Characterization of Phosphate Accumulation in Lolium multiflorum for Remediation of Phosphorus

Characterization of Phosphate Accumulation in Loliummultiflorum for Remediation of Phosphorus-Enriched SoilsN I L E S H C .S H A R M A A N D S H I V E N D R A V .S A H I *Biotechnology Center,Department of Biology,Western Kentucky University,Bowling Green,Kentucky 42101Deterioration in water quality caused by the movement of excessive soil P has created a condition necessary for the development of a sustainable P remediation technology.In this investigation,the phytoremediation potential of Gulf and Marshall ryegrass (Lolium multiflorum )grown in a greenhouse was determined under varying conditions of soil P concentration,pH,and temperature.Both genotypes demonstrated P accumulations g 1%shoot dry weight depending on soil P concentrations (0-10g of P/kg of soil),with higher shoot P in Gulf than Marshall ryegrass.An increase in plant biomass was proportional to the increasing concentrations of P up to a level of 10g of P/kg of soil.The effect of soil pH on plant uptake of P was noticeable with a significant rise in shoot P in acidic soil (pH 5.6)as compared to soil with pH 7.8.Significant differences were observed in the biomass productivity and shoot Paccumulation at varying temperatures in both grass types.The patterns of acid phosphomonoesterase and phytase activities in plant roots were interesting,activities being 2-fold higher in alkaline soil than acidic soil in bothgenotypes.The effect of P supply on the enzyme activity was also distinct,as plants growing in a high P concentration showed higher activity (nearly 30%)than those growing under P deficiency conditions (with no addition of P).These results indicate that Gulf and Marshall ryegrass can accumulate high P under optimal conditions and thus reduce soil P concentrations in successive cropping.IntroductionFarm soils located in areas of intensive farming have received phosphorus (P)applications in excess of the P quantity removed by crop harvest,resulting in elevated soil P concentrations (1).Animal manure applications to pastures have resulted in relatively high P runoff,even when manure is applied at recommended rates.There is a concern that high P soils represent an increased risk for nonpoint source pollution of surface waters (2,3).High proportions of P (80-90%)in runoff are in the soluble form,which is the most readily available form for algal uptake (4).Eutrophication of freshwater is thus a growing environmental problem world-wide,and excess P is well-documented as its most common cause in many aquatic systems (5).Phosphorus runoff from poultry and swine farms has also been implicated in the emergence of a dinoflagellate,Pfiesteria piscicida ,in water-ways on the eastern coast of the U.S.(6).This toxin-producing pathogen is another concern for the aquaculture industry as well as human health.Reduction of P inputs to surface water is necessary and thus is receiving much attention these days.Increased emphasis on soluble P losses from cropland has expanded the use of chemical amendments to immobilize P in soils.Salts of Fe,Ca,and Al have been used to decrease P solubility in P rich manures and runoff from manure-amended soils (7,8).These chemical amendments reduced P runoff significantly.However,P immobilization in soil by these amendments may not be stable on a long-term basis (9)and instead result in higher soluble phosphates as in the case of Ca and ferric phosphate dissolution under certain normal soil conditions (10).Although the use of Al salts to precipitate P in manure or soil is considered a better choice (10),these applications may also affect soil chemistry on a long-term basis.The stability of the P complexes formed with Al-oxides,as it relates to P lability in the environment,is uncertain (11).Likewise,the application of biosolids is also not considered the best management practice to halt P loss from soils (12).Alternatively,plant-assisted extraction of phosphate (Pi)could be an attractive strategy.Mining of soil P,which includes harvesting P taken up from the soil by a crop grown without external P application,has been proposed as a possible management strategy for P enriched soils (9,13-15).Phytoremediation is an inexpensive,nonintrusive,and often highly effective technique (16).Plant-based clean-up strategies offer a number of advantages over traditional clean-up methods,as well as over other bioremediation technolo-gies.There are several reports of metal hyperaccumulators that are immensely useful in phytoremediation (16,17).Plants,generally referred to as metal hyperaccumulators,have the inherent potential to survive and accumulate excessive amounts of metal ions in their biomass without incurring damage to basic metabolic functions (16).However,the ability of vegetation to assist in the remediation of P remains largely unknown.Some researchers suggest that for P phytoremediation to be effective,plants should have a high biomass and accumulate P significantly higher (g 1%DW)than the common plants do (9).P remediation potentials of a number of crops were evaluated in a pot and field study indicating a differential pattern of phosphate (Pi)uptake by those crops (18).Other studies also indicate the usefulness of phytoremediation using stargrass (14)and perennial ryegrass (15)for P impacted soils.Current P uptake rates are low for common row crops and forage grasses used to assimilate P from soil (19).Therefore,factors such as foliar P concentration and biomass yield are crucial for the application of a plant type in P phytoremediation.Both soil and crop management practices may thus require optimiza-tion for the P hyperaccumulator plant to compete with other plant species.Annual ryegrass (Lolium multiflorum )is a closely related and interfertile species with perennial ryegrass (Lolium perenne ),and both are grown all over the world as key forage grasses (20).These are among the most palatable and highly digestible grasses for livestock.In a hydroponic study,Marshall and Gulf ryegrass,two cultivars of L.multiflorum ,demonstrated a large accumulation of P (>2%shoot DW)when grown in the medium enriched with KH 2PO 4without displaying signs of toxicity (21).Therefore,the aim of this study was to assess the efficacy of Marshall and Gulf ryegrass in the remediation of P impacted soils under greenhouse conditions.Plant uptake of P depends on the availability of orthophosphates (Pi)in soil solution,and their forms change*Corresponding author phone:(270)745-6012;fax:(270)745-6856;e-mail:shiv.sahi@.Environ.Sci.Technol.2005,39,5475-548010.1021/es050198t CCC:$30.25©2005American Chemical SocietyVOL.39,NO.14,2005/ENVIRONMENTAL SCIENCE &TECHNOLOGY95475Published on Web 06/11/2005according to soil pH(22).Temperature is another crucial factor that affects plant growth,particularly root growth, influencing the physiology of P uptake(23).In this backdrop, these ryegrass cultivars were characterized for shoot P accumulation under varying conditions of soil P concentra-tion,pH,and temperature.Recent investigations also indicate the involvement of root acid phosphatases in P nutrition of plants(24-26);thus,the role of plant acid phosphomo-noesterase and phytase activities for P assimilation in Marshall and Gulf ryegrass was also examined. Experimental ProceduresSeed Germination.Seeds of Marshall grass and Gulf ryegrass (L.multiflorum cultivars),provided by USDA Lab,Starkville, MS were sterilized with sodium hypochlorite(1%v/v)and rinsed several times with sterile deionized water.They were then transferred to water-agar(0.8%)medium in Magenta boxes and maintained at25(2°C under a12:12light/dark regime in a growth chamber.Ten day old seedlings isolated from agar medium were rinsed with deionized water before transplantation.Growth of Seedlings in P Enriched soil.The pot experi-ment was carried out in a greenhouse using flats(volume of 2.5L)filled with2kg of soil.To simulate P impacted soils, pot soils(Table1)were enriched with the application of0-20 g of KH2PO4/kg of soil,8weeks before the transplantation of seedlings.Soils were also mixed with sand(4parts soil and 1part sand)to reduce the compaction.The soil sample used in this study belonged to the Pembroke series and had characteristics of a Mollic epipedon(Ap horizon)s dark brown silt loam that was neutral to slightly alkaline(Table1).The addition of0-20g of KH2PO4/kg of soil results in the extraction of4.9-197mg of water soluble P/kg of soil(Table 1).Five clumps,each with five seedlings,were transplanted in each flat.Each treatment was replicated4times.Pots were randomized in a complete block design.The plants were kept in a greenhouse with16h of sunlight,and they were watered4times a week or as required.The temperature varied from18to20°C at night and from22to25°C during the day unless otherwise indicated.Pot plants were fertilized with modified Hoagland mixture(21)every week and harvested after5-14weeks.For the measurement of biomass growth, harvested plant parts(aerial parts2cm above the ground) were dried in an oven at70°C for3days,or until the weight stabilized,and then measured in g/pot.Determination of Plant and Soil P.Following5-14weeks of growth,plants from different treatments were harvested and washed thoroughly with deionized water,divided into root and shoot biomass,and air-dried.The ground samples were then weighed and placed in15mL Teflon beakers.Three mL of concentrated HNO3was added to the sample,and the beaker was placed on a hotplate set at100°C overnight,until it evaporated to dryness.The samples were allowed to cool and were made up gravimetrically to a volume of20mL with 2%HNO3.A VG Elemental Plasma Quad(model PQZ)ICP-AES was used for all data acquisition.Analyses were performed using an external calibration procedure,and internal standards were included to correct for matrix effects and instrumental drift corrections(27).Six to eight weeks after the P applications,soil samples(2g)from each treatment were stirred in5mL of deionized water for24h and spun on a tabletop centrifuge(7710g;15min),and the supernatant was filtered through a2.5µm sieve.The filtrate was then assayed for P as described previously.Elemental analysis of soil samples was also performed using ICP-AES(Table1).Effect of Soil pH on P Accumulation.The soil pH was adjusted by adding different quantities of elemental sulfur or lime to achieve pH values of5.6,6.5,and7.8.The amended soils were allowed to equilibrate for a period of2weeks in the greenhouse undergoing three cycles of saturation with water and air-drying before being remixed and planted(17). Plants were grown in the pH-adjusted soils containing2.5 g of KH2PO4/kg of soil,in the manner described previously, and harvested after6weeks.Each treatment was replicated 3times.Effect of Temperature on Shoot Dry Mass and P Accumulation.Plants were transferred to soil containing2.5 g of KH2PO4/kg of soil and grown in a plant growth chamber set at a varying temperature regimen[20and16°C,24and 20°C,28and24°C,and32and28°C(day and night)]with a16:8h light/dark cycle under200-250µmol m-2s-1cool fluorescent illumination.Each treatment was replicated3 times.Controls without application of KH2PO4were also set up against each temperature treatment.Plants were harvested after6weeks for determination of biomass growth and P accumulations in roots and shoots,as described previously.Phosphomonoesterase and Phytase Assays.Plants were harvested after5weeks of growth in soils containing either 0or2.5g of KH2PO4and washed thoroughly with deionized water followed by a rinse in a2-morpholinoethanesulfonic acid,monohydrate(MES)buffer solution(pH5.5).Roots were separated,chilled on ice,and homogenized with a mortar and pestle in15mM MES buffer(pH5.5,0.5mM CaCl2‚H2O, and1mM EDTA).The buffer was added at a ratio of1:5(root fresh weight/extraction buffer volume).The extract was centrifuged(13000g;15min at4°C),and the supernatant was used for the enzyme assay.For the assay of phosphomonoesterase activity,the enzyme extract(50µL)was incubated in a total volume of 500µL of15mM MES buffer(pH5.5,0.5mM CaCl2)in the presence of10mM p-nitrophenyl phosphate and disodium salt(Sigma-Aldrich,St.Louis,MO)(25,26).The assay was conducted over30min,and reactions were terminated by equal volumes of0.25M NaOH.The enzyme activity was calculated from the release of p-nitrophenol(pNP),deter-mined at412nm(relative to standard solutions)by a UV-vis spectrophotometer(model Ultrospec3000,Pharmacia Biotech).To assay for phytase activity,500µL of enzyme extract was incubated in a total volume of1mL of15mM MES buffer(pH5.5,0.5mM CaCl2)in the presence of2mM myo-inositol hexaphosphoric acid(Sigma-Aldrich,St.Louis,MO)TABLE1.Characteristics of Soil Used for the Study of P Remediationapplied(g of P/kg of soil)WSP a(mg/kg)pH b(g/kg)Ca(g/kg)Zn(g/kg)Fe(g/kg)Mn(g/kg)sand c(g/kg)silt(g/kg)clay(g/kg)O.M.d(g/kg)0 4.9a7.832.70.1412.80.8680-100700180152.524.3b5.044.1c1096.3d20197.0ea Water soluble phosphorus(WSP)was extracted after application of0-20g of KH2PO4/kg of soil.The values in the column having different letters were significantly different(P<0.05).b Determined in1:1soil/water mixture.c Physical characteristics as in standard Pembroke silt loam.d Organic matter(O.M.).54769ENVIRONMENTAL SCIENCE&TECHNOLOGY/VOL.39,NO.14,2005(25,26).The assay was conducted over 60min,and reactions were terminated by equal volumes of ice-cold 10%trichlo-roacetic acid (TCA).Solutions were subsequently centrifuged to remove precipitated material,and the phosphate con-centration of the solutions was determined by measuring the absorbance at 882nm using the molybdenum-blue reaction (28).Phosphate determinations were recorded at a fixed time within 1h following the addition of the color reagent to samples,to minimize possible interference.The enzyme assays were conducted at 26°C using three replicates.Phosphomonoesterase and phytase activities were expressed in mU g -1of root fresh weight (FW),where 1U is defined as the release of 1µmol of Pi min -1under the assay conditions.Statistical Analyses.The data were analyzed by two-way analysis of variance where the F ratios were significant (P <0.05),using SYSTAT (version 9for Windows,1999,Systat Software Inc.,Richmond,CA).Means of plant P,soil WSP,and plant biomass were tested for significant differences.Results and DiscussionGrowth and P Accumulation on P Enriched Soils.The biomass of plants increased with increasing concentrations of soil P until the concentration reached a level of 20g of P/kg of soil,where growth was affected (Table 2).A significant increase (P <0.05)in biomass with respect to control and also among the treatments was observed in both grass species supplied with P up to 10g/kg of soil,while a decrease in biomass was significant (P <0.05)at 20g of P/kg of soil.In respect of shoot biomass growth on high P,Marshall ryegrass displayed better adaptation,a trend consistent with earlier hydroponic studies on Gulf and Marshall ryegrass (21).Both crops accumulated increasing amounts of P (P <0.05)in their shoots and roots with an increase in soil P (Figures 1and 2).P accumulations in Gulf ryegrass varied from 8200to 13000mg/kg of shoot dry weight (Figure 1),while P accumulations in Marshall ryegrass were 7800-11000mg/kg of shoot dry weight depending on soil P concentrations (Figure 2).A significant difference in the pattern of P accumulation in these plant types was observed with respect to root P,which was higher in Marshall than Gulf in most of the treatments (Figures 1and 2).In another experiment,plants grown in the presence of 2.5g of P/kg were harvested over a period of 5-14weeks to study the pattern of variation in the P accumulation over time.Gulf ryegrass showed a steady pattern of shoot P accumulation with no significant difference up to 12weeks (Figure 3),whereas Marshall ryegrass displayed a significant decrease in shoot P at all P concentrations at 12weeks of harvest (Figure 4)as compared to shoot P harvested at 6weeks (Figure 2).An earlier study by Delorme et al.(18)pointed to the phosphate phytoremediation potentials of a few crops [Indian mustard (Brassica juncea ),canola (Brassica napus Westar),corn (Zea mays ),collard (Brassica oleracea L.Acephala Group),alfalfa (Medicago sativa ),and soybean (Glycin max L.)etc.]grown in greenhouses.This study indicated the lowestP accumulation in the canola shoot (0.2%of tissue dry weight)and the highest in collard and corn shoots (0.6%and 0.5%of tissue dry weight,respectively).Likewise,root P was recorded in the range of 0.2-0.5%dry weight for collards and corn.In the previous study,most of the plant species had higher root P than shoot P,which is not desirable for phytoremediation.In the present study,both ryegrasses demonstrated a greater P accumulation potential in shoots as well as roots (Figures 1and 2).The shoot-to-root ratio of P accumulation was also greater than 1in both plant types.Three cool-season turf grasses:Kentucky bluegrass (Poa pratensis ),tall fescue (Festuca arundinaceae ),and perennial ryegrass (Lolium perenne )were also investigated for phos-phate removal capacity from enriched soils (29).Shoot P differed significantly among these three grasses ranging from 0.3to 0.45%of dry mass.These results also showed that genetic differences in P absorption might exist among turf grasses at both interspecific and intraspecific levels.Aggres-sive grasses such as the Brachiaria species and a tropical forage legume (Arachis pintoi )known for P-uptake efficiency,when grown in greenhouses with different sources of soil P at the rate of 20-100kg/ha,demonstrated much less foliar P concentration than these annual ryegrasses (30).TheseTABLE 2.Shoot Biomass Growth of Annual Ryegrass Grown in Soils Enriched with 0-20g of P/kg of Soil for 6Weeksshoot biomass (g of dry weight/pot)treatments (g of P/kg of soil)Gulf ryegrass Marshall ryegrass 00.68a a0.45a 2.5 1.11b 1.68b 5 1.38b 1.99b 10 2.70c 3.45c 200.59a0.98aaValues are the mean of four replicates,and,within each column,those not followed by the same letter are significantly different (P <0.05).FIGURE 1.P accumulation in Gulf ryegrass grown in soils enriched with 0-10g of P/kg of soil for 6weeks.Values represent four replicates (standard error of themean.FIGURE 2.P accumulation in Marshall ryegrass grown in soils enriched with 0-20g of P/kg of soil for 6weeks.Values represent four replicates (standard error of the mean.VOL.39,NO.14,2005/ENVIRONMENTAL SCIENCE &TECHNOLOGY95477observations suggest that annual ryegrass has a much greater potential for removing excess P from soil.The fate of manure P changes in the soil over time,the majority of P fractions being locked with soil components (31).Since plants can access only water soluble forms of P,particularly orthophosphates (22),soils in this experiment were enriched with P levels up to 20g/kg,which yield a maximum of 197mg of water soluble P/kg of soil (Table 1).Furthermore,water soluble P is the dominant form of P in water runoff causing water-related problems.Effect of pH on P Accumulation.The form of P most readily accessed by plants is orthophosphates (Pi)and their forms in soil solution change according to soil pH (22).The p K values for the dissociation of H 3PO 4into H 2PO 4-and then into HPO 4-are 2.1and 7.2,respectively.Thus,below pH 6.0,most Pi will be present as the monovalent H 2PO 4-species,whereas H 3PO 4and HPO 4-will be available only in trace amounts (22).Plant uptake is also affected by fixation of P by soil components,which is greatest in the presence of Fe-and Al-hydroxylated surfaces and,at a higher pH,calcium carbonate (31).Therefore,to study how varying soil pH conditions in the Pembroke silt loam influence P uptake in Gulf and Marshall ryegrass,plants were grown in P enriched (2.5g of P/kg of soil)soils maintained at pH 5.6,6.5,and 7.8.A significant increase in shoot P was observed in both grass types at pH 5.6with respect to accumulation at pH 7.8(Figure 5A,B).However,the difference in shoot P between pH 6.5and 7.8was significant (P <0.05)in Gulf but not in Marshall ryegrass.The pattern in Marshall was also different with regards to root P (Figure 5B),which was maximal at pH 7.8and declined with decrease in pH,probably a lower pH favoring translocation of Pi from root to shoot.Most studies on the pH dependence of Pi uptake in higher plants have found that uptake rates are highest between pH 5.0and 6.0,where plant assimilable H 2PO 4-dominates (22).Effect of Temperature on Shoot Dry Mass and P Accumulation.This experiment was designed to study the effect of changing temperature that may be encountered by the crops during different seasons on the dry mass produc-tivity and corresponding P uptake.Variations in the shoot dry matter and P accumulations were significant (P <0.05)at different temperature regimes in these grasses (Table 3).Biomass growth in Marshall ryegrass was greater than Gulf ryegrass at all temperatures,which is consistent with the results of previous experiments in this study (Table 2)and also with earlier studies involving solution culture (21).As differences in biomass growth are greater,the total P removal capacity of these plants will also be significantly different.Reports suggest that the air or soil temperature may influence dry mass accumulation as well as P uptake in plants (23,32).Cool soil temperatures generally result in reduced P uptake from soil reserves by plant roots.Root growth was greatly affected in maize seedlings by decreasing the soil temperature (32).Even soils with high levels of P may not provide adequate P to plants when the temperature is suboptimal during the cool season.Annual ryegrass is generally cultivated as a winter crop in the temperate climates,but this study suggests that it can be grown also during the summer when the temper-ature exceeds 30°C,while serving the purpose of P mining.Phosphomonoesterase and Phytase Activities in Plants.Acid phosphatases (E.C.3.1.3.2)are required for mineraliza-tion of organic forms of soil P to release phosphate for plant uptake (33).Phosphatases with various substrate specificities (e.g.,phosphomono-and phosphodiesterases)have been characterized in plant roots.More recently,phytases (E.C.3.1.3.26),which are phosphomonoesterases with a high specific activity against phytate,have also been described in roots (24,25).In the present study,both grass types were assayed for the activities of acid phosphomonoesterase and phytase in the roots when grown in acidic and slightly alkaline soils under P sufficiency or P deficiency conditions.The results indicate that phosphomonoesterase and phytase activities were more or less similar in both Marshall and Gulf grasses when grown in acidic soils but that activities were significantly higher in Marshall than Gulf when grown in alkaline soil (Table 4).Phosphomonoesterase activity in annual ryegrass was significantly higher than the corre-sponding values reported for wheat grown in sterile medium containing various sources of P (25).Phytase activity in annual ryegrass was lower in acidic soils;however,the activity was greater in the alkaline soil (Table 4)relative to phytase activity of wheat roots grown in the medium containing high P (25).The phytase activity expressed in terms of a percent of the total phosphomonoesterase activity was low (0.7-1.0%)in annual ryegrass but greater than Arabidopsis (26)and pasture grasses (34).Plants having a high phytase activity in their roots can hydrolyze phytates,which account for a large proportion of unavailable soil P pool,and can thus deplete the excess P source more efficiently (26).The enzyme activities in annual ryegrass also varied with respect to soil pH (Table 4).The activities were about 2-fold higher in alkaline soil than acidic soil in the case of both enzyme types.The possibility of P immobilization with Ca underalkalineFIGURE 3.P accumulation in Gulf ryegrass grown in soils enriched with 2.5g of P/kg of soil over time (5-14weeks).Values represent four replicates (standard error of themean.FIGURE 4.P accumulation in Marshall ryegrass grown in soils enriched with 0-20g of P/kg of soil for 12weeks.Values represent three replicates (standard error of the mean.54789ENVIRONMENTAL SCIENCE &TECHNOLOGY /VOL.39,NO.14,2005conditions may necessitate conditions for plants to express a high enzyme activity.The effect of P supply on the activity of enzymes was also significant in annual ryegrass where both phosphomo-noesterase and phytase activities were higher in P rich plants than P deficient plants.This feature,although not uncom-mon,was not compatible to many studies that showed enhanced activities,particularly of phytase in plant root extracts (24,34).However,in an elaborate study involving several temperate pasture legumes and grass species,Hayes et al.(34)found that the P deficiency had not resulted in anenhanced root acid phosphatase activity with most of the species.This study also demonstrated that the legume species (Trifolium spp.and Medicago polymorpha )had higher levels of phytase activity when P was supplied as compared to the grass species.Therefore,enhanced P uptake in annual ryegrass cannot be directly correlated with the determined enzyme activities,but the interesting pattern in their activities may be one of the unique features that influences P nutrition and accumulation in these plants.This investigation demonstrates the ability of L.multi-florum to assimilate and remove soil P at an enhanced rate (g 1%shoot dry weight)under optimal soil and culture conditions.When,in search for a suitable plant for P phytoremediation,the P removal capacity of several plants including pasture grasses was examined (14,18),none showed a level of P accumulation comparable to the annual ryegrass accumulations.In a field study,star grass (Cynodon nlemfuensis )was shown to remove the majority of applied P at a certain combination of P and K applications in soil and was thus considered a good candidate for mining P (14).The P removal rate,in this study,was calculated on the basis of total dry matter (DM)yield and the quantity of P applied per hectare.But the amount of P per kg of DM was much less in stargrass (0.24g)than annual ryegrass (8-10g).Koopmans et al.(15)performed a pot experiment in the greenhouse where perennial ryegrass (L.perenne ),a close relative of annual ryegrass,was cropped on a P enriched sandy soil over a long period,and observed P accumulation varying up to 7g per kg of DM,a value comparable to annual ryegrass accumulation.The molecular mechanism of P nutrition in plants under the P sufficiency condition is not well-understood;however,much information is available on the acquisition of P under P deficiency (33).The genes coding for P transporters have been characterized under P deficiency conditions when the plant shoot senses the signal;the mechanism being known as high affinity P transport system.The low affinity P transport system,which is a constitutive mechanism,operates in the P sufficiency condition,as in the Arabidopsis (Pho2)mutant (35).The P uptake rate of pho2is nearly twice (1.5%shoot DM)that of wild-type plants in the presence of high concentrations of P.The low-affinity phosphate transporter gene from this plant was cloned and characterized (36).It is also interesting to note that ryegrass,in the present study,does not demonstrate P toxicity symptoms when grown on excessive P (up to 10g/kg of soil).It thus appears that ryegrass may have an efficient P sequestration mechanism to avoid P toxicity similar to Arabidopsis .Further studies ongeneticFIGURE 5.(A)P accumulation in Gulf ryegrass grown in soils (pH 5.6-7.8)enriched with 2.5g of P/kg of soil for 6weeks.Values represent five replicates (standard error of the mean.(B)P accumulation in Marshall ryegrass grown in soils (pH 5.6-7.8)enriched with 2.5g of P/kg of soil for 6weeks.Values represent five replicates (standard error of the mean.TABLE 3.Effect of Temperature on Shoot Biomass and P Accumulation in Annual Ryegrass Grown in P Enriched Soil abiomass(g of dry weight/pot)P(mg/kg of shoot dry weight)treatment temperature(°C)Gulf ryegrass Marshall ryegrass Gulf ryegrass Marshall ryegrass 200.84a b 1.00a 7900a 7500a 24 1.11b 1.68b 8200a 7800a 28 1.38c 1.46b 9400b 8500b 320.60a0.85a9100c8300ba P was applied at the rate of 2.5g/kg of soil.b Values are the mean of three replicates,and,within each column,those not followed by the same letter are significantly different (P <0.05).TABLE 4.Acid Phosphomonoesterase and Phytase Activities of Root Extracts in Annual Ryegrass Grown in P Enriched a Acidic and Slightly Alkaline Soils for 5Weekstreatmentsacidphosphomonoesteraseactivity (mU g -1root FW)phytase activity (mU g -1root FW)Acidic soil (pH 5.7)Gulf ryegrass control (P -)371(19.6b2.5(0.62Gulf ryegrass (P +)460(38.73.2(0.85Marshall ryegrass control (P -)397(14.1 2.3(0.81Marshall ryegrass (P +)431(53.2 3.0(0.33Alkaline soil (pH 7.8)Gulf ryegrass control (P -)549(83.3 3.6(0.32Gulf ryegrass (P +)722(87.57.6(1.01Marshall ryegrass control (P -)693(59.35.8(0.91Marshall ryegrass (P +)883(46.67.0(1.14aP was applied at the rate of 2.5g/kg of soil.b Values are the meanof three replicates (standard error of the mean (SEM).VOL.39,NO.14,2005/ENVIRONMENTAL SCIENCE &TECHNOLOGY95479。