode ChlD and ChlI Subunits of mg Chelatase, a Key Enzyme for Chlorophyll Synthesis and Chloroplast Development. Plant Mol. Biol. 2006, 62, 325–337. [CrossRef][PubMed] 34. Rao, R.N.; Udaykumar, M.; Farquhar, G.D.; Talwar, H.S.; Prasad, T.G. Variation in carbon isotope discrimination and its relationship to specific leaf area and ribulose-1, 5-bisphosphate carboxylase content in groundnut genotypes. Funct. Plant Biol. 1995, 22, 545–551. [CrossRef] 35. Ehleringer, J.R.; Hall, A.E.; Farquhar, G.D. Stable Isotopes and Plant Carbon/Water Relations; Academic Press: San Diego, CA, USA, 1993. 36. Li, C. Carbon isotope composition, water-use efficiency and biomass productivity of Eucalyptus microtheca populations under different water supplies. Plant Soil 1999, 214, 165–171. [CrossRef] 37. Flexas, J.; Niinemets, U.; Galle, A.; Barbour, M.; Centritto, M.; Diaz-Espejo, A.; Douthe, C.; Galmes, J.; Ribas-Carbo, M.; Rodrigues, P.L.; et al. Diffusional Conductances to CO2 as a Target for Increasing Photosynthesis and Photosynthetic Water Use Efficiency. Photosynth. Res. 2013, 117, 45–59. [CrossRef] [PubMed] 38. Pallardy, S.G. Physiology of Woody Plants, 3rd ed.; Academic Press: San Diego, CA, USA, 2008. Forests 2017, 8, 388 15 of 15

39. Flexas, J.; Diaz-Espejo, A.; Conesa, M.A.; Coopman, R.E.; Douthe, C.; Gago, J.; Galle, A.; Galmes, J.; Medrano, H.; Ribas-Carbo, M.; et al. Mesophyll Conductance to CO2 and Rubisco as Targets for Improving Intrinsic Water Use Efficiency in C3 Plants. Plant Cell Environ. 2016, 39, 965–982. [CrossRef][PubMed] 40. Ferrio, J.P.; Pou, A.; Florez-Sarasa, I.; Gessler, A.; Kodama, N.; Flexas, J.; Ribas-Carbo, M. The Péclet Effect on

Leaf Water Enrichment Correlates with Leaf Hydraulic Conductance and Mesophyll Conductance for CO2. Plant Cell Environ. 2012, 35, 611–625. [CrossRef][PubMed] 41. Nilsen, E.T.; Orcutt, D.M.; Hale, M.G. The Physiology of Plants under Stress; Wiley: New York, NY, USA, 1996. 42. Daldoum, D.M.A.; Hammad, G.H. Performance of Acacia senegal (L.) Wild Seedlings Growth under some Tree Manures and NPK Fertilizers in Nursery Site. J. Environ. Sci. 2015, 31, 303–311. [CrossRef] 43. Fernández, M.; Novillo, C.; Pardos, J.A. Effects of Water and Nutrient Availability in Pinus pinaster Ait. Open Pollinated Families at an Early Age: Growth, Gas Exchange and Water Relations. New For. 2006, 31, 321–342. [CrossRef] 44. Stovall, J.P.; Fox, T.R.; Seiler, J.R. Short Term Changes in Biomass Partitioning of Two Full Sib Clones of Pinus taeda L. Under Differing Fertilizer Regimes over 4 months. Trees 2012, 26, 951–961. [CrossRef] 45. Dvorak, W.S.; Uruena, H.; Moreno, L.A.; Goforth, J. Provenance and family variation in Sterculia apetala in Colombia. For. Ecol. Manag. 1998, 111, 127–135. [CrossRef]

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