Plnt Stress 29 Glol Science Books Effect of Slinity on Puccinelli distns (L.) Prl. Treted with NCl nd Folirly Applied Glycineetine Roslind Scli 1* Elisett Oddo 2 Filippo Sino 1 Frncesc Grisfi 2 1 Diprtimento di Ingegneri e Tecnologie Agroforestli, vile delle Scienze ed. 4 9128 Plermo, Itly 2 Diprtimento di Scienze Botniche, vi Archirfi 38-9123 Plermo, Itly Corresponding uthor: * r.scli@unip.it ABSTRACT Turfgrsses generl ppernce is much ffected y environmentl stresses ecuse the species used for this purpose re prticulrly exigent in terms of technicl inputs nd wter need. In the Mediterrnen re, sometimes irrigtion is provided y using wste wter which my contin high concentrtions of dissolved slts which cn cuse slt stress injury nd poor turf qulity. Puccinelli distns (L.) Prl. is hlophyte cool seson grss tht seems to hve high slinity tolernce when cultivted in sodic soils or in NCl-rich hydroponic cultures. We investigted the response of P. distns to slinity in soil culture in controlled growth environment. The effect of different concentrtions of NCl (85, 275 nd 6 mm) on shoot nd root growth nd chloride content ws ssessed. After determining the resistnce of P. distns to the highest slinity level, we tested the efficiency of the osmoprotectnt glycineetine (GB) in reducing slt stress effects y mesuring some physiologicl prmeters. The results showed very good dptility of P. distns to slinity conditions. When irrigted with slt solution t the highest concentrtion (6 mm), plnts showed reduction in growth rte nd iomss production tht seemed to e relieved y GB ppliction. With GB ppliction, lef reltive wter content nd iomss production were similr to the control. However, GB ppliction did not result in generlly etter turf qulity compred to untreted plnts, so it did not give relevnt dvntge in reducing technicl nd lor inputs with respect to the mintennce of P. distns turf. Keywords: chlorophyll content, glycineetine, Puccinelli distns, slt stress, turfgrss Arevitions: DW, dry weight; FW, fresh weight; GB, glycineetine; RWC, reltive wter content; TW, turgid weight INTRODUCTION Despite the economic importnce nd continuous pulic demnd for turf res, the current drinking wter shortges in ll Mediterrnen res clerly set limits on the mount nd qulity of wter ville for lndscpe irrigtion, so non-potle wter sources my e used for this purpose (Mrcum 24). However, effluent wter or low qulity groundwter cn often contin high concentrtions of dissolved slts. The detrimentl effects of slinity on turfgrss growth include osmotic stress, ion toxicity, nd nutritionl disturnces (Greenwy nd Munns 198; Luchli 1986; Cheesemn 1988). Soil slinity, in low to moderte concentrtions, minly reduces growth due to its osmotic effect (Greenwy nd Munns 198; Munns nd Termt 1986). At higher concentrtions, slts my ccumulte in the leves to toxic level, resulting in scorching or firing of leves (Bñuls nd Primo-Millo 1992; Storey nd Wlker 1987). The detrimentl effects of stress on growth nd lef firing re prolem for turf mintennce, s density nd color re importnt trits tht chrcterize turf qulity. Sodium (N + ) nd chloride (Cl - ) re the two key ions responsile for oth osmotic nd ion-specific dmge tht significntly reduce growth nd yield (Munns nd Tester 28). Slt tolernt plnts hve the ility to minimize these detrimentl effects with series of ntomicl, morphologicl, nd physiologicl dpttions, such s n extensive root system nd slt secreting glnds on the lef surfce (Liphschitz nd Wisel 1974; Oross nd Thomson 1982; Mrcum nd Murdoch 199; Mrcum et l. 1998). Some growth indictors hve een relted to slinity tolernce, such s root length nd/or weight in turfgrsses cultivted in liquid medium (Mrcum 1999; Alshmmry et l. 24; Mrcum 24). Some uthors hve found tht slinity growth curves for shoot nd root cn e considered the most importnt prmeter to clssify slinity resistnce of hlophytes nd their ecotypes (Lee et l. 24, 25); some others hve mesured gs exchnge prmeters nd chlorophyll content to relte them with the reduction of iomss production (Hmeed nd Ashrf 28). Limited informtion is ville to compre slinity responses of shoot nd root in solution culture versus soil culture systems. Glycineetine (GB) is n osmoregultor produced nturlly y mny hlophytes (Guy et l. 1984), which is involved in reducing cellulr dmge under stress conditions (Rjsekrn et l. 1997; Chen nd Murt 28). Severl uthors hve studied GB s n osmoprotectnt in the dpttion to wter, slt nd cold stress (Guy et l. 1984; Kishitni et l. 1994; Xing nd Rjshekr 21; Girij et l. 22). Mäkelä et l. (1996) reported tht plnts re le to use folir-pplied GB nd trnslocte it to lmost ll plnt prts, especilly developing orgns. Thus, folir pplictions increse the levels of GB in plnts tht re unle to synthesize this compound (Agom et l. 1997). P. distns is hlophyte cool seson grss dpted to wide rnge of soils nd climtic conditions, le to settle lso on slty soils (Trsoff et l. 27). The choice of turf species tht responds well to slty wter irrigtion nd of nturl osmoregultor tht cn e pplied to reduce slt stress effects could e key-point for turfgrss specilists. The ims of this study were: to determine the ehvior of turf of P. distns irrigted with sline wter nd to verify whether GB could e used to reduce effects of NCl stress, chieving etter turf qulity compred to untreted plnts. In first experimentl tril we mesured lef height, Received: 8 July, 29. Accepted: 9 Decemer, 29. Originl Reserch Pper
Plnt Stress 3 (1), 49-54 29 Glol Science Books fresh nd dry weight, Cl - mount, totl osmolyte mount in roots, shoots nd leves of P. distns grown in soil filled pots under controlled conditions, irrigted with different NCl concentrtions (85, 275 nd 6 mm). In second experimentl tril we tested the effect of folir ppliction of GB on plnts irrigted with 6 mm NCl. MATERIALS AND METHODS Plnt mteril nd tretments P. distns seeds (15 g m -2 ) were sown in 465.5 cm 3 pots. The soil mixture used ws 45% snd, 45% orgnic rich soil (C/N=25), 5% pet nd 5% orgnic mnure (swine nd equine, Jolly Flor Pellet, C/N=12.5) (ph 6.5). This orgnic rich soil is typiclly used in nurseries when optimum growth conditions re required, nd with the ddition of snd it ws dpted for turfgrss. Mintennce fertiliztion ws provided y dding slow relesing fertilizer (OSMOCOTE plus, 15: 9: 11: 2 w/w N: P 2 O 5 : K 2 O: MgO trces of B, C, Cu, Fe, Mn, Mo, S nd Zn; Scotts ) to the soil mixture. The growth-chmer ws set up t 25 ± 1 C with 12 h photoperiod provided y neon fluorescent lmps (PAR 45.5 μmol m -2 s -1 t the lef surfce). The first experimentl cycle ws crried out to test the response of P. distns to different NCl concentrtions. Pots were irrigted with tp wter until the 4 th dy fter sowing, when slt tretments were strted. For slt tretments, pots were irrigted once week with 2 ml of NCl solution, until the 9th dy fter sowing. In the first experimentl cycle, the three NCl concentrtions tested were: 85, 275 nd 6 mm. Control pots were irrigted with tp wter throughout the experiment. In the second experimentl cycle the effect of folir ppliction of GB ws tested. Sowing nd growth conditions were the sme s ove, nd slt tretment ws provided with 6 mm NCl solution. GB tretment ws provided y hnd-sprying ech pot with 1 ml of.1 M GB solution (Agom et l. 1997), once week from the 4 th to the 9 th dy fter sowing. Throughout the experiments, wter stress ws voided y checking the weight of ll pots dily nd irrigting ech one to field cpcity with tp wter if necessry. Irrigtion with wter or sline solution ws lwys done slowly, to void percoltion nd leching of NCl from the pots. Destructive nd non-destructive growth nlysis All plnts were cut once week, t height of 5 cm from the soil surfce, eginning from the 4 th dy fter sowing. To determine growth rte, plnt height ws mesured efore ech weekly cut; lef iomss excised from individul pots during ech cut ws weighed for the determintion of clipping fresh weight (FW), dried t 7 C overnight nd weighed to determine clipping dry weight (DW). The lef reltive wter content (RWC) of lef smples excised during the lst cut for ech pot ws mesured. The lef portions were weighed (fresh weight FW), floted on wter for two hours to llow turgidity to e regined, weighed gin (turgid weight, TW) nd dried overnight t 7 C to determine dry weight (DW). The reltive wter content ws clculted s (FW DW)/(TW- DW). At the end of the experiment, leves nd shoots were removed nd weighed for the determintion of totl iomss fresh weight. Root length ws mesured s root extension (in cm) from the stemses to the frthest extending root. Roots were wshed with deionized wter, lotted dry, weighed to determine fresh weight, nd then dried t 7 C for 24 h to determine root dry weight. Generl overll condition of plnts ws monitored nd percentge of lef firing ws ttriuted t the end of oth the experimentl cycles. The turf qulity nd lef firing on pots were visully estimted with the ttriution of percentge of chlorotic leves respect the totl. Sp nlysis nd chloride content A 1 g smple of the iomss tken t the end of the experiment from ech pot ws used for the extrction of folir sp. The lef smple ws put in plstic hypodermic syringe, frozen in liquid nitrogen nd fter thwing sp ws expressed nd collected in n Eppendorf vil. The sp smples were centrifuged (Eppendorf Microfuge) to precipitte cell deris nd 5 μl of the superntnt were used for nlysis. The totl osmollity of cell sp (expressed per grm of lef fresh weight) ws mesured with cryoscopic osmometer (Osmomt 3 GONOTEC). The quntity of chloride ion (s meq per grm of lef fresh weight), ws determined y titrtion on the sme sp smples (Vogel 1989). A smple of 1g of fresh roots per pot ws used for the extrction of sp s descried ove for leves nd the Cl - ion content of the sp ws determined y titrtion. Soil of ech pot ws dried t 15 C nd then used for the determintion of Cl - y titrtion (Vogel 1989). Chlorophyll content At the end of the second experimentl cycle,.1 g of lef iomss for ech pot were extrcted in 8% cetone (Arnon 1949) nd chlorophyll content ws determined y spectrophotometric nlysis (Beckmn Coulter DU8), sornce of the superntnt ws red t 652 nm. An identicl quntity of leves from ech pot ws tken nd dried overnight t 7 C to determine dry weight tht ws used to express the totl chlorophyll content results. SEM To check for the presence of slt glnds, lef surfce ws oserved y scnning electron microscopy (Leic Cmridge LEO 42) on.5 cm long lef segments, dried overnight t 6 C, fixed on luminum stus nd gold metlized under vcuum. Experimentl design nd dt nlysis Pots were rrnged in complete rndomized design, with five replictes per thesis. Men vlues nd stndrd devitions were clculted nd the significnce (P<.5) of differences etween sets of dt ws tested using one-wy ANOVA, followed y Tukey s test in cses involving significnt F-vlues (Systt softwre, SYSTAT Inc.). Correltion etween different prmeters ws crried out pplying Person correltion (SPSS softwre, SPSS Inc.). RESULTS AND DISCUSSION A 9-dy experimentl cycle ws set up to study the effects of different NCl solutions on P. distns in terms of growth nd Cl - ion ccumultion. Mesurements of the mount nd distriution of Cl - were chosen ecuse this is n essentilly free ion in the circulting solution nd immeditely ville for the plnts (White nd Brodley 21), while soil ction exchnge cpcity my reduce the uptke of N +. Furthermore there is evidence tht in glycophytes such s pepper, the tissue concentrtion of N + ws lower thn tht of Cl - (Silv et l. 28) nd tht monocotyledonous hlophytes tend to tke up less N + in the shoot thn dicotyledonous hlophytes (Tester nd Dvenport 23). Vrition in lef firing, shoot nd root weight re highly correlted, nd re therefore useful prmeters to predict slinity tolernce (Mrcum nd Murdoch 199; Mrcum 26). On the other hnd, plnt height, lef reltive wter content, chlorophyll content nd totl produced iomss re physiologicl prmeters tht cn give informtion on the overll generl condition of plnts nd their metolic efficiency. Destructive nd non-destructive growth nlysis Tle 1 shows the plnt height reched efore ech weekly cut nd the weight of lef iomss removed. On the 17 th dy fter irrigtion with slty wter, corresponding to the second week of tretment, plnts wtered with 6 mm NCl solution hd lower growth rte respect the control, while plnts treted with 85 mm NCl solution hd growth rte higher thn the controls (P<.5). This ehvior is in 5
Effect of slt nd glycineetine tretments on Puccinelli distns. Scli et l. Tle 1 Plnt height reched efore every weekly cut nd lef weight of removed iomss, dt re the mens of five replictes. Tretments CONTR 85 mm 275 mm 6 mm Lef+shoot weight Plnt height Lef+shoot weight Plnt height Lef+shoot weight Plnt height Lef+shoot weight Plnt height cut I.2 ±.8 13 ± 4.22 ±.1 14 ± 1.25 ±.6 14 ± 2.17 ±.4 11 ± 1 II.12 ±.1 1.3 ±.7.13 ±.2 12 ± 1*.11 ±.3 1.6 ±.8.6 ±.1 9.7 ±.4 III.18 ±.5 12.4 ±.9.22 ±.4 11.9 ±.4.19 ±.5 12 ± 1.16 ±.3 1 ± 1 IV.8 ±.3 9. ±.5.9 ±.3 9. ±.8.2 ±.4 9 ± 3.2 ±.5 9 ± 1 V.11 ±.3 9.3 ±.3.4 ±.3 1 ± 1.17 ±.1 11 ± 1.12 ±.2 11 ± 1 VI.1 ±.5 1 ± 1.3 ±.3 1 ± 1.12 ±.2 1 ± 1.12 ±.4 1 ± 2 VII.5 ±.2 7 ± 1.7 ±.1 8.2 ±.6.8 ±.1 8 ± 1,6 ±,2 6.8 ±.8 g cm * this vlue is significnt for P<.5 Root to shoot rtio (g) 1.2 1.8.6.4.2 Control 85 mm 275 mm 6 mm Fig. 1 Root to shoot fresh weight rtio of the plnts t the end of the first experimentl cycle. Dt re the mens of five replictes, stndrd devition ws clculted on the sis of the rtio root to shoot for ech replicte. Tukey s pired test of the rtio ws not significnt. Folir fresh weight (g) 2.5 2 1.5 1.5 Control GB NCl+GB NCl Fig. 2 Men weight of the fresh folir (shoot + lef) iomss, hrvested on the 9 th dy fter sowing, t the end of the second experimentl cycle, fter 6 weeks of weekly tretments with slt nd GB depending on the thesis. Different letters indicte significnt differences t P<.5 using Tukey s test. greement with other studies reporting tht in mny hlophyte species high slinity levels cuse reduction of plnt growth, wheres low slt concentrtion initilly promotes growth rte (Luchli 1986; Jungklng et l. 23). Lef men height nd clipping weight of susequent cuts were slightly higher for plnts treted with 85 mm nd 257 mm NCl when com pred to controls nd 6 mm NCl, ut these differences were not sttisticlly significnt. Fresh weight of shoot + leves removed on dy 9 th fter sowing, fter 6 weeks of tretments, is not sttisticlly different mong the four tretments. Totl fresh shoot + lef iomss removed t the end of the first experiment ws slightly lower in the cse of irrigtion with the highest concentrtion of NCl, 6 mm. These results confirm tht P. distns my e considered n osmotic tolernt species, ccording to the definition used y Rjendrn et l. (29) for c Folir dry weight (g).35.3.25.2.15.1.5 Control GB NCl+GB NCl Fig. 3 Men weight of the dry folir (shoot + lef) iomss hrvested on the 9 th dy fter sowing, t the end of the second experimentl cycle fter 6 weeks of weekly tretments with slt nd GB depending on the thesis. Different letters indicte significnt differences t P<.5 using Tukey s test. plnts which mintin under slt stress conditions growth rtes similr to those of control plnts. The root to shoot fresh weight rtio clculted t the end of the first cycle showed higher vlues with incresing NCl concentrtions (Fig. 1), though the differences were not sttisticlly significnt. Alshmmry et l. (24) reported similr ehviour for oth Puccinelli distns nd Festuc rundince: incresed rooting my e n dptive mechnism to overcome the osmotic nd nutrient deficiency stresses present under sline conditions y incresing root soring surfce t the expense of ove-ground iomss. The results of the first experimentl cycle showed tht P. distns ws le to tolerte irrigtion with the highest NCl concentrtion tested, showing 3% of lef firing tht could e considered cceptle nd interesting for further tests. For this reson second 9 dy experimentl cycle ws crried out to see if folir pplictions of GB could reduce the effects of slt stress resulting from irrigtion with 6 mm NCl. Fig. 2 shows tht, t the end of the second experimentl cycle, NCl-treted plnts hd the lowest fresh iomss. GB tretment incresed lef nd shoot fresh weight oth in slt stressed nd non-stressed plnts. In non stressed plnts, GB lso incresed dry iomss, while this did not occur in slt stressed plnts (Fig. 3), where GB ppliction ppered to ffect tissue wter content. This ws lso confirmed y the higher reltive wter content in the NCl+GB tretment with respect to the slt tretment (Fig. 4). This effect of GB hs een reported lso in other species such s tocco (Agom et l. 1997) nd Kidney en (Lopez et l. 22). Tretment with GB of NCl-irrigted plnts, however, did not ffect lef firing, tht ws gin out 3% respect to the control. c c 51
Plnt Stress 3 (1), 49-54 29 Glol Science Books.7.6 c Lef Reltive Wter Content (g).5.4.3.2 d.1 Control GB NCl+GB NCl Fig. 4 Lef reltive wter content t the end of the second experimentl cycle. Different letters indicte significnt differences t P<.5 using Tukey s test. 35. 3. 25. Osmoles of slts/gfw Osmoles of slt/g DW.4.35.3 moles of Cl-/g FW mmoles of Cl-/g DW 2. 15. 1. 5.. Control 85 mm 275 Mm 6 mm mmoles/g.25.2.15.1.5 Fig. 5 Totl osmollity of lef sp extrcted t the end of the first experimentl cycle reported oth on fresh nd dry tissue weight sis. Folir sp nlysis nd Cl - content Totl osmolrity nd Cl - content of lef sp re shown in Figs. 5 nd 6 expressed oth on fresh weight nd dry weight sis. The comprison of the totl osmolrity with the quntity of chloride ion per grm of lef fresh weight shows tht plnts treted with 6 mm NCl hd higher folir sp osmolrity, ut the mount of chloride ion ws not significntly different from control plnts. These dt suggest tht treted plnts hve the cpcity to void ccumultion of the toxic ion Cl - in the leves, through mechnism of exclusion either y sequestrtion in or trnsloction to the roots (Rjendrn et l. 29). The increse in totl osmolrity my counter the osmotic stress cused y irrigtion with slty solution, so tht slt treted plnts my improve wter uptke thnks to osmotic djustment. Tle 2 shows the correltion mong ll mesured prmeters: NCl concentrtion of the irrigtion solution nd osmolrity per grm of lef fresh weight re highly correlted, s the mount of Cl - mol g -1 of dry soil with the totl osmolrity per grm of lef fresh weight (P<.1) confirming the effect of incresing soil slinity on the osmotic djustment of the leves. Root fresh weight is lso positively correlted with the totl osmolrity per grm of folir fresh weight (P<.5). No correltion ws found etween Cl - ions in shoot fresh weight nd NCl concentrtion of the solutions pplied to the soil, this could further support the hypothesis tht in P. distns in Cl - toxicity is voided y exclusion from the leves. Totl chlorophyll content t the end of the second experiment is shown in Fig. 7. In greement with dt reported y Hmeed et l. (28) for Cynodon dctylon, chlorophyll content ws significntly lower in slt stressed plnts nd GB ppliction did not increse chlorophyll content either in slt stressed or control plnts. Pictures with SEM were tken to check if slt glnds were present on P. distns lef surfce nd if the different tretments hd ny effect on lef morphology. Slt glnds were not found in ny of the tretments, in greement with Alshmmry et l. (24). Figs. 8 nd 9 show the detil of lef from control plnt nd NCl 6 mm+gb plnt respectively, prllel veins re evident nd no slt excreting structure cn e seen in either smple. CONCLUSIONS Control 85 mm 275 mm 6 mm Fig. 6 Cl - content of lef sp extrcted t the end of the first experimentl cycle, reported oth on fresh nd dry tissue weight sis. Tukey's pired test showed no sttisticl differences ut correltion with the other prmeters ws nlysed in Tle 2. Dt otined showed very good dptility of P. distns to different slinity levels: t the end of the first tril there were no significnt differences in the growth prmeters exmined. The Cl - mounts found in folir sp were not significntly different mong the four tretments, proly 52
Effect of slt nd glycineetine tretments on Puccinelli distns. Scli et l. Tle 2 Correltion tle of the mesured prmeters (Person s). Averge root lenght 1 Men weight root.146 1 Averge shoot height.871 -.33 1 Men weight cut.875.421.892 1 Totl slt content per g of LFW -.219.925(*) -.48.45 1 Cl - moles per g of LFW.375.793.46.76.568 1 Men weight shoot -.425 -.628.48 -.273 -.552 -.21 1 Cl - moles per g of soil (DW) -.46.759 -.668 -.258.94(*).252 -.573 1 mmolrity -.384.725 -.685 -.285.91(*).182 -.624.995(**) 1 Averge root lenght * Correltion is significnt t the.5 level (1-tiled). ** Correltion is significnt t the.1 level (1-tiled). Men weight root Averge shoot height Men weight cut Totl slt content per g of LFW Cl - moles per g of LFW Men weight shoot Cl - moles per g of dried soil mmolrity 35 3 μg/gdw 25 2 15 1 5 Control GB NCl+GB NCl Fig. 7 Totl chlorophyll content t the end of the second experimentl cycle, expressed on lef dry weight sis. Different letters indicte significnt differences t P<.5 using Tukey s test. Fig. 8 Scnning electron microscopy photogrph showing the lef surfce of control plnt. Fig. 9 Scnning electron microscopy photogrph showing the lef surfce of NCl + GB-treted plnt. ecuse of the ility of the plnt to trnslocte nd/or sequester Cl - in the root cells. When treted with GB, P. distns showed etter overll physiologicl condition: plnts hd the cpcity to produce more fresh iomss respect to the slt tretment nd hd higher lef reltive wter content. However, the percentge of lef firing ws not reduced nd the chlorophyll content ws not incresed with respect to the slt stressed plnts, so tht the physiologicl enefits were not relted to etter generl spect of the turf. Moreover, from prcticl point of view, the fct tht GB treted plnts produced more iomss would e disdvntge ecuse of the need for more frequent cuts of turf. GB pplictions therefore did not seem to give relevnt dvntge in reducing technicl inputs required y P. distns turf. P. distns shows good response when irrigted with slty wter, ut the physiologicl mechnisms of this resistnce still need to e investigted nd defined. Ongoing field experiments re eing crried out to vlidte our results nd test the effects of GB on this species when sujected to longer period of slt stress. ACKNOWLEDGEMENTS The uthors thnk Dr Annmri Mnnino for help with the Scnning Electron microscopy. The work ws supported in prt y funding from the Università di Plermo (fondi di Ateneo). 53
Plnt Stress 3 (1), 49-54 29 Glol Science Books REFERENCES Alshmmry SF, Qin YL, Wllner SJ (24) Growth response of four turfgrss species to slinity. Agriculturl Wter Mngement 66, 97-11 Agom PC, Peltonen-Sinio P, Hinkknen R, Pehu E (1997) Effect of folir ppliction of glycineetine on yield components of drought-stressed tocco plnts. Experimentl Agriculture 33, 345-352 Arnon DI (1949) Copper enzymes in isolted chloroplsts, polyphenoloxidse in Bet vulgris. Plnt Physiology 24 (1), 1-15 Bñuls J, Primo-Millo E (1992) Effects of chloride nd sodium on gs exchnge prmeters nd wter reltions of citrus plnts. Physiologi Plntrum 86, 115-123 Cheesemn JM (1988) Mechnisms of slinity tolernce in plnts. Plnt Physiology 87, 745-755 Chen THH, Murt N (28) Glycineetine: n effective protectnt ginst iotic stress in plnts. 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