HORTSCIENCE 53(11):1562–1569. 2018. https://doi.org/10.21273/HORTSCI13280-18 When crops are subjected to soil salinity levels exceeding their tolerance levels, plant Kunzea growth declines and crop yields decrease. For Salinity Tolerance of Muntries ( example, strawberry (sensitive) exhibits a re- pomifera duced number of leaves and leaf area at F. Muell.), a Native Food Crop 30 mM NaCl and a 20% reduction in fruit yield (Garriga et al., 2015). A significant in Australia decrease in growth of date palm (tolerant) –1 is observed at 7.3 dS·m , and fruit yield is Chi M. Do, Kate L. Delaporte, Vinay Pagay, and Carolyn J. Schultz1 reduced by 25% (Department of Agricul- School of Agriculture, Food and Wine, Waite Research Institute, The ture and Food, 2016). Olive is an example of University of Adelaide, PMB1, Glen Osmond, SA, 5064, Australia a moderately tolerant fruit crop that shows relatively minor impacts at high salinity (7.5 Additional index words. alternative fruits, homeostasis, potassium, salinity stress, sodium dS·m–1), with 20% to 30% reduction in oil chloride, sustainable agriculture and fresh-fruit yield compared with nonsalt- stressed plants (Al-Rawi and Al-Mohemdy, Abstract . Identifying productive food crops that tolerate moderate soil salinity is critical 2001). In citrus (lime and lemon, both sensi- Kunzea pomifera for global food security. We evaluate the salinity tolerance of tive crops), moderate salinity (50 mM NaCl) (muntries), a traditional Indigenous food plant that grows naturally in coastal regions reduces leaf number, area, and thickness of southern Australia and thrives on relatively low rainfall. A range of saline irrigation by 20% (Alireza et al., 2013). treatments were tested on four genotypes: tap water, 50, 200, 300, and 400 mM NaCl In addition to changes in growth param- [Maarten’s Favorite (MF)] and up to 200 mM NaCl (MP1, SES2, and CJ1). After a 10- eters, many plants show changes in ion week saline irrigation treatment at 50 mM NaCl, SES2 appeared to have the highest salt accumulation in roots and/or leaves or shoots, tolerance of all genotypes based on no significant change in the number of secondary with contrasting trends for sodium and po- branches. At 50 mM NaCl, sodium accumulated significantly in roots but not the leaves of tassium accumulation. There is evidence that three genotypes, suggesting an active shoot exclusion mechanism. At 200 mM NaCl, plant reduced levels of K+ have a negative impact growth decreased, Na+ and ClL generally accumulated to significantly higher levels in + on stomatal function (Andres et al., 2014; leaves, compared with 50 mM NaCl, whereas potassium (K ) levels were unchanged. At Barrag an et al., 2012; Liu et al., 2000) and high NaCl (300 and 400 mM), MF showed severe growth retardation with leaf symptoms many other physiological processes (Shabala K. pomifera, appearing in week 9. Our results indicate that two genotypes of SES2 and and Pottosin, 2014). CJ1, are moderately salt tolerant based on modest reductions in three growth parameters Assessing genotypic variability for salin- at 50 mM NaCl, compared with MF and MP1. Further evaluation of the natural diversity ity tolerance is a key approach to identifying of this species should reveal a range of diverse mechanisms of salinity tolerance thus the most saline tolerant lines for existing and providing a new fruit crop for moderately saline soils. Chemical names: NaCl (sodium new crops. Salt tolerant genotypes of existing chloride). crops, such as wheat (Munns et al., 2012) and strawberries (F. chiloensis f. patagonica) (Garriga et al., 2015), have been identified Approximately 800 million ha worldwide in a low production year (Al-Rawi and Al- by screening wild accessions. Other efforts to are affected by saline soils (salinity) (FAO, Mohemdy, 2001; Ben-Gal, 2011; Chartzoulakis, increase global food security include identi- 2016) caused by both natural processes over 2005; Murkute et al., 2005; Tripler et al., 2011). fying new crops that can grow productively long periods of time and more rapidly by To reduce the impacts of salinity on global food under saline conditions, such as quinoa (Che- human practices such as land clearing, chang- production, a variety of complementary research nopodium quinoa) and saltbushes (Atriplex ing cropping systems, and irrigation (Parihar efforts are underway, including understanding sp.) (Jacobsen et al., 2012). et al., 2015). In Australia, 5.7 million ha the mechanisms of salinity tolerance, introduc- One crop plant that has not yet been had high salinity levels in 2000, and it is ing new plant cultivars with increased salinity assessed for salinity tolerance is Kunzea estimated that this number will triple to 17 tolerance, and evaluating diverse plant species pomifera (muntries; Myrtaceae), one of 13 million ha by 2050, with 11 million ha of that are naturally adapted to saline conditions key Australian native food crops (Clarke, agricultural land affected (Australian Bureau (Loescher et al., 2011; Roy et al., 2014). 2013; Do et al., 2017; Sultanbawa, 2016). of Statistics, 2010). Globally, salinity is re- Food crops have varying tolerances to K. pomifera produces berries called muntries sponsible for economic losses with nominal salinity and are typically classified as either that are naturally sweet, contain high levels values of $27.3 billion (Qadir et al., 2014) and salt sensitive or salt tolerant at the two of antioxidants, and have high consumer yield losses as high as 80% for some crops extremes [U.S. Department of Agriculture acceptance in a range of products (Schultz (USDA), 2017]. Select fruit crops have been et al., 2009). K. pomifera grows naturally in categorized as salt sensitive (very low salin- coastal regions of South Australia and Vic- ity: <1.3 dS·m–1 10 mM NaCl 500 mg·L–1 toria, Australia (Ryder et al., 2008). Although Received for publication 7 June 2018. Accepted for NaCl), for example, strawberry (Fragaria the commercial production industry of mun- publication 27 Aug. 2018. ·ananassa Duch.) and grapefruit (Citrus tries is still in its infancy, several plant This work was supported by Glenn and Joan · Dennis (Mt Pleasant, South Australia) and Ray paradise); moderately sensitive (low salin- selections have been made and are currently –1 and Pat Rogers (McLaren Flat, South Australia) ity: 1.3–3.0 dS·m 10–25 mM NaCl 500– growing in orchards in South Australia. A –1 who generously provided their knowledge and time 1500 mg·L NaCl), for example, grape (Vitis recent study of plants at two orchards iden- and allowed us to conduct this research using vinifera) and plum (Prunus domestica); mod- tified 15 unique genotypes and demonstrated propagation material from their orchards. We thank erately tolerant (moderate salinity: 3.0–6.0 high DNA sequence divergence between Maarten Ryder and Michelle Wirthensohn for in- dS·m–1 25–60 mM NaCl 1500–3500 these selections (Do et al., 2018). K. pomifera sightful comments on the manuscript. Chi M. Do is mg·L–1) for example, olive (Olea europaea) is assumed to have a degree of tolerance to recipient of a joint Vietnam International Educa- andfig(Ficus carica); and tolerant (high salin- salt spray based on its ability to consistently tion Department and the University of Adelaide ity: >6.0 dS·m–1 >60 mM NaCl >3,500 produce fruit in ‘‘front-line’’ coastal environ- scholarship. · –1 1Corresponding author. E-mail: carolyn.schultz@ mg L ; extremely high salinity: 25.2–33.8 ments that occur within their natural range; –1 adelaide.edu.au. dS·m 300–400 mM, seawater), for example, however, its tolerance to saline irrigation This is an open access article distributed under the date palm (Phoenix dactylifera)(Department water is unknown. Salinity assessment of CC BY-NC-ND license (http://creativecommons. of Human Services—South Australia, 1999; K. pomifera plants was listed as a research org/licenses/by-nc-nd/4.0/). USDA, 2017). priority in a recent evaluation of the Australian
1562 HORTSCIENCE VOL. 53(11) NOVEMBER 2018 | BREEDING,CULTIVARS,ROOTSTOCKS, AND GERMPLASM RESOURCES native food industry (Clarke, 2012). The aims ‘‘6 mm Premium’’ (Mount Gambier, South of this study were to establish a reference point Australia) with the following composition: for salt stress in K. pomifera. We did this by fine lime (2.0 kg·m–3), dolomite (1.0 kg·m–3), investigating the tolerance of four geno- gypsum (1.0 kg·m–3), potassium nitrate (0.4 types of K. pomifera to a broad range of soil kg·m–3), copper sulphate (0.03 kg·m–3), su- salinity levels and assessing physiological perphosphate (0.5 kg·m–3), ferrous sulphate performance. The analyses provide infor- (0.5 kg·m–3), urea (0.2 kg·m–3), micromix 240 mation on potential mechanisms of salinity (0.3 kg·m–3), Osmoform 38N-400102 (ICL tolerance in K. pomifera. Specialty Fertilizers, 0.5 kg·m–3), Osmoform NXT 4003 (ICL Specialty Fertilizers, 0.75 Materials and Methods kg·m–3), 3- to 4-month Osmocote exact mini 16–3.5–9.1 + TE (ICL Specialty Fertilizers, Design of experiments. A pilot experi- 2.5 kg·m–3), Kwik-Wet 225 (1 kg·m–3), with ment was conducted to establish methods and pH 6.2 and EC 1.2 dS·m–1. Additionally, appropriate application of saline irrigation Wettasoil (Amgrow, Lidcome, Australia) was solutions; two genotypes of K. pomifera were applied 1 week before NaCl treatments. tested, Maarten’s Favorite (MF) and MP1. NaCl treatments. Irrigation solutions of Plants, in draining pots, were irrigated with tap water (control) and up to 200 mM NaCl tap water (‘‘control’’), 50, 100, and 200 mM were applied directly to the plants each time NaCl. No growth parameters were measured (2–3 times per week and 250 mL saline and only EC (1:5) of potting mix and Na+ and solution per plant) for a 10-week period Fig. 1. Plant growth parameters: main stem, primary K+ content of leaves and roots are reported (Pilot: 3 Mar. 2016 to 10 May 2016; Expts. (1 ) branches and secondary (2 )branches. (see Results). Two experiments were sub- 1 and 2: 17 July 2016 to 26 Sept. 2016). For sequently conducted in parallel using four treatments of 300 and 400 mM NaCl applied diverse plant genotypes: MF, MP1, SES2, to MF only, an incremental concentration collected for fresh weight determination be- and CJ1 (Do et al., 2018). Expt. 1 evaluated was applied to minimize osmotic shock. For fore separating into leaves and stems. Fresh an expanded range of physiological and the treatment of 300 mM NaCl, 200 mM NaCl weights were recorded and leaves and stems morphological responses of MF plants to was applied for week 1, while 300 mM NaCl were dried separately at 65 C for 2 to 3 d a broad range of salinity treatments (control, was applied from week 2 to week 10. For the until constant weight. 50, 200, 300, and 400 mM NaCl). Expt. 2 treatment with 400 mM NaCl, 200 and For root samples, potting mix was re- compared the same responses of different 300 mM NaCl were applied to plants in weeks moved as much as possible. A subsample of plant genotypes (MP1, SES2, and CJ1) treat- 1 and 2, respectively, and 400 mM NaCl was roots was collected and washed using tap ed with tap water (control) and 50 and applied to plants in weeks 3 to 10. Electrical water, then rinsed using reverse osmosis 200 mM NaCl, except for CJ1 where no conductivity (EC1:5) of tap water was 0.8 water. Roots were placed on filter papers control treatment was performed because dS·m–1 (10 mM equivalent NaCl), and the (No. 5, Whatman) for 3 to 5 h at ambient insufficient plants were available, and it was solutions with additional NaCl (50, 100, 200, temperature until no water was left on the assumed that the other genotypes would pro- 300, and 400 mM NaCl) measured 5.2, 8.9, filter paper. Fresh weights were recorded and vide approximate control values. All exper- 17.8, 25.2, and 33.8 dS·m–1, respectively. the roots were then dried at 65 C for 2 to 3 d iments were conducted in a glasshouse Measurements of relative growth reduction until no weight change was observed. (South Australia Research Development In- in plant genotypes. Vegetative growth of plants Determination of Na+ and K+ content. stitute) at 23 ± 3 C with three to five pots per was measured at week 9 of the 10-week Approximately 0.1 g of dried leaves and treatment and one plant per pot (Pilot Exper- saline irrigation treatment (Expts. 1 and 2 roots were digested into 10 mL 1% HNO3. iment, n = 4–5; Expts. 1 and 2, n = 3–5). only). Plant growth parameters measured The samples were incubated at 80 C over- Plant propagation and establishment. consisted of the length of the longest main night (Berger et al., 2012). The digested Plants were propagated from 3- to 5-cm stem, the number of 1 branches containing samples were diluted 1:30 and 1:40 with vegetative cuttings; two plant genotypes, 2 branches, and the number of 2 branches Milli-Q water for samples of leaves and roots, MP1 and MF, were propagated by a commer- (Fig. 1). Relative growth reduction (%) was respectively. The diluted samples were used cial nursery (State Flora, South Australia), calculated as the mean of each of the in- to determine sodium and potassium levels whereas the SES2 and CJ1 genotypes were dicated growth parameters (50 mM NaCl) using a flame photometer (Model 420; Sher- propagated in the experimental glasshouse. compared with controls {100 – [100 · mean wood, UK). The results were calculated as All plants were 2 years old at the time of the (measurement at 50 mM NaCl)/mean (mea- micromoles per gram dry weight (mmol·g–1 experiment. These genotypes were chosen surement of the control)]} (Obermeier et al., DW) using a calibration curve generated because they are genetically diverse (Do 2013). The assessment of sensitivity of rela- from 5.8, 11.7, 17.4, 23.2, 29.0, and 34.8 et al., 2018) and differ in a variety of tive growth (%) was based on Cassaniti et al. mg·L–1 NaCl. horticultural traits, flavor (MF and MP1), (2009), although the applied treatments were Measurement of Cl- content. Cl– content and large fruit size (SES2), whereas CJ1 is slightly different: sensitive genotypes ($75% was determined using the method of Munns a small fruited, recent selection from a coastal growth reduction); moderately sensitive geno- (2010) with modification for a smaller vol- exposure in a low rainfall area of South types (between 50% and 75% growth reduc- ume, as follows. Mercuric thiocyanate [Hg Australia (Do et al., 2018). tion); moderately tolerant genotypes (between (SCN)2, 0.834 g] was dissolved in 100 mL To obtain plants with high uniformity 25% and 50% growth reduction) and tolerant methanol, then diluted with 200 mL metha- (Expts. 1 and 2), all plants were pruned to genotypes (#25% growth reduction). nol. The solution of Hg(SCN)2 was mixed one or two main stems (20–25 cm long), each Determination of chlorophyll content. well before filtering (No. 5 Whatman). Ferric having 2 to 4 primary branches (5 to 10 cm Chlorophyll content was determined in week nitrate [Fe(NO3)3 · 9H2O, 40.4 g] was dis- long) and repotted into round free-draining 9 of the 10-week saline irrigation treatment solved in 100 mL distilled water, then con- plastic pots (diameter 120 mm and height (Expts. 1 and 2 only) using a SPAD meter centrated HNO3 (5 mL) was added into the 150 mm). Plants for the pilot experiment (Model 502Plus; Konica Minolta Sensing ferric nitrate solution before diluting with were also repotted (90 · 90 mm cross- Inc.) with auto calibration settings. Milli-Q water to 200 mL and mixed. The section and height 180 mm) but were pruned Collection of shoot and root material for ferric nitrate solution was mixed well and less (retaining up to five main stems and 70– ion analysis. Subsamples of shoots and roots filtered (No. 5 Whatman). One hundred fifty 90 cm of each stem). Plants were grown for were collected separately for each plant. Five milliliters of mercuric thiocyanate solution 3 months. Potting mix used was Bio Gro to 10 shoots (5–10 cm each) per plant were was combined with the 150 mL ferric nitrate
HORTSCIENCE VOL. 53(11) NOVEMBER 2018 1563 solution; the mix was then diluted to 1 L Expt. 1: Evaluation of responses of MF using distilled water. Finally, 1.5 mL of the genotype to a broad range of saline irrigation diluted mercuric thiocyanate and ferric nitrate (control, 50, 200, 300, and 400 mM NaCl). mix was combined with 50 mLdigested1% Growth of MF plants. Growth of MF geno- HNO3 samples of leaves or roots and 450 mL type was inhibited in a NaCl concentration- 1% HNO3, and absorbance at 480 nm was dependent manner as shown in several determined using a spectrophotometer (CECIL growth parameters (Fig. 3A–E). EC1:5 of Model 2000; Cambridge, UK). The results potting mix increased with increasing exter- were calculated as micromoles per gram dry nal NaCl treatments (mean ± SD of 1.0 ± 0.7, weight (mmol·g–1 DW) using a calibration 5.91 ± 1.7, 8.1 ± 1.8, 13.3 ± 1.6, and 14.1 ± curve generated from 5, 20, 40, 80, and 1.4 dS·m–1 for control, 50, 200, 300, and 140 mg·L–1 NaCl. 400 mM NaCl treatments, respectively), Measurement of electrical conductivity showing an opposite trend to plant growth (EC1:5) of potting mix. Approximately 120 to (Fig. 3A–E). Plant growth and EC1:5 of the 150 g of potting mix was collected after re- potting mix were negatively correlated (lin- moving roots. Fresh weight was recorded, and ear regression) with R2 for main stem length then the mix was dried at 80 Cfor3to4duntil (0.79, P = 0.043), the number of 1 branches there was no change in weight. Ten grams of containing 2 branches (0.79, P = 0.044) and dried potting mix was mixed with 50 mL Milli-Q the number of 2 branches (0.71, P = 0.072). water before shaking at 200 rpm for 1 h. EC1:5 Plant growth reduced significantly when was determined using an EC meter (SmartChem- treated with 200 mM NaCl, compared with Lab, TPS, Brendale, Australia) as per Imada et al. control plants, but declined more dramati- (2015). cally for all three growth parameters when Statistical methods. Data were analyzed plants were exposed to 300 and 400 mM NaCl using either a one-way or two-way analysis of (Fig. 3A and C–E). Main stem length reduced variance for effects on different genotypes and 1.8-fold (Fig. 3A and C), and more dramatic NaCl treatments. Significance between means reductions were observed in the number of 1 was compared using Tukey’s multiple compar- branches containing 2 branches (7.0-fold) ison test (P < 0.05). Linear regression was (Fig. 3A and D) and the number of 2 performed between the growth characteristics branches (8.0-fold) (Fig. 3A and E) between (mean values) and the NaCl concentration. R2 control plants and plants treated with the and P values are reported. GraphPad Prism highest concentration of NaCl (400 mM). version 7 for MS Windows was used for all At week 9, most plants irrigated with calculations (GraphPad Software; La Jolla, CA). 200 mM NaCl had a few older leaves that were brown/pigmented on the main stem and Results primary branches, and the apical leaves were less expanded in all directions. At week 9, the Pilot experiment: Evaluation of responses MF plants treated with 300 mM NaCl de- of MF and MP1 to saline irrigation (control, veloped minor leaf symptoms, such as epi- 50, 100, and 200 mM NaCl). Saline irrigation nastic leaves (5/5 plants) and red margins on treatments applied to MF and MP1 plants the leaves of the primary apices (5/5 plants, Fig. 2. The effect of NaCl treatment on potting mix + + resulted in an increase in EC1:5 of the potting Fig. 3F). At 400 mM NaCl two plants had salinity and cation content (Na and K ) of two mix (Fig. 2A), suggesting the 10-week NaCl primary branches with leaves that looked K. pomifera genotypes, MP1 and MF. NaCl treatment was effective in applying a salt dehydrated based on leaf color change from treatments were control, 50, 100, and 200 mM stress. No leaf symptoms were observed in bright green to gray/green (Fig. 3G). In all NaCl corresponding to measured EC1:5 of any of the plants. Na+ accumulated in roots plants, most of the leaves were still bright irrigation solutions, 0.8, 5.2, 8.9, and 17.8 · –1 and leaves of both MF and MP1 genotypes, green, with no significant difference in rela- dS m , respectively. (A) Electrical conductiv- · –1 + ity (1:5) of potting mix (dS m ) and concen- with leaf Na increasing 8.1-fold and 4.7- tive water content (%, P = 0.1069). A trations (mmol·g–1 DW) of (B)Na+ and (C)K+ fold at 200 mM NaCl compared with the significant difference was detected in chloro- in leaves and roots (above and below the x axis, controls, respectively. Sodium accumulated phyll content (P = 0.0176), with a reduction respectively). Data are means of four to five to higher levels in roots of plants watered in chlorophyll content (SPAD values) evident replicates ± SD. Letters (a different one for each with 200 mM NaCl (Fig. 2B). No significant from the control treatment (58.5 ± 2.2) to the genotype and tissue) are used to indicate decrease in leaf potassium was measured for 400 mM-treated plants (51.3 ± 3.5), although different tests and subscripted numbers are either genotype, although a significant re- none of the pairs of means was statistically used to indicate significant differences among duction in root potassium (1.7-fold) was significant. treatments according to Tukey’s multiple com- parison test (P # 0.05). observed for both MF and MP1 at 200 mM At week 10, some of the symptoms in the NaCl compared with the controls (Fig. 2C). 400 mM NaCl-treated MF plants worsened, Thus, the pilot experiment demonstrated that and two plants had primary stems that had sues under saline conditions (Fig. 3J). Plants the 200 mM NaCl irrigation treatment applied died (Fig. 3H). The stress was so severe that treated with the highest salinity concentration a relatively mild salt stress, affecting leaf and approximately half the plants within each of (400 mM NaCl) accumulated significantly root Na+ levels, but not leaf K+, with both the 300 and 400 mM NaCl treatments died more Na+ than control plants, and the fold genotypes responding in a similar fashion. within 1 week of repotting following root change in leaves (29.8-fold) was greater than To extend these preliminary findings, two harvesting and resumption of normal (nonsa- the fold change in roots (4.5-fold) (Fig. 3J). parallel experiments were conducted, the first line) watering, whereas most plants treated with No significant difference in Na+ levels was (Expt. 1) on a specific genotype, MF, with 0, 50, and 200 mM NaCl survived soil removal, observed in leaf and roots of plants grown at a broad range of saline irrigations, and the root harvesting, and repotting (data not shown). 50, 200, and 300 mM NaCl (Fig. 3J). second, Expt. 2, which used three treatments Even at 50 and 200 mM NaCl, a noticeable A similar increasing trend was observed (up to 200 mM) and three genotypes (MP1, decline in healthy white roots was evident when in the chloride content in leaves and roots at SES2, and CJ1). Insufficient plants were the pots were removed (Fig. 3I). different levels of salinity stress (Fig. 3K). available to test all genotypes across the Na+ and Cl- content in leaves and roots of Chloride content in leaves and roots in- expanded range of NaCl concentrations. MF. Sodium content increased in plant tis- creased in plants treated with 400 mM NaCl,
1564 HORTSCIENCE VOL. 53(11) NOVEMBER 2018 Fig. 3. Response of K. pomifera MF plants to a broad range of saline irrigation treatments. (A) MF plants in pots at week 9 of a 10-week treatment (control, 50, 200, 300, and 400 mM NaCl treatments). (B) MF plants after removing potting mix, (C) main stem length, (D) number of 1 branches containing 2 branches, (E) number of 2 branches, (F) plant 21 at Week 9 (300 mM NaCl treated, inset with close-up of leaf symptoms), (G) five plants from the 400 mM NaCl treatment (week 9, with close-up of leaf symptoms below), (H) two plants at week 10 of 400 mM NaCl treatment (harvest), (I) root growth before removal of potting media, and content (mmol·g–1 DW)of(J)Na+,(K)Cl-,and(L)K+ in leaves and roots (above and below the X axis, respectively) of MF plants after 10 weeks of treatment. Data are means of three to five replicates ± SD. Letters above the bars (C–E) indicate significant differences among treatments according to Tukey’s multiple comparison test (P # 0.05). In F and G, white arrows indicate leaves with symptoms on the margins, * indicates dead leaves, and black arrows indicate dehydrated/graying leaves. In J–L, letters a (for shoots) and b (for roots) are used to indicate different tests, and subscripted numbers are used to indicate significant differences among treatments.
13.7-fold (from 306 ± 50.2 to 4182 ± 1442 (Fig. 3K). Chloride was 1.4·, 3.8·, and K+ content in leaves and roots of MF mmol·g–1 DW in leaves) and 5.4-fold (from 2.0· higher in leaves than in roots of plants plants. K+ decreased significantly in roots 387.6 ± 294.3 to 2102 ± 715.3 mmol·g–1 DW exposed to 200, 300, and 400 mM NaCl, and leaves of MF with saline irrigation at in roots) compared with control plants respectively. 200 mM NaCl and above (Fig. 3L). The K+
HORTSCIENCE VOL. 53(11) NOVEMBER 2018 1565 content of leaves and roots of plants exposed for SES2 in the 50 mM NaCl treatment. All 50 mM NaCl was 3.1-fold, 7.0-fold, and 4.1- to 400 mM NaCl reduced 0.7-fold and 5.7- three growth parameters remained relatively fold higher for MP1, SES2, and CJ1, re- fold, respectively, compared with control stable at 50 mM NaCl in SES2 compared with spectively (Fig. 4D). plants [reduced from 967.1 ± 169.5 to 713.8 ± controls (Fig. 4A–C), whereas a decrease was The saline irrigation treatments also 121.2 mmol·g–1 DW (leaves) and from 394.0 ± observed in the number of 1 branches con- resulted in an increase in Cl- content in leaves 164.4 to 69.0 ± 34.1 mmol·g–1 DW (roots)] taining 2 branches (Fig. 4B) and the number of all three plant genotypes (Fig. 4E). CJ1 (Fig. 3L). The K+ content of leaves and roots of of 2 branches for MP1 (Fig. 4C). At 200 mM accumulated significantly more Cl- in leaves plants grown at 200, 300, and 400 mM NaCl NaCl, the number of 1 branches containing than MP1 at 200 mM NaCl (Fig. 4E). In roots, (Fig. 3L) was not significantly different. 2 branches reduced 3-fold for both MP1 there was a trend toward higher Cl- content in In summary, increasing salinity resulted and SES2 plants (compared with controls) roots at 50 mM NaCl (significant for SES2), in significant changes in plant growth and ion and 2-fold in CJ1 plants (compared with the but levels decreased (nonsignificantly) for all accumulation in MF plants. Growth of MF 50 mM NaCl treated plants) (Fig. 4B). The genotypes at 200 mM NaCl (Fig. 4E). + + plants dramatically declined at the highest EC1:5 of the potting mix increased with In contrast to accumulation of Na ,K salinity treatments of 300 and 400 mM NaCl. increasing concentration of NaCl in the irriga- content decreased significantly in roots of + - Na and Cl accumulated in both leaves and tion water as expected. EC1:5 values were MP1 and SES2 plants treated with 50 mM roots of MF plants whereas K+ levels de- consistent with those obtained in Expt. 1 (data NaCl, 3.4-fold and 1.8-fold compared with creased, particularly in roots. not shown). control plants [from 517.7 ± 5.3 to 153.8 ± Expt. 2: evaluation of three additional K. Content of Na+,Cl- and K+ in plant tissues 41.6 (MP1) and from 408.1 ± 134.4 to pomifera genotypes (MP1, SES2, and CJ1) of MP1, SES2, and CJ1 genotypes. Treatment 223.4 ± 76.6 (SES2) mmol·g–1 DW]. Leaf with saline irrigation (control, 50, and with NaCl resulted in a significant increase in K+ content was significantly lower for SES2 at 200 mM NaCl). Growth of MP1, SES2, and Na+ in the roots, compared with the controls, 50 mM NaCl compared with the control plants, CJ1 plants. At 50 mM NaCl, the main stem of MP1 at 200 mM NaCl (1.9-fold) and but for MP1 there was no significant difference length of all three genotypes was similar to SES2 at 50 mM NaCl (2.0-fold) (Fig. 4D). (Fig. 4F). At 200 mM NaCl, CJ1 showed the control plants (Fig. 4A). Stem length The levels of Na+ in the root of CJ1 at 50 and a different trend (an increase) in leaf K+ decreased 0.3 to 0.4 fold in MP1 and SES2 200 mM NaCl were similar to the levels of relative to 50 mM NaCl, and this response (from control to 200 mM NaCl) and 0.4-fold MP1. All three genotypes had a significant was significantly different from that of the in CJ1 (from 50 to 200 mM NaCl) (Fig. 4A). increase in Na+ in the leaf at 200 mM NaCl other two genotypes (Fig. 4F). Branching [number of primary (1 ) branches compared with controls (MP1 and SES2) or On the basis of the modest percentage containing secondary (2 ) branches, and the 50 mM NaCl (SES2 and CJ1) (Fig. 4D). For reduction in relative growth, SES2 and CJ1 number of 2 branches] was inhibited with example, the increase in Na+ content in leaves may be considered moderately tolerant ge- increasing salinity in most genotypes except of plants grown at 200 mM compared with notypes (25.3% and 29.3% reduction in the
Fig. 4. Growth, development, and ion content (Na+,Cl-, and K+) of three K. pomifera genotypes in response to moderate salinity. (A) Main stem length, (B) number of 1 branches containing 2 branches, (C) number of 2 branches, content (mmol·g–1 DW) of (D)Na+,(E)Cl-, and (F)K+ in leaves and roots (above and below the X-axis, respectively) of MP1, SES2, and CJ1 plants after 10 weeks of treatment (control and 50 and 200 mM NaCl). Data are means of three to five replicates ± SD. Letters (a different letter for each genotype/tissue combination) are used to indicate different tests and subscripted numbers are used to indicate significant differences between NaCl concentrations according to Tukey’s multiple comparison test (P # 0.05); x1 and x2 represent significant differences between plant genotypes at 50 mM NaCl treatments and y1 and y2, between plant genotypes at 200 mM NaCl treatments.
1566 HORTSCIENCE VOL. 53(11) NOVEMBER 2018 number of 2 branches, respectively), while three growth parameters in SES2 and CJ1 and maintain cells with normal metabolism MF1 and MP1 may be considered moderately remained relatively stable compared with the and functioning (Shabala and Pottosin, sensitive genotypes (55.7% and 59.3% re- other genotypes [assuming that the estimated 2014). In this study, the higher K+ content duction in the number of 2 branches, re- control value for CJ1 is realistic (see Table 1), in leaves compared with roots could be due to spectively) (Table 1). Therefore, based on which will need to be confirmed in future a homeostasis of K+ in leaves. One genotype growth parameters, the salinity tolerance of experiments]. At 200 mM NaCl, deleterious (MP1) maintained stable leaf K+ content at four K. pomifera genotypes may be ranked as effects on growth parameters were observed 200 mM NaCl, two genotypes had a modest SES2 CJ1 > MF MP1. across all genotypes, although MP1 had decrease in leaf K+ (MF and SES2), and one a slightly higher number of 2 branches so genotype had an increased K+ content (CJ1) Discussion could be considered more tolerant of higher (Figs. 3I and 4F). The mostly stable K+ levels of salinity. content in leaves of K. pomifera indicates These experiments have evaluated the Effect of salinity on accumulation of ions active transport of K+ from roots to shoots as salinity tolerance of four K. pomifera geno- in K. pomifera plant tissues. In all four K. has been suggested for Juncus spp. (Al types. The findings suggest that genotypic pomifera genotypes, a similar accumulation Hassan et al., 2016). More evidence for leaf variation exists for traits that are responsive pattern of Na+ was observed under low and K+ homeostasis was observed in the pilot to saline irrigation in this small yet genotyp- moderate salinity (control and 50 mM NaCl), experiment, where plants with more canopy ically diverse sample. where Na+ mainly accumulated in roots (less severely pruned) were used. Plants in Reduction in plant growth. Because there (Figs. 3J and 4D) suggesting that both sodium the pilot experiment were multistemmed as are limited reports of quantitative growth exclusion and tissue tolerance (Munns and observed in natural environments, and this assessment of K. pomifera either in the field Tester, 2008) may be contributing to salinity apparently provided a sodium dilution effect or in pots (Page, 2004), it was necessary to tolerance in K. pomifera. At moderate salin- because leaf Na+ accumulation was 2-fold decide which characteristics to measure. At ity (50 mM NaCl), Na+ could be either lower for multistemmed MF (Fig. 2A) com- low, moderate, and high salinity levels (con- maintained in root vacuoles, effluxed from pared with single-stemmed MF plants trol, 50 and 200 mM, respectively), reductions cells to the apoplast, or excreted to the soil. If (Fig. 3J), and for multistemmed MP1 plants in three traits were observed: main stem any or all of these situations occur, Na+ (Fig. 2A) compared with single-stemmed length, number of 1 branches containing 2 transport to leaves would be negligible, MP1 plants (Fig. 4D). Leaf K+ was un- branches, and number of 2 branches accounting for the low Na+ content (not changed in the multistemmed MF plants, (Figs. 3C–E and 4A–C). This study showed significantly different from the controls) in even at 200 mM NaCl (Fig. 2C) compared that the number of 2 branches was the most leaves relative to roots. However, at high with the significant K+ reduction seen in responsive parameter to increasing saline salinity (200 mM NaCl), the Na+ levels in the single-stemmed MF plants (Fig. 3L). This irrigation based on a significant reduction in shoots increase significantly, but the levels in suggests that plants in the field may be two genotypes at 50 mM NaCl (MF and MP1), roots remained relatively unchanged, and tolerant to higher levels of saline irrigation followed by the number of 1 branches excess Na+ was likely delivered to leaves treatments than those grown in pots, which containing 2 branches (significant reduction through the transpiration stream. We there- should be tested in the future. in MP1 at 50 mM NaCl), and main stem length fore hypothesize that Na+ is compartmental- A few comments are provided on the (significant reduction in all four genotypes at ized in leaf vacuoles to prevent high toxicity technical aspects of these experiments. Large 200 mM NaCl) (Figs. 3C–E and 4A–C). Other to the cytoplasm (Munns and Tester, 2008; error bars were observed in some experiments growth traits were measured, including number Roy et al., 2014) because only minor leaf and we conclude that these were likely due to of 1 branches, number of freshly emerged symptoms were observed at 200 mM NaCl. ‘‘real differences’’ in the tissues samples rather leaves, length of 1 branches and of 2 At the highest salinity levels in this trial than low replication because the samples with branches; these parameters were either not (300 and 400 mM NaCl), Na+ accumulated to the largest error bars, SES2 and CJ1, at 200 mM significant (number of 1 branches) or were higher levels in leaves compared with roots (Fig. 4D and E) had four and five replicates, difficult to measure objectively. of MF plants [1.2-fold higher (300 mM) and respectively. One possible explanation for this Growth reductions during the ion- 1.9-fold higher (400 mM)], whereas the ratio is the use of a variable number of older and accumulation independent phase have two at high salinity (200 mM NaCl) was 0.76. At younger leaves between samples. This could be broad consequences: reduction in cell divi- extremely high salinity levels, plants devel- tested in the future by comparing the ion sion and cell elongation (Fricke and Peters, oped yellow-brown senescent leaves at the content of leaves on the main, primary and 2002; Passioura and Munns, 2000). To de- end of week 9, and the MF plants deteriorated secondary stems, and ensuring that similar termine which processes are affected in quickly in week 10 of NaCl application, with proportions of each are taken when sampling. saline-treated K. pomifera, future studies leaf drop producing leafless regions of stem Another difference that could be imple- could measure internode length on the main (Fig. 3G and H). Future efforts to identify mented in future experiments would be to stem (Abassi et al., 2014) and epidermal cell genotypes that are more salt tolerant than ensure that micronutrient activity is main- size (Wu et al., 2015). SES2 and CJ1 should consider measuring tained by calculating the concentration of On the basis of the categories of relative different traits such as leaf color (yellow/ micronutrients needed to compensate for the tolerance of plants to saline water (see In- brown), epinasty, and length of leafless stems impact of NaCl on for example calcium troduction) and compared with other fruit (due to leaf loss) when K. pomifera is availability (Jha et al., 2010). This can be crops grown in these salinity ranges, K. exposed to high salinity levels. done using programs such as Visual Minteq pomifera may be considered moderately tol- Maintaining potassium homeostasis in the Version 2.3 (KTH, Department of Land and erant to salinity (up to 50 mM NaCl) as the cytoplasm is vital to avoid sodium toxicity Water Resources Engineering, Stockholm, Sweden). We elected not to do this in these Table 1. Relative growth reduction (%) of K. pomifera genotypes exposed to 50 mM NaCl compared with experiments to mimic the effect of using saline nonsalt-treated K. pomifera.z groundwater, which is generally used as is. An important consideration for future Relative growth reduction at 50 mM NaCl (%) experiments is to measure the salinity of Genotype Main stem length 1 branches with 2 branches No. 2 branches leachate at each watering to establish the soil MF 19.7 34.9 55.7 MP1 0.0 44.6 59.1 salinity build up during the experiment. We SES2 0.0 0.0 25.3 allowed the salinity to build up during these CJ1 1.4 14.2 29.3 experiments to mimic the effects of salt build z Calculation for each genotype and growth trait was {100 – [100 · mean (measurement at 50 mM NaCl)/ during the hot, dry summers of southern mean (measurement of the control)]}. For CJ1, a control value was estimated from the mean control values Australia. New crops are often watered with- of MF, MP1, and SES2. Bold indicates values where growth reductions are less than 30%. out consideration of the need for additional
HORTSCIENCE VOL. 53(11) NOVEMBER 2018 1567 water to ensure that irrigation water reaches different natural environments. Funct. Plant Nations (FAO). 25 Mar. 2017.
1568 HORTSCIENCE VOL. 53(11) NOVEMBER 2018 marketing approach using muntries, an Austra- Australian Native Plants: Cultivation and Uses Agriculture Agricultural Research Service. 25 lian native berry. Food Austral. 61:335–341. in the Health and Food Industries. CRC Press, Mar. 2017.
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