Distribution of recently fixed photosynthte in switchgrss plnt-soil system D.R. Chudhry 1,2, J. Sxen 1, N. Lorenz 1, R.P. Dick 1 1 School of Environment nd Nturl Resources, Ohio Stte University, Columbus, USA 2 Discipline of Wstelnd Reserch, Centrl Slt nd Mrine Chemicls Reserch Institute, Council of Scientific nd Industril Reserch, Gujrt, Indi ABSTRACT The use of switchgrss (Pnicum virgtum L.) s n energy crop hs gined gret importnce in pst two decdes due to its high biomss yields on mrginl lnds with low griculturl inputs nd low mintennce requirements. Informtion on the lloction of photosyntheticlly fixed C in the switchgrss-soil system is importnt to understnd the C flow nd to quntify the sequestrtion of C in soils. The lloction of 13 C lbeled photosynthtes in shoot, root, soil, nd in microbil biomss crbon (MBC) of rhizosphere nd bulk soil of 45 dys old, greenhouse grown-switchgrss ws exmined during 20 dys 13 C-CO 2 pulse lbeling period. The totl 13 C recovered in the plnt-soil system vried from 79% fter 1 dy to 42% fter 20 dys of lbeling. After lbeling, 54%, 40%, nd 6% excess 13 C resided in shoot, root nd soil, respectively on dy 1; 27%, 61% nd 11%, respectively on dy 5 nd 20%, 63% nd 17%, respectively dy 20 fter lbeling. The mximum incorportion of 13 C from roots into the MB of rhizosphere soil occurred within the first 24 h of lbeling. The excess 13 C vlues of rhizosphere soil nd rhizosphere MBC were significntly higher thn excess 13 C vlues of bulk soil nd the bulk soil MBC, respectively. The proportion of excess 13 C in soil s MBC declined from 92 to 15% in rhizosphere soil nd from 79 to 18% in bulk soil, for 1 dy nd 20 dys fter lbeling, respectively. The present study showed the effectiveness of 13 C lbeling to exmine the fte of recently photosynthesized C in soil-plnt (switchgrss) system nd dynmics of MBC. Keywords: Pnicum virgtum L.; 13 C pulse lbeling; microbil biomss; rhizodeposition; crbon lloction; C sequestrtion Switchgrss (Pnicum virgtum L.) is summer perennil C4 grss ntive to North Americn Tllgrss Pririe, primrily used for soil conservtion, forge, urbn lndscping, nd wild life hbitt (Keshwni nd Chng 2009). Recently, there hd been n incresed interest to use switchgrss to produce biofuels (Keshwni nd Chng 2009). The mjor benefits of switchgrss includes its wide geogrphicl distribution, high biomss yields on mrginl griculturl soils, nturl resistnce to mny pests nd plnt diseses, nd very low requirements for irrigtion nd fertiliztion (Keshwni nd Chng 2009). Additionlly, switchgrss cn hve 20 30 times greter C storge in soil thn other crops (McLughlin nd Wlsh 1998). The flux of recently fixed crbon (C) from plnt to soil is one of the lest understood nd poorly quntified prts of the C cycle (Cheng 1999, Kuzykov nd Domnski 2000). Plnt roots secrete exudtes, vriety of compounds tht cn ccount for up to 5 to 21% of photosyntheticlly fixed C trnsferred to the rhizosphere (Mrschner 1995). Root exudtes re importnt in regulting the structure nd functioning of the microbil community in the rhizosphere (Wlker et l. 2003). However, there re very few studies on the ssimiltion of photosyntheticlly fixed C into the rhizosphere microbil biomss, trnsloction below ground nd exudtion by rhizospheres (Norton et l. 1990, Kuzykov nd Domnski 2002, Butler et l. 2004, Yevdokimov et l. 2006). Understnding the process controlling C fluxes between plnt roots, microbil biomss, soil nd tmosphere is importnt for predicting nd mnging C sequestrtion in soils. Supported by the Deprtment of Science nd Technology, Government of Indi in the form of BOYSCAST fellowship to D.R. Chudhry. PLANT SOIL ENVIRON., 58, 2012 (6): 249 255 249
To investigte the fte nd prtitioning of fixed C from CO 2 into rhizosphere nd bulk soil, continuous or pulse-chse 13 C or 14 C lbeling techniques re sensitive in quntifying lbeled C in plnt nd soil frctions (Phillips nd Fhey 2005, Yevdokimov 2006, Plin et l. 2009). For isotopic trcking studies 13 C is preferred over 14 C becuse of its lower discrimintion of 13 C reltive to 12 C during photosynthesis, greter sfety, nd lck of regultory brriers for use nd disposl (Svejcr et l. 1990). So fr, the prtitioning nd fte of photosyntheticlly fixed C in the switchgrss rhizosphere hs not been studied. Therefore, the objective of this investigtion ws to determine the fte of photosytheticlly fixed C ( 13 C) in the switchgrss plnt, roots, soil, nd MBC of the rhizosphere. MATERIAL AND METHODS Soil nd greenhouse experimentl conditions. A Crosby silt lom ( Stgnic Luvisol in the FAO/ WSR clssifiction nd fine, mixed, mesic Aeric Ochrqulf in the USDA clssifiction) soil ws collected in July 2009 from the Wtermn Diry Frm of the Ohio Stte University, Columbus, USA. The initil totl C nd N contents of the soil were 1.32% nd 0.086%, respectively, nd ph ws 7.2. Field moist soil pssed through 4 mm sieve, homogenized nd used for the experiment. A greenhouse study ws conducted using blck plstic pots tht were filled with 1250 g (dry weight bsis) soil. Switchgrss (Pnicum virgtum L., vriety Cve in Rock) seeds were germinted on moist filter pper in petri pltes. Three seedlings were trnsferred to ech pot nd thinned two plnts per pot fter trnsplnting. Plnts were fertilized 0 nd 15 dys with nutrient solution (100 mg/l) of 2.0, 0.44 nd 1.65% of N, P, nd K, respectively. Soil wter content ws mintined t two thirds field cpcity by irrigting every 2 3 dys intervls. In greenhouse the verge dily temperture ws 22 24 C, nd the photoperiod ws 12 h of illumintion per dy. Pulse chse 13 C lbeling. The plnts were grown for 45 dys nd then lbeled with 13 CO 2 or 12 CO 2. The lbeling ws done in two ir tight chmbers (s described below). One chmber hd unlbeled 12 C-NHCO 3 nd the other with 13 C-NHCO 3 (99.9 tom% 13 C; Cmbridge isotope lbortory Inc., Andover, USA). To initite 13 CO 2 lbeling, 4 ml of 1.5 mol/l HCl ws dded to beker contining 9.91 mg of 13 C s NHCO 3 (per pot). Once the CO 2 concentrtion fell below 102 μmol/mol, HCl ws dded to n djcent beker contining 9.91 mg of 13 C s NHCO 3 (second time). Similrly, HCl ws dded in third beker (third time). At 1, 5, 10, nd 20 dys fter lbeling 3 replicte pots were destructively smpled. Soil (bulk nd rhizosphere), stems, roots nd microbil biomss were nlyzed for totl C nd 13 C content. The lbeling ws done in two chmbers constructed of wooden frme (0.61 m 0.65 m 0.90 m) with Teflon sheeting windows on 5 out of 6 sides (excluding the bottom side). One 12VDC cooling fn (10 cm dimeter, 4.2 W, 85 cfm) ws instlled to ensure proper circultion. Ice pcks were plced inside the chmber to minimize excessive heting nd to condense excess humidity. A hole ws drilled t the bottom of the chmber nd fitted with rubber septum for use in lbeling. Two holes were drilled on the top of the chmber with ports to fit tubing for CO 2 nlyzer nd CO 2 inside the chmber ws continuously monitored with LI-6250 portble CO 2 nlyzer (LI-COR Inc., Lincoln, USA). Supplementl lighting ws plced to ensure optiml rtes of photosynthesis during lbeling. Hrvesting procedure. The root-soil systems were shken in plstic continer until pproximtely 80% of the initil soil ws collected nd this portion ws considered s bulk soil. The remining soil tht ws ttched to the root system of plnts ws defined s rhizosphere soil. Root frgments remining in the bulk nd rhizosphere were crefully removed with help of forceps. Both types of soil smples were stored in plstic bgs t 20 o C till further use. Plnts nd soil sub-smples were dried in oven t 50 o C for 72 h. The dried plnt smples were weighed nd ground with Wiley mill to pss 100 mesh sieve nd stored t 4 C. Subsmples of fresh soil were ground to pss 100 mesh sieve with mortr nd pestle. These smples were used for determintion of totl C nd δ 13 C. Microbil biomss crbon (MBC). Soil ws nlyzed for microbil biomss crbon (MBC). MBC ws extrcted using fumigtion-extrction procedure of Vnce et l. (1987) s modified by Bruulsem nd Duxbury (1996). Smples were nlyzed for totl C nd δ 13 C bundnce s described below. An effective concentrtion (K EC ) fctor of 0.45 ws used to estimte MBC (Vnce et l. 1987). Isotopic nlysis nd clcultions. Plnt, soil nd K 2 SO 4 extrcts were nlyzed for totl C nd 13 C bundnce with CHN EA 1108 elementl nlyzer (Crlo Erb Instruments, Lkewood, USA) coupled to Delt V Advntge Isotopic Rtio Mss Spectrometer (IRMS) (Thermo Electron 250 PLANT SOIL ENVIRON., 58, 2012 (6): 249 255
Corportion, Bremen, Germny). By convention, 13 C bundnce ws expressed to Pee Dee Belemnite stndrd s either δ 13 C or tom frction 13 C excess. The isotopic signl for C ws expressed s δ 13 C versus the interntionl stndrd Pee Dee Belemite (PDB): δ 13 C ( ) = [(R smple R stndrd )/R stndrd ] 10 3 Where: δ 13 C is the prts per thousnd, or per mil ( ), R smple nd R stndrd re the 13 C: 12 C rtio of smple nd stndrd (0.0112372), respectively. Smple δ 13 C ( ) ws converted to milligrms C isotope using procedure described by Boutton (1999). The soil/plnt δ 13 C vlue first were converted to the bsolute isotope rtio ( 13 C/ 12 C) of smple (R): R smple = 13 C/ 12 C = [(δ 13 C/1000) + 1] R stndrd The frctionl bundnce (A) of 13 C reltive to 13 C + 12 C ws then relted to R smple by the eqution: A = 13 C/( 13 C + 12 C) = R smple /(R smple + 1) Frctionl bundnce nd totl C (mg) of smple were used to clculte 13 C of the smple: mg 13 C smple = A totl C content of smple (mg) The enrichment level of the smple (mg 13 C smple ) in excess of nturl bundnce (mg 13 C nl, nonlbeled) ws clculted s: Excess mg 13 C smple = mg 13 C smple mg 13 C nl Excess mg 13 C of ech pool (shoot, root nd soil) ws clculted s the product of excess mg 13 C smple nd pool mss. Recovery of 13 C ws defined s the percentge of excess mg 13 C of ech pool of the totl mg 13 C dded to the lbeling chmber. Sttisticl nlysis. Anlysis of vrince (ANOVA) ws used to evlute time effects using the SAS sttisticl softwre pckge (SAS Institute, 1996). For ll smples, rhizosphere nd bulk soils were nlyzed seprtely becuse they were not independent of ech other. Difference between rhizosphere nd bulk soils chrcteristics were nlyzed using pired t-test. Significnt differences re reported t the P < 0.05 level. Dt re reported s men of three replictes. RESULTS Plnt biomss nd crbon content. The totl biomss of switchgrss incresed from 3.73 to 4.70 g/pot during the chse period (Tble 1). The increse in plnt biomss ws lso reflected by n increse in totl C ccumultion, which incresed from 1707 to 2347 mg/pot during the chse period (Tble 1). The dry weight root nd shoot biomss ws similr. Initilly, higher C ccumultion ws observed in shoot until 5 dys fter lbeling. However, 10 dys fter lbeling, higher C ccumultion ws noticed in roots. Fixed 13 C-CO 2 recovery in plnt nd soil. The recovery of 13 C in shoot, nd root biomss decresed during the chse period (Figure 1), which ws significnt in cse of shoot only. One dy fter lbeling, the 13 C recovery ws highest in shoot biomss (43%), followed by root biomss (33%) nd soil (4%). However, 5 dys fter lbeling, root (29 27%) demonstrted the highest 13 C recovery until the end of lbeling period (20 dys) followed by shoot (13 8%) nd soil (5 7%) (Figure 1). Totl 13 C recovered in plnt-soil system vried from 79% fter 1 dy to 42% fter 20 dys of lbeling (Figure 1). Throughout the chse period, the δ 13 C vlues in the shoot nd root were significntly higher thn the shoots nd roots of the unlbeled control plnts (shoots: 9.78, roots: 10.00 verge of whole chse period). The verge δ 13 C vlue for shoot ws 1306 1 dy fter lbeling nd declined to 201 20 dys fter lbeling (dt not shown). The dynmics of δ 13 C vlues in the root followed similr trend to the shoot, where verge δ 13 C of the root residue ws 1099 fter 1 dy of lbeling tht declined to 572 fter 20 dys of lbeling. Pulse lbeling resulted in excess 13 C content of 13 nd 10 mg/pot for shoot nd root, respectively, fter 1 dy of lbeling tht decresed to 3 nd 8 mg/pot by the end of lbeling period (20 dys), respectively (dt not shown). The decrese in Tble 1. The plnt biomss nd crbon content of switchgrss plnt during lbeling period Dys fter lbeling Plnt biomss (mg pot 1 ) Crbon (mg pot 1 ) shoot root totl shoot root totl 1 1.93 (0.01) 1.80 (0.10) c 3.73 (0.11) c 904.54 (15.77) c 802.36 (21.05) 1706.90 (55.88) c 5 2.00 (0.09) 1.92 (0.13) bc 3.92 (0.22) bc 968.97 (17.34) bc 907.37 (30.76) 1876.34 (64.83) bc 10 2.08 (0.03) 2.29 (0.10) b 4.38 (0.11) b 1033.03 (12.28) b 1125.63 (28.41) 2158.66 (27.44) b 20 2.37 (0.12) 2.33 (0.13) 4.70 (0.24) 1093.85 (5.38) 1253.14 (40.04) 2346.99 (75.04) Vlues in prenthesis re stndrd error. Vlues within column with the sme letters re not significntly different t P < 0.05 PLANT SOIL ENVIRON., 58, 2012 (6): 249 255 251
Recovery of 13 C ( ) Recovery of 13 C(%) 50 40 30 20 10 b Soil Shoot Root 0 0 1 5 10 15 20 Time fter lbeling (Dys) (dys) excess 13 C content of switchgrss ws significnt in cse of shoot only. Totl C content of rhizosphere soil ws higher thn tht of bulk soil, lthough, the differences in C content between rhizosphere nd bulk soil were not significnt (Figure 2). Throughout the chse period, the δ 13 C vlues of both the rhizosphere nd bulk soils were higher thn the unlbeled plnted control soils (δ 13 C of unlbelled rhizosphere soil grown with switchgrss vried from 22.44 to 23.94 nd bulk soil 29 to 30.10 ). The δ 13 C vlues in the rhizosphere nd bulk soil did not chnge significntly during the 20 dys chse period. The δ 13 C vlue in the rhizosphere soil declined stedily during the chse period, from n verge of 9.49 to 20.00. Wheres, δ 13 C b Soil Shoot Root b Figure 1. Recovery of 13 C in the plnt-soil system (shoot, root nd soil) of switchgrss during 20 dys chse period. Error brs indicte stndrd error. Smpling points with the sme lower cse letters within vrible cross smpling dtes re not significntly different t P < 0.05 of bulk soil incresed from 21.63 to 16.67 during the chse period. Significnt differences in excess 13 C were observed between rhizosphere nd bulk soil on dy 1 nd dy 5 of the lbeling period (Figure 2). During the lbeling period, rhizosphere soil on n verge contined more thn three times excess 13 C thn in bulk soil. 13 C incorportion in microbil biomss. The MBC (lbeled nd unlbeled soil) in rhizosphere nd bulk soil of switchgrss did not chnge significntly during the chse period (Figure 3). The MBC content of rhizosphere soil ws significntly higher thn in bulk soil throughout the chse period. The mximum incorportion of 13 C from roots into the MBC of rhizosphere soil occurred within the first 24 h of lbeling. Lter, excess () Crbon (g (g/kg) -1 ) 16.0 15.5 15.0 () Bulk soil soil 14.5 14.0 * 5 Bulk soil Excess 13 C (mg/kg) Excess 13 C (mg kg -1 ) 4 * 3 2 1 0 0 1 5 10 15 20 Time fter lbeling (dys) Time fter lbeling (Dys) * Figure 2. Totl crbon () nd excess 13 C content in bulk nd rhizosphere soil during 20 dys chse period. Error brs indicte stndrd error. Smpling points with the sme lower cse letters within vrible cross smpling dtes re not significntly different t P < 0.05, *indictes significnt difference between bulk nd rhizosphere soil t P < 0.05 252 PLANT SOIL ENVIRON., 58, 2012 (6): 249 255
13 C content of rhizosphere soil declined from 3.10 mg/kg fter 1 dy to 0.55 mg/kg fter 20 dys of lbeling. In bulk soil there were no significnt chnges in microbil excess 13 C during the lbeling period. There ws strong correltion (r = 0.78) between the excess 13 C of rhizosphere soil nd bulk soil. The proportion of the 13 C tht resided in the MBC pool of rhizosphere soil declined from 92% fter 1 dy of lbeling to 30, 37 nd 15% fter 5, 10 nd 20 dys of lbeling, respectively. Their respective decline in bulk soil ws from 79% fter 1 dy of lbeling to 46, 22 nd 18% fter 5, 10 nd 20 dys of lbeling, respectively. DISCUSSION Crbon lloction. The C sink ctivity in roots is reported to be greter in the younger plnts (Keith et l. 1986, Plt nd Gregory 1997). Switchgrss hs n extensive, deep root system nd hs been shown to produce more root biomss thn corn (Ze mys L.) (Frnk et l. 2004). The roots of young switchgrss plnts in the present study demonstrted greter sink strength for C by storing more of the photosynthte in roots thn in shoots (Figure 1). Similrly, M et l. (2001) found C storge in roots of switchgrss ws 2.2 times higher thn in shoots. In the present study, there ws less thn 100% 13 C recovery, with 79% of the totl 13 CO 2 dded () Microbil excess 13 C (mg/kg) Microbil biomss C (mg/kg) Microbil biomss C (mg kg -1 ) Microbil excess 13C (mg kg -1 ) 500 450 400 350 300 250 200 150 2.5 2.0 1.5 1.0 0.5 () * * Bulk soil 3.5 * Bulk soil 3.0 b* Bulk soil 0.0 0 1 5 10 15 20 * b* Time fter lbeling (dys) Time fter lbeling (Dys) * b* recovered in the vrious plnt nd soil frctions during the lbeling period. Similr observtions were mde on sugr mple (Acer scchrum) nd yellow birch (Betul llegheniensis), where 76 nd 73% of ssimilted 13 C ws recovered fter shortterm 13 C pulse lbeling, respectively (Phillips nd Fhey 2005). This is likely due to respirtion of shoot nd roots, which ws shown by Leke et l. (2006) even in continuous flow sturted system. Gregory nd Atwell (1991) found 15 to 25% of ssimilted 14 CO 2 ws respired in 50-dy old whet or brley plnts within 24 h fter lbeling begn. As expected we found the recovery of fixed CO 2 in the plnt systems declined over time. This ws from 79% of totl 13 C dded fter 1 dy of lbeling to 42% by the end of experiment (20 dys fter lbeling) (Figure 1). In 2-dy 13 CO 2 pulse lbeling of grsslnd vegettion, Ostle et l. (2000) within 24 h found decline in 13 C ssimiltion by 76.4 nd 61.65% in shoot nd root, respectively. This rpid decline in pulse derived C in plnt structurl components cn be ttributed to losses of CO 2 -C by root nd shoot respirtion nd from root exudte-c s well s from dilution effect by uptke of unlbeled 12 CO 2 -C (Ostle et l. 2000). The distribution of recovered 13 C showed tht 54, 40 nd 6% of 13 C ws recovered fter 1 dy; 27, 61 nd 11% fter 5 dys; nd 20, 63 nd 17%, fter 20 dys of lbeling for shoots, roots, nd soil, respectively (Figure 1). Butler et l. (2004) found n verge of Figure 3. Microbil biomss crbon () nd microbil excess 13 C in bulk nd rhizosphere soil during 20 dys chse period. Error brs indicte stndrd error. Smpling points with the sme lower cse letters within vrible cross smpling dtes re not significntly different t P < 0.05; *indictes significnt difference between bulk nd rhizosphere soil t P < 0.05 PLANT SOIL ENVIRON., 58, 2012 (6): 249 255 253
70%, 20%, nd 10% retined in boveground biomss, roots, nd soil, respectively, in ryegrss (Lolium multiflorum Lm.) system fter 8 dys of exposure to 13 CO 2. Our results show the importnce of chrcterizing C fixtion over the growing seson s the distribution does chnge with time. Comprisons with other plnt species the fte of fixed C, especilly those tht would be used for biofuels, is n importnt prmeter in designing mngement systems for optimizing C sequestrtion. The belowground trnsloction of ssimilted C in our study rnged from 46 to 80% during the lbeling period where mjority of it ws recovered in roots (Figure 1). This is similr to the mount of totl ssimilted 13 C distributed belowground in psture plnts (50 to 80%) found by Kuzykov nd Domnski (2000); where hlf ws found in roots nd bout one-third lost s root nd microbil respirtion, with the remining incorported into MBC nd soil orgnic mtter. Wheres, Šntrůčková et l. (1999) when mesuring the effect of doubling the tmospheric CO 2 levels (700 µmol/mol) on crbon budget in hydroponiclly grown (in snd) winter whet plnts, observed bout 30% of dily ssimilted C trnslocted to roots, 60% llocted into new biomss leves, nd 10% lost in respirtion of leves t night over the 5 34 dys of verged experimentl life spn. Hlf of C tht ws trnsported to roots ws lost in root respirtion nd the remining hlf in equl proportions ws utilized for root biomss nd relese into the rhizosphere. Three-fifth of the C relesed in rhizosphere ws lost in microbil respirtion (Šntrůčková et l. 1999). 13 C-derived rhizodeposition. Severl investigtions on vriety of crops species hve shown tht lbeled CO 2 cn be found in MBC within hours fter the plnt hs been exposed to the C isotope (Cheng et l. 1993, Rttry et l. 1995, Lu et l. 2002). This indictes tht recently ssimilted C moves rpidly through plnts to the roots nd surrounding soil nd is redily vilble to microorgnisms. Time of mximum C incorportion into MBC fter exposure to lbeled CO 2 from studies done so fr is from 1 to 5 dys (Gregory nd Atwell 1991, Kuzykov et l. 2001, Lu et l. 2002). Our results with switchgrss re consistent with these studies where trnsloction of 13 C to rhizosphere MBC occurred rpidly, which ws highest t 24 h fter lbeling nd declined therefter (Figure 3). Initilly fter 1 dy, 92% of the rhizosphere soil nd 79% of the bulk soil of the 13 C were ws found in MBC. However this declined to 15% nd 18%, respectively 20 dys fter lbeling (Figure 3). This shows the importnce of rhizosphere microorgnisms in controlling the fte of root C exudtes. However, on percentge bsis of the mount of MBC is reltively low. In our study, between 2.7% nd 5.1% of the plnt-soil system 13 C ws in the MBC pool on dy 1 nd 20, respectively (dt not shown). This is in comprison to somewht lower MB 13 C vlues reported for the rice rhizosphere 0.15 to 0.94% (Lu et l. 2002) nd greter vlues of 1 to 5% for whet nd mize rhizosphere (Merckx et l. 1985, Liljeroth et l. 1990, Mrtin nd Merckx 1992, Vn Ginkel et l. 2000). Significntly higher MBC nd microbil excess 13 C ws observed in rhizosphere soil thn in bulk soil throughout the chse period (Figure 3). Higher MBC is generlly observed in rhizosphere soil thn in bulk soil (Butler et l. 2004). This is probbly due to decresing rtios of soluble-to-insoluble rhizodeposits with incresing distnce from the roots (Whipps 1984). The discussion bove shows the importnce of the rhizosphere MB in converting rhizo-deposited C to soil orgnic C in the bulk soil. This is further reinforced by the high correltion between 13 C levels in the rhizosphere soil nd the bulk soil. And tht MB- 13 C declined in the bulk soil, while totl 13 C in the soil slowly ccumulted over the experimentl period, further demonstrtes movement of C through rhizosphere MB nd into surrounding bulk soil. REFERENCES Boutton T.W. (1999): Stble crbon isotope rtio of nturl mterils: I. Smple preprtion nd mss spectrometric nlysis. In: Colemn D.C., Fry D. (eds): Crbon Isotope Techniques. Acdemic Press, Sn Diego, 155 171. Bruulsem T.W., Duxbury J.M. (1996): Simultneous mesurement of soil microbil nitrogen, crbon, nd crbon isotopic rtio. Soil Science Society of Americ Journl, 60: 1787 1791. Butler J.L., Bottomley P.J., Griffith S.M., Myrold D.D. (2004): Distribution nd turnover of recently fixed photosynthte in ryegrss rhizospheres. Soil Biology nd Biochemistry, 36: 371 382. Cheng W. (1999): Rhizosphere feedbcks in elevted CO 2. Tree Physiology, 19: 313 320. Cheng W., Colemn D.C., Crrol C.R., Hoffmn C.A. (1993): In situ mesurement of root respirtion nd soluble C concentrtions in rhizosphere. Soil Biology nd Biochemistry, 25: 1189 1196. Frnk A.B., Berdhl J.D., Hnson J.D., Liebig M.A., Johnson H.A. (2004): Biomss nd crbon prtitioning in switchgrss. Crop Science, 44: 1391 1396. 254 PLANT SOIL ENVIRON., 58, 2012 (6): 249 255
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