13 Carbon Isotope Discrimination in Roots and Shoots of Major Weed Species of Southern U.S Rice Fields and Its Potential Use for Analysis of Rice–Weed Root Interactions Author(s): David R Gealy and Glenn S Gealy Source: Weed Science, 59(4):587-600 2011 Published By: Weed Science Society of America DOI: http://dx.doi.org/10.1614/WS-D-10-00140.1 URL: http://www.bioone.org/doi/full/10.1614/WS-D-10-00140.1 BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and environmental sciences BioOne provides a sustainable online platform for over 170 journals and books published by nonprofit societies, associations, museums, institutions, and presses Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use Usage of BioOne content is strictly limited to personal, educational, and non-commercial use Commercial inquiries or rights and permissions requests should be directed to the individual publisher as copyright holder BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research Weed Science 2011 59:587–600 13 Carbon Isotope Discrimination in Roots and Shoots of Major Weed Species of Southern U.S Rice Fields and Its Potential Use for Analysis of Rice–Weed Root Interactions David R Gealy and Glenn S Gealy* Assessing belowground plant interference in rice has been difficult in the past because intertwined weed and crop roots cannot be readily separated A 13C discrimination method has been developed to assess distribution of intermixed roots of barnyardgrass and rice in field soils, but the suitability of this approach for other rice weeds is not known 13C depletion levels in roots and leaves of rice were compared with those of 10 troublesome weed species grown in monoculture in the greenhouse or field Included were C4 tropical grasses: barnyardgrass, bearded sprangletop, Amazon sprangletop, broadleaf signalgrass, fall panicum, and large crabgrass; C4 sedge, yellow nutsedge; and C3 species: red rice, gooseweed, and redstem Rice root d13C levels averaged , 228%, indicating that these roots are highly 13C-depleted Root d13C levels ranged from 212% to 217% among the tropical grasses, and were 210% in yellow nutsedge, indicating that these species were less 13C depleted than rice, and were C4 plants suitable for 13C discrimination studies with rice Among the C4 species, bearded sprangletop and yellow nutsedge were most and least 13C depleted, respectively d13C levels in shoot and root tissue of pot-grown plants averaged 6% greater for C4 plants and 9% greater for rice in the field than in the greenhouse In pots, shoots of rice typically were slightly more 13C depleted than roots A reverse trend was seen in most C4 species, particularly for broadleaf signalgrass and plants sampled from field plots Corrections derived from inputs including the total mass, carbon mass, carbon fraction, and d13C levels of roots and soil increased greatly the accuracy of root mass estimates and increased slightly the accuracy of root d13C estimates (, 0.6 to 0.9%) in samples containing soil Similar corrective equations were derived for mixtures of rice and C4 weed roots and soil, and are proposed as a labor-saving option in 13C discrimination root studies Nomenclature: Barnyardgrass, Echinochloa crus-galli (L.) Beauv.; bearded sprangletop, Leptochloa fusca (L.) Kunth var fascicularis (Lam.) N Snow; Amazon sprangletop, Leptochloa panicoides (J Presl) A S Hitchc.; broadleaf signalgrass, Urochloa platyphylla (Nash) R D Webster; fall panicum, Panicum dichotomiflorum Michx.; large crabgrass, Digitaria sanguinalis (L.) Scop.; yellow nutsedge, Cyperus esculentus L.; gooseweed, Sphenoclea zeylanica Gaertn.; redstem, Ammannia coccinea Rottb.; red rice, Oryza sativa L.; rice, Oryza sativa L Key words: Stable carbon isotope, 13C/12C isotope ratio, d13C, 13C depletion, C3 photosynthetic pathway, C4 photosynthetic pathway, crop–weed root interference, tropical japonica rice, indica rice 13 C isotope discrimination analysis was recently used to determine the levels and distribution of roots of weedsuppressive rice and barnyardgrass in soil (Gealy and Fischer 2010) Barnyardgrass is an aggressive tropical grass that greatly affects rice production worldwide 13C is a naturally occurring, stable isotope that is present in about 1.1% of the atmospheric CO2 (West et al 2006) The availability of this technique for rice–weed root interaction studies represents a significant step forward because of the inherent complexities and difficulties in sampling, extricating, and quantifying intermixed rice and weed roots under flooded field conditions This isotope analysis approach is feasible because barnyardgrass uses the C4 photosynthetic pathway (Giussani et al 2001; Sage 2004; Smith and Brown 1973), whereas rice uses the C3 pathway C3 plants fix a lower percentage of 13C, and therefore are 13C-depleted in all plant organs compared with C4 plants because of inherent differences in the photosynthesis processes and anatomy of these two plant types (Ehleringer 1991; Farquhar et al 1989) C4 photosynthesis occurs in three monocot families, including the Poaceae (Giussani et al 2001; Waller and Lewis 1979) and the Cyperaceae (Muasya et al 2002), and in 16 dicot families including Amaranthaceae and Portulacaceae (Sage 2004) Factors that change stomatal conductance or photosynthetic capacity (e.g., light, water deficit, vapor pressure deficit) in typical C3 plants can alter the ratio of the CO2 partial pressures in the leaf interior substomatal cavities and the ambient air surrounding the leaf (i.e., Pi/Pa), which alters discrimination against 13C (Badeck et al 2005; Dingkuhn et al 1991) Thus, lower discrimination against 13C can result from lower leaf CO2 conductance, greater CO2 incorporation capacity, or both (Farquhar et al 1982) Changes in leaf CO2 conductance due to stress typically affects 13C discrimination differently in C4 plants from that in C3 plants C4 plants concentrate CO2 into bundle sheath cells even when stomata are partially closed and shade (as well as water or nutrient stress, and genetic variation) can induce leakiness of the bundle sheath cells to CO2 (Clay et al 2009; Farquhar et al 1982; Pansak et al 2007) 13 C isotope discrimination analysis, often measured as 13 d C, an expression of the 13C/12C isotope ratio relative to a fixed standard, has been used previously to determine the proportions of roots of C3 and C4 species in a number of field systems (Derner et al 2003; Eleki et al 2005; Gealy and Fischer 2010; Svejcar and Boutton 1985; Svejcar et al 1988) In other applications, 13C discrimination analysis has been used in rice to improve water use efficiency or transpiration efficiency (Dingkuhn et al 1991; Impa et al 2005; Kondo et al 2004; Scartazza et al 1998; Xu et al 2009) and to explain suppression of a weed species under temporary water stress (Fischer et al 2010) Examination of genetic associations of d13C levels with crop productivity traits in mapping populations of rice have indicated quantitative trait loci for d13C on five (Xu et al 2009) or on six (Laza et al 2006) of DOI: 10.1614/WS-D-10-00140.1 * Plant Physiologist, U.S Department of Agriculture Agricultural Research Service, Dale Bumpers National Rice Research Center, 2890 Highway 130 East, Stuttgart, AR 72160 Second author: Principal Professional Staff, Johns Hopkins University Applied Physics Laboratory, Laurel, MD Corresponding author’s E-mail: david.gealy@ars.usda.gov Gealy and Gealy: 13 Carbon isotope discrimination in rice–weed root interactions N 587 the 12 rice chromosomes 13C discrimination analysis has also been used to explain the effects of stress on grain crop yield loss (Clay et al 2001, 2005) Numerous weed species including barnyardgrass are problematic in rice fields in the southern United States (Smith 1988), but the prospects of using 13C isotope analysis to evaluate their root interactions with rice have not been explored in detail (Gealy et al 2005; Gealy and Fischer 2010) Among these species are other C4 grasses (Giussani et al 2001; Sage 2004; Smith and Brown 1973; Waller and Lewis 1979) such as bearded sprangletop, Amazon sprangletop, broadleaf signalgrass, fall panicum, and large crabgrass Additional common or troublesome weed species in rice include biotopes of red rice, gooseweed, redstem, and yellow nutsedge Although these species can be controlled to some degree in rice using registered herbicides (Scott et al 2010), they are among the most common and troublesome weeds in this crop in the southern United States (Smith 1988; Webster 2008) Simple extrapolations from standard concentration curves can provide good estimates of intermixed rice and C4 weed root quantities using 13C isotope discrimination analysis (Gealy and Fischer 2010) Inconsistent or incomplete soil removal from roots during processing, however, can introduce unpredictable errors Vigorous, extended washing/rinsing action usually removes most of the soil residue, but potentially increases time and resource requirements Further, the precise point at which soil has been adequately and uniformly removed for optimum results is difficult to determine in real time Thus, even after extensive washing procedures are completed, roots may retain unpredictable and sizeable levels of soil Evidence of this soil residue phenomenon is apparent in analyses of carbon content that show that carbon fraction (C fraction) levels in root samples tend to be much more variable and lower than those in shoot samples (Gealy and Fischer 2010) These observations suggest that derivations of soil correction calculations for root mass and d13C values might be developed on the basis of knowledge of the carbon composition of the plants and soil With the exception of barnyardgrass, little is known of the suitability of major weed species to 13C isotope depletion root interaction techniques in flooded rice systems Thus, the objectives of this research were to: (1) quantify d13C levels in roots of numerous troublesome weed species and rice cultivars grown as monocultures in flooded soil in field and greenhouse environments; (2) compare d13C levels in roots with those in shoots; and (3) develop mathematical corrections for d13C and root mass values in soil-contaminated samples Materials and Methods Pot Study in Greenhouse and Field Barnyardgrass, bearded sprangletop, broadleaf signalgrass, and fall panicum were chosen for a pot study to determine their d13C levels and assess the feasibility of using these grass weeds in 13C discrimination/root interaction studies with rice The rice cultivars ‘Lemont’ (Bollich et al 1985), a tropical japonica southern long grain, and ‘PI 312777’ (T65*2/TN 1; ‘WC 4644’), a weed-suppressive Asian indica (Gealy et al 2003; Gealy and Fischer 2010), were included as standards Seedlings of these weed and rice species were selected from natural stands and drilled rows, respectively, in rice research field plots that had been planted May 24, 2007 and emerged 588 N Weed Science 59, October–December 2011 June Plants in the four- to six-leaf stage were transplanted on June 18 to individual pots (, 20-cm diameter and , 24-cm depth) filled to , 83% capacity (, cm below the rim) with DeWitt silt loam soil (fine smectitic, thermic, Typic Albaqualfs) having a pH of 5.8 and an organic matter content of 1.2% Pots containing individual weed species or rice cultivar were randomly assigned to one of two groups placed under substantially different environmental conditions: ‘‘field environment’’ and ‘‘greenhouse environment.’’ The field pots were placed in bar ditches on the interior perimeter of the rice research field at the University of Arkansas Division of Agriculture Rice Research and Extension Center (RREC) (34u28980N, 91u259120W) near Stuttgart, AR On June 25, nitrogen fertilizer was applied to each pot as urea at a rate of , 110 kg N/ha On June 25, a permanent flood of 8- to 10-cm depth was established and maintained for the remainder of the growing period The upper rim of each pot was placed at approximately the level of the soil surface in the research plots, allowing water to flow naturally into and submerge the pots while plots were flooded Unwanted weeds were removed by hand At harvest, the aboveground portion of each plant (the shoots) was cut from roots at the soil surface The greenhouse pots were placed in a greenhouse equipped with a multistage evaporative cooling system that was thermostatically controlled to maintain minimum night temperatures above 21 C and maximum daytime temperatures below 35 C Daytime temperatures, however, sometimes exceeded 38 C during the hottest periods of the summer Midday irradiance levels in the greenhouse (photosynthetic photon flux density max , 400 mEm22s21) were only about one-quarter to one-third of the ambient levels in the field primarily because of deployment of ceiling shades intended to maintain greenhouse temperatures within tolerable limits No supplemental lighting was used; thus, day lengths were the same as ambient in the field A constant flood (, cm) was maintained in pots by adding deionized water as needed All other aspects of plant growth, culture, and sampling were the same as for the field pots Similar to the methods described by Gealy and Fischer (2010), roots from the entire soil/root mass in each pot were extracted and cleaned thoroughly Expanded Species Survey in Field In 2007 and 2008, an expanded group of weed species was sampled from natural stands present in drill-seeded, irrigated weed research plots at the RREC These areas were managed using the same general practices described previously for weed-suppressive rice experiments (Gealy and Fischer 2010) The species consisted of the original C4 tropical grass weed species used in the pot study and six additional weed species that have typically been among the most common and troublesome weed species in rice in the southern United States (Webster 2008) These included Amazon sprangletop, large crabgrass, yellow nutsedge, gooseweed, redstem, and the red rice biotypes AR-1995-StgB (PI653422) awned blackhull, AR-1994-8 (PI653425) awned blackhull, AR-1994-11D (PI653417) awned, LA-1995-LA3 (PI653420) awned brownhull, and AR-1995-StgS (PI653423) awnless strawhull (Gealy et al 2009; GRIN 2010) Rice entries included those from the pot study, the additional tropical japonicas ‘Wells’ (Moldenhauer et al 2007; long grain), ‘CL 141’ (imidazolinone-resistant, proprietary BASF cultivar; long grain), and ‘Bengal’ (Linscombe et al 1993; medium grain), the indica accession ‘4593’ (PI 615031; GRIN 2010), and locations d13C data were analyzed using the SAS Proc Mixed procedure The shoot–root difference in d13C levels in each plant sample was compared by subtracting the root value from the shoot value A value , indicates that the root value is higher (less 13C-depleted) than the shoot value An LSmeans test (P 0.05) was performed to determine which shoot and root values were significantly different from one another (i.e., shoot–root differences not equal to zero) Shoot–root differences for C fraction and mass values were similarly calculated and analyzed statistically The experimental design for the expanded species survey was a randomized complete block with the yr of the study serving as blocks with four subsampled plants per block d13C data were analyzed using the SAS Proc Mixed procedure An LSmeans test of the root–shoot difference that yielded a value different from zero at P 0.05 indicated that d13C values in roots and shoots were different for a particular species C fraction and mass data were analyzed using the SAS Proc GLM procedure and the mean differences were determined using Duncan’s multiple range test at P 0.05 ‘XL723’ (proprietary RiceTec hybrid) Individual rice, red rice, gooseweed, and redstem plants were obtained from areas receiving a season-long flood, while the other species were obtained from intermittently flooded or wet areas near levees adjacent to these areas Mature plants were collected, typically after rice harvest Although the intent was to retrieve the complete root systems, very long or fine roots could not always be extricated completely Plants were divided into roots and shoots The roots were washed and rinsed to remove soil as described by Gealy and Fischer (2010) After discovering that the root-cleaning procedures used in field and pot studies in 2007 sometimes failed to remove soil completely from plants, additional time and vigor of agitation were used to clean roots in 2008 Plant Tissue Analysis Roots and shoots from all experiments were dried to constant mass at 60 C and weighed to the nearest 0.1 g All shoots and the largest root masses were then ground in a large Wiley mill1 with 2-mm screen openings to produce coarsely ground tissue This material was mixed thoroughly and a total of , 30 g of representative tissue was removed in numerous subsamples, combined, and reground using a smaller Wiley mill2 with 1-mm screen openings, resulting in powdered tissue Root samples weighing less than 30 g were ground only in the smaller Wiley mill The 13C and C fraction levels in these plant tissues were quantified at the University of Arkansas Stable Isotope Laboratory using the procedure described by Gealy and Fischer (2010) Briefly, subsamples were weighed to an accuracy of 0.0001 mg, combusted in an elemental analyzer in a stream of helium, and resultant CO2 gas was analyzed by an isotope ratio mass spectrometer Raw 13C/12C isotope ratios were acquired by comparison with a reference gas injection and were normalized by comparison with in-house isotope standards traceable to international references The C fractions of samples were determined via instrument response to known standards One third to one half of the samples processed through the combustion/mass spectrometer procedure consisted of isotope standards to ensure proper calibration (Gealy and Fischer 2010) 13 C/12C isotope ratios were expressed relative to the international Pee Dee Belemnite (PDB) limestone fossil standard as d13C (Farquhar and Lloyd 1993; O’Leary 1993): ÂÀ Á à d13 Csample (0=00)~ Rsample {Rstandard =Rstandard |1,000 Corrections of Root d13C Values and Root Mass for Soil Contamination A mathematical expression to correct for the effect of soil contamination on estimated root mass of a single plant species was derived from a mixing equation describing the C fractions of the sample, root, and soil components A related expression that corrects for the effect of soil contamination on the sample root d13C level was derived independently Carbon Fraction Mixing Equation Root samples obtained from field soils contain carbon from the root tissues and from the soil that remained after washing These carbon masses can be expressed as follows: where Mc is the total carbon mass in the root sample, Mc1 is the carbon mass of the root, and Mcs is the carbon mass of the soil These carbon masses also can be expressed as the product of (total mass of each component in the mixture) (C fraction of that component) Thus: ½3Š f M ~f1 M1 zfs Ms ½1Š where f, f1, and fs are the respective C fractions, and M, M1, and Ms are the respective masses of the total sample, root component, and soil component (g) in the sample mixture Note that for simplicity and internal consistency with variable names that were used in separately derived Equations 10–25, we used the suffix ‘‘s’’ to designate soil and the number or to designate a plant species A variable name without one of these suffixes indicates that it refers to the sample mixture Substituting (M M1) for Ms and rearranging produces the corrected value for root mass (M1) expressed in terms of M and the component C fractions   f {fs ½4Š M1 ~M f1 {fs where d13Csample is the isotope ratio (in parts per thousand; %) relative to the PDB standard Rsample and Rstandard are the 13 12 C/ C molar abundance ratios of the plant sample and the PDB standard (Rpd; 0.0112372), respectively (Eleki et al 2005) Average d13C values for C3 and C4 plants were reported to be approximately 227% and 213%, respectively (Boutton 1996) The negative value indicates a lower 13C/12C ratio in plants than in the PDB standard Vogel (1980) considered d13C values for C4 plants to range within 29% to 216% and C3 plants to range within 222% to 234% For classification purposes in the present study, plants with d13C values 217% were included with the C4 plants And by definition: Statistical Design and Analysis The experimental design for the pot study was a randomized complete block with four replications and the two experiments were considered to be Gealy and Gealy: ½2Š Mc ~Mc1 zMcs ½5Š Ms ~M {M1 13 Carbon isotope discrimination in rice–weed root interactions N 589 Using methods described in the ‘‘Plant tissue analysis’’ section, M and f were determined for each root sample, and the fs value that was obtained from samples of root-free field soil was determined to be 0.008335 (considered a constant in this study) An approximation was used to determine the f1 values In the context of this correction procedure, f1 was set equal to the value of the shoot C fraction (f1 shoot) from the same plant This approximation was reasonable, because root and shoot C fractions were nearly equal in a subset of rice and C4 plant samples that had been vigorously rewashed to remove soil from roots In the cases in which f was f1 shoot, the f value was typically substituted for f1, forcing M M1 (via Equation 4) An alternative approach not used here would be to define f1 as the largest f value for that species, assuming that well-cleaned monoculture root samples were available for comparison Simple d13C Mixing Equation A d13C mixing equation was derived using carbon mass inputs along the lines of those used for C fraction mixing above (Equations and 3) Thus:         Mc1 Mcs f1 M1 fs Ms d~d1 zds ½6Š zds ~d1 Mc Mc fM fM where d, d1, and ds, are d13C levels in the carbon present in the total sample, root component (unknown), and soil component, respectively Using methods from the ‘‘tissue analysis’’ section above, d values were determined for each sample The ds of root-free soil samples was determined to be 221.27% (considered a constant in this study) Other variables were as defined previously Substituting M M1 for Ms as before, and rearranging, yields a second expression of M1:   df {ds fs ½7Š M1 ~M d1 f1 {ds fs Combine Equations and by factoring out M1, rearrange, and solve for d1, which is the soil-corrected d13C value for root tissue in the sample mixture:   df (f1 {fs ){ds fs (f1 {f ) d1 ~ ½8Š f1 (f {fs ) This expression for d1 in Equation was derived using a simple d13C mixing analogy and is a close approximation to the exact expression derived from a true mixing analogy on the basis of actual 13C/12C isotope ratios instead of the relative d13C values Over the broad range of input values used in the present studies, this approximation yielded d13C values nearly identical (to at least three decimal places) to those calculated using a more complex exact derivation on the basis of the actual carbon isotope ratios (Supplemental Appendix 1A) A spreadsheet containing the formulas for these equations can be accessed from Supplemental Appendix The approximation in Equation produces soil-corrected d13C values (d1) very close to those from the exact expression because the value of the Rpd standard and all other R values used in the exact expression are very small (i.e., 0.0112372 or less) We calculated this error to be in the range of , 0.0180 to 0.0281% (for d12ds differences of and 10%, respectively; data not shown) A more simplified approximation of d1 can be derived from Equation 8: 590 N Weed Science 59, October–December 2011   (d{ds )(fs )(f1 {f ) d1 ~dz f1 f ½9Š This equation generally yielded the same result as Equation when f 0.1 and fs of contaminating soil ,, f1 For instance, the fs 0.008335 for the low-organic-matter soil in the present study will produce acceptable results over a wide range of conditions However, an fs 0.05, as may occur in higher-organic-matter soils, could result in significant errors when using Equation Soil Supplementing Experiment To demonstrate the effect of soil contamination on the measured levels of d13C and C fraction in roots, root samples of monoculture Wells rice or barnyardgrass from the 2008 field study that had previously been shown to be nearly soil-free (i.e., similar C fractions in roots and shoots) were mixed with soil Pure soil used for supplementing experiments had a C fraction (fs ) of 0.008335 and a d13C of 221.27% Root samples were ground to a powder using the small Wiley mill with a 1-mm screen (as described above) and supplemented with pulverized, dry field soil (described above) at planned levels of 0, 12.5, 25, 50, 75, and 87.5% (g/g; soil/[roots+soil]) As actually prepared, the rice mixtures contained 0, 12.2, 26.3, 49.2, 75.1, and 87.3% soil (one subsample) and the barnyardgrass mixtures contained 0, 12.5, 25.4, 50.0, 73.6, and 87.0% soil (average of two subsamples) Samples were mixed thoroughly and submitted to the University of Arkansas Stable Isotope Laboratory for d13C and carbon content analysis as described in the ‘‘tissue analysis’’ section above Equations and were used to compare the mathematically corrected values for root mass and d13C, respectively, with those obtained for samples via laboratory analysis Expected d13C values for soil levels higher than those measured experimentally (i.e., 88, 94, 97, 98.5, 99.25, 99.625, and 99.81% soil) were simulated by solving Equation (after rearrangement) for d13C of the sample (d) at the sample C fractions (f ) equivalent to the respective soil% values above (all other variables held constant) General Correction Equations for Estimation of Rice and C4 Weed Root Mass in Samples with Soil Contamination Extending the logic we had used previously to produce corrections for the mass (Equation 4) and d13C (Equation 8) values of single-species root samples containing soil, we developed another set of mathematical expressions to correct for soil in sample mixtures containing unknown amounts of C3 rice and C4 weed roots To ensure the highest level of accuracy for results across the broadest range of variable inputs, we derived the relevant equations for this C3–C4–soil mixture from the exact expressions of the carbon isotope ratios (i.e., not d13C values) In a more complex system, attempting to simultaneously distinguish among three different species in root mixtures, Polley et al (1992) used a mixing approach to account for inherent species differences in C fraction Definitions for the variable names are similar to those for Equations 2–9 above: f and fs are the respective C fractions and M and Ms are the respective masses of the total sample and soil component (g) in the sample mixture; f1 and f2 are the respective C fractions and M1 and M2 are the respective masses of the C3 root component and C4 root component in the sample mixture Similarly, d, d1, d2, and ds are d13C   fi Mi C 12i ~ Ri z1 levels in the carbon contained in the total sample, the C3 and C4 root components, and the soil component, respectively We derived the appropriate expressions for M1 and M2 on the basis of two basic equations The first equation expresses the sample C fraction in terms of its component C fractions, similar to the approach used in Equation Thus: f M ~f1 M1 zf2 M2 zfs Ms Substituting expressions for 13C components from Equation 19 into the numerator of Equation 15, and the expressions for 12 C components from Equation 20 into the denominator of Equation 15, produces Equation 21, which is an expression of the sample R value as a function of the component R values (i.e., it is a mixing equation for the 13C/12C ratios) ! ! !1 f1 M1 f2 M fs ðM {M1 {M2 Þ B 1z z 1z z C 1z Rs B C R1 R2      C ½21Š R~B  B f1 M f2 M2 fs ðM {M1 {M2 Þ C @ A z z Rs z1 R1 z1 R2 z1 ½10Š Expressing Ms in terms of the other mass components: Ms ~M {M1 {M2 ½11Š f M ~f1 M1 zf2 M2 zfs ðM {M1 {M2 ị ẵ12 Thus: f ~f1       M1 M2 M {M1 {M2 zf2 zfs M M M Simplifying and rearranging yields:   M ðf {fs ịzM2 fs {f2 ị M1 ~ f1 {fs ẵ13 This can be rearranged to produce a second, independent expression for M1:   1  f2 ðR2 {R Þ fs ðR{Rs Þ fs ðRs {R Þ z zM M B R2 z1 Rs z1 R z1 C C ½22Š   s M1 B ~ @ A fs ðRs {R Þ f1 ðR{R1 Þ z Rs z1 R1 z1 ½14Š This is an independent expression for M1 based on a mixing equation for C fraction The second basic equation is the expression of the ratio (R) of 13C/12C in the sample (i.e., the 13C/12C mass fraction ratio)   C 131 zC 132 zC 13s ½15Š R~ C 121 zC 122 zC 12s The two expressions for M1 (Equations 22 and 14) are set equal, M1 is factored out, and the equation solved for M2, yielding the soil-corrected root mass of plant type (i.e., C4): M2 ~M  f ðR {R Þ    R {R z f1 ðR{R1 Þ ðf {fs Þ{ fs ðRs sz1 Þ ðf1 {fs Þ R1 z1 [23] @    ÁA f2 ðR2 {R Þ fs ðR{Rs Þ fs ðRs {R Þ f1 ðR{R1 Þ À ðf1 {fs Þ{ Rs z1 z R1 z1 fs {f2 R2 z1 z Rs z1 s This equation can be rearranged by grouping common terms, and further simplified to the following form: where i refers to plant type 1, i refers to plant type 2, and i refers to soil We know that 12C and 13C isotopes comprise essentially 100% of the carbon mass in our samples Thus:   C 12i zC 13i ½17Š fi ~ Mi M2 ~M   1[24] {R ðf {f1 Þ{ f1 ðR1z1 Þ ðf {fs Þ R1 @ A      f2 ðR2 {R Þ R {R {R ðf1 {fs Þz fs ðRs sz1 Þ ðf2 {f1 Þ{ f1 ðR1z1 Þ ðf2 {fs Þ R2 z1 R1 B fi M i C C C 13i ~B @ A z1 Ri fs ðRs {R ị Rs z1  Ri :Rpd ẵ1zdi =1,000ị ẵ25 Note that this is a generalized rearrangement of Equation Substituting this definition for the R values in Equation 23 or Equation 24 yields equations that express 13C/12C ratios in terms of Rpd, a fixed constant, and the familiar d13C term The mass of rice roots (M1) can be obtained from the same general equations (Equation 23 or Equation 24) after exchanging the fi and Ri indices for plant and plant (i.e., the original f1 and R1 values become f2 and R2, respectively, whereas the original f2 and R2 values become f1 and R1, respectively) The fs and f values and the Rs and R values are left unchanged This maneuver temporarily redefines plant as plant and vice versa, which facilitates the calculation of the root mass of the other species (M1) Soil mass (Ms) was calculated as before using Equation 11 Equations 10–25 were derived with Rpd and other R values expressed on both a molar abundance ratio basis and a mass C ½19Š Substituting C12iRi (as rearranged from Equation 16) for C13i in Equation 19, and rearranging, yields the equivalent expression for 12C mass: Gealy and Gealy:  For any 13C/12C ratio (Ri), its d13C value (di) can be expressed relative to the R value of the PDB standard (Rpd) according to the definition: which applies to plant type 1, plant type 2, and soil Combining Equations 16 and 17, we obtain:  1 C 13i z1 B C Ri C ½18Š fi ~B @ A Mi 13 s Rs z1 By definition, for any carbon-containing component of type i: C 13i Ri ~ ½16Š C 12i Rearranging Equation 18 yields an expression for the mass: ½20Š 13 Carbon isotope discrimination in rice–weed root interactions N 591 Table d13C levels in weed and rice samples from pots maintained in greenhouse or field environments, and the application of a mathematical correction for soil contamination in roots.a,b d13C Growth environment Species Barnyardgrass Bearded sprangletop Broadleaf signalgrass Fall panicum Lemont rice PI 312777 rice Greenhouse Field Greenhouse Field Greenhouse Field Greenhouse Field Greenhouse Field Greenhouse Field Shoot Root Corrected rootc Shoot minus corrected rootc,d Corrected rootc,d (species main effect) Shoot minus corrected rootc,d,e (species main effect) -(%) -213.8 ab 213.8 a 213.8 0.05 213.1 a 20.23 ab 213.0 a 212.9 a 212.4 20.51 215.8 c 216.8 b 216.7 0.94 215.8 c 0.58 a 214.7 b 215.0 ab 214.9 0.23 213.8 ab 215.1 ab 215.0 1.23* 215.0 bc 1.56* a 213.0 a 215.0 ab 214.9 1.90* 213.4 a 213.7 a 213.6 0.21 213.8 ab 0.55 a 213.2 a 214.3 ab 214.0 0.89 230.9 e 228.6 c 228.8 22.12* 228.1 d 21.76* b 228.8 d 226.9 c 227.4 21.40* 23.02* 229.0 d 21.71* b 232.1 f 228.7 c 229.0 229.4 d 228.2 c 228.9 20.39 Species Species environment environment interaction not interaction ns significant (ns) at P 0.05 at P 0.05 a Plants were grown in flooded pots in soil in the field or greenhouse during 2007 Values in columns are the estimated means according to an LSmeans test Values followed by the same letter were not different according to LSmeans (P 0.05) Corrected root d13C values were calculated using Equation d For the difference ‘‘shoot minus corrected root,’’ a value indicates that root value is lower (d13C is more negative; tissue is more 13C-depleted) than shoot value * indicates that the ‘‘shoot-corrected root’’ difference within that species is different from zero (i.e., shoot and root values are different from one another) according to an LSmeans test (P 0.05) e Main effect means for growth environment ‘‘Corrected root’’; field 218.8% and greenhouse 219.5% (P ,, 0.05) ‘‘Shoot-corrected root;’’ field 0.12% and greenhouse 20.45% (P 0.118) b c fraction basis Rpd molar abundance ratio 13C/12C 0.0112372 (Eleki et al 2005), and Rpd mass fraction ratio (Rpd molar abundance ratio)(13/12) Because calculated results were , identical to four decimal places, both models were considered equally acceptable A more complete explanation of the various steps used to derive the equations in this section is presented in Supplemental Appendix 1B A spreadsheet containing formulas to calculate results for Equation 23 can be accessed from Supplemental Appendix A less cumbersome and simplified approximation of the exact expression of soil-corrected root masses shown in Equation 23 was developed using a simple mixing model for d13C values It is presented as Equation 14c.12 in Supplemental Appendix 1C Results and Discussion 13 d C Levels in Roots and Shoots of Rice and Weeds: Pot Study The d13C levels in roots of the tropical C4 grass weeds were readily distinguished from those in rice cultivars in both the field and greenhouse (Table 1) Soil-corrected d13C values for the C4 grass roots ranged from 212.4% to 216.7% and the uncorrected values were slightly lower, ranging from 212.9% to 216.8% Among these four weed species, root and shoot d13C levels in barnyardgrass and fall panicum were highest, whereas those in bearded sprangletop were lowest Soilcorrected values for rice roots averaged , 228.5% and the uncorrected sample averages were slightly greater at 228.1% Root d13C levels in tropical japonica Lemont rice were usually similar to those in indica PI 312777 rice, but shoot d13C levels in the greenhouse were 4% (or 1.2%) lower in PI 312777 than in Lemont The d13C levels in rice in the field and greenhouse in the present studies generally were similar to those in 592 N Weed Science 59, October–December 2011 nonstressed rice described earlier (Scartazza et al 1998; Zhao et al 2004) Our data clearly confirm these four grass weeds and rice to be C4 and C3 plants, respectively Root d13C soil-corrected values averaged up to 2.1% higher in C4 grasses and 2.5% lower in C3 rice compared with noncorrected values (Table 1) These divergent trends for the corrected values of C4 grasses and rice are consistent with the fact that the d13C level in our soil (, 21.27%; as described in Materials and Methods) was between that of the two plant types These results for C4 and C3 plants were generally consistent with those estimated previously (Gealy and Fischer 2010), where root d13C levels in monoculture C4 barnyardgrass and rice averaged 213.1% and 228.5%, respectively Similarly, d13C levels in nonstressed Leptochloa fusca (L.) Kunth (Kallar grass), a bearded sprangletop C4 relative, were 214.7% (Akhter et al 2003) d13C levels in both root and shoot tissues were greater (i.e., less 13C-depleted) in the field than in the greenhouse, exhibiting increases of about 6% for C4 plants and 9% for rice (Table 1) On calm, sunny days, air in the rice field canopy may have become CO2 depleted (Gealy, unpublished data) compared with the well-mixed ambient air introduced into the greenhouse Plants were probably not fully light saturated in the greenhouse where they were at O lower irradiance levels than in the field Corn under low light conditions has been reported to have greater 13C discrimination levels than if grown under full sun (Clay et al 2009) Therefore, low light conditions in the greenhouse may have contributed to discrimination differences seen in our plants when compared with field values In both pot environments, shoot d13C levels closely mirrored those in the roots The 13C levels in C4 grass species (Table 1) generally were similar or lower (more 13C- Table d13C levels in additional weed species and rice cultivars growing in or near rice field plots in 2007 and 2008, and the application of a mathematical correction for soil contamination in roots.a,b d13C c Species Bearded sprangletop Amazon sprangletop Barnyardgrass Broadleaf signalgrass Fall panicum Crabgrass Yellow nutsedgec Redstem Gooseweed AR-1995-StgB awned red rice Wells long-grain rice 4593 indica rice Bengal medium-grain rice XL723 hybrid rice Shoot Root Corrected rootd Shoot minus corrected rootd,e - (%) -214.7 c 217.2 d 217.1 d 2.45* a 213.9 bc 215.2 cd 215.1 cd 1.19* a 212.4 a 213.5 bc 213.5 bc 1.08* ab 212.9 ab 214.2 bc 214.2 bc 1.30* ab 212.5 a 213.9 bc 213.8 bc 1.29* ab 212.5 a 211.9 ab 211.9 ab 20.56 bc 212.3 a 210.4 a 210.3 a 21.94* c 228.1 d–f 228.6 e 228.7 e 0.66 ab 227.2 d 226.8 e 226.9 e 20.25 bc 229.1 f 228.9 e 229.0 e 20.12 bc 227.9 de 227.5 e 227.9 e 0.01 abc 228.3 ef 227.0 e 227.0 e 21.29* bc 227.6 de 226.8 e 227.3 e 20.31 bc 228.1 d–f 228.1 e 228.4 e 0.36 abc Additional O sativa entries from same field locationc AR-1995-StgS awnless red rice 228.1 0.1 (2007 only) (n 4) AR-1994-8 awned red rice 229.4 0.8 (2008 only) (n 4) AR-1994-11D awned red rice 229.6 0.6 (2008 only) (n 4) LA-1995-LA3 awned red rice 229.8 0.9 (2008 only) (n 4) Lemont rice (2008 only) 227.9 0.5 (n 4) CL 141 rice (2007 only) 227.5 0.1 (n 4) PI 312777 rice (2008 only) 227.9 0.1 (n 4) 228.0 0.7 (n 4) 227.8 0.4 (n 4) 228.6 0.1 (n 4) 228.7 0.3 (n 4) 227.9 1.2 (n 3) 227.1 0.5 (n 4) 225.9 1.3 (n 4) 228.2 (n 227.8 (n 228.7 (n 228.7 (n 228.0 (n 227.8 (n 226.0 (n 6 6 6 0.7 4) 0.4 4) 0.1 4) 0.3 4) 1.2 3) 0.5 4) 1.3 4) 0.03 0.72 (n 4) 21.63 0.63 (n 4) 20.97 0.67 (n 4) 21.15 1.08 20.10 0.84 (n 3) 0.37 0.55 (n 4) 21.90 1.33 (n 4) a Plants were grown in or near flooded rice field plots in 2007 or 2008 (or both years) Values in columns are the estimated means according to an LSmeans test in Proc Mixed Values followed by the same letter were not different according to LSmeans (P 0.05) The additional O sativa entries (bottom section of table) that were evaluated in yr only were not included in the statistical analysis with other data Only the means and standard deviations of subsamples were calculated c The d13C of yellow nutsedge nutlets averaged 211.53% (data from 2008 only; not included in statistical analysis; not corrected for soil contamination) The additional O sativa entries were obtained from same field location as species above, evaluated yr only, and were not included in statistical analysis Plant growth environment: rice cultivars, red rice lines, redstem, and gooseweed obtained from flooded rice fields; bearded sprangletop from flooded rice fields or area adjacent to rice field levees; all other plant species from areas adjacent to rice field levees d Corrected root d13C values were calculated using Equation e For the difference, ‘‘shoot minus corrected root,’’ a value indicates that root value is lower (d13C is more negative; tissue is more 13C-depleted) than shoot value * indicates that the shoot-corrected root difference within that species is different from zero (i.e., shoot and root values are different from one another) according to an LSmeans test (P 0.05) b d13C Levels in Roots and Shoots of Rice and Weeds: Expanded Species Field Survey In the species common to both experiments, d13C levels in the expanded field survey generally followed trends similar to those in the pot study d13C levels for roots of C4 plants were lowest in bearded sprangletop (217.1%), followed by Amazon sprangletop, intermediate in broadleaf signalgrass, fall panicum, and barnyardgrass, and greatest in crabgrass and yellow nutsedge (210.3%) (Table 2) Rajagopalan et al (1999) reported similar high d13C levels (28.2% to 211.5%) in the cellulose of relatives of yellow nutsedge (Cyperus spp.) growing in peat bogs In contrast to the pot study, root d13C levels in nearly all of the C4 grass species in the expanded species survey were lower than in shoots, ranging from 8.7% (1.1%) for barnyardgrass to 16.7% (2.5%) for bearded sprangletop Yellow nutsedge differed from most of the other C4 weed species in that its root d13C levels were 15.8% (1.9%) higher than in shoots, which was similar to the trend for rice (Table 2) Both root and shoot d13C levels were similar among the four rice cultivars in the field plots (Table 2) Root d13C levels depleted) in roots than in shoots, and this difference averaged , 12% (1.6%) in broadleaf signalgrass Data from Badeck et al (2005) indicated that d13C levels in roots were sometimes greater and sometimes less than in shoots of C4 species (n 10) In contrast to the C4 weeds, rice d13C levels averaged , 6% (1.7%) greater (less 13C-depleted) in roots than in shoots (Table 1) Previous reports have also indicated that rice leaves and shoots generally were more 13C-depleted than roots (Badeck et al 2005; Klumpp et al 2005; Scartazza et al 1998; Zhao et al 2004) A compilation of , 400 comparisons of 13C depletion in numerous species showed that roots of C3 plants were, on average, 1.08% less 13Cdepleted compared with leaves (Badeck et al 2005) The corrected root d13C values in rice were at least 78% (12.3%) lower than in the four weed species (on the basis of species main effect means), whereas noncorrected root d13C values in rice were at least 70% (11.8%) lower than in these weeds (on the basis of the species environment interaction means) (Table 1) Clearly, C3 and C4 plant roots can be distinguished in our rice field soils containing low levels of organic carbon Gealy and Gealy: 13 Carbon isotope discrimination in rice–weed root interactions N 593 were greater than shoot d13C levels in 4593 indica rice only, but a similar trend was noticed in PI 312777 indica rice (2008 only) (Table 2) This tendency toward greater d13C levels in roots than in shoots of rice was even more pronounced in the pot experiments (Table 1) Earlier studies have also reported a tendency toward lower shoot d13C levels (greater 13C discrimination) in indica than in japonica rices (Dingkuhn et al 1991; Kondo et al 2004; Peng et al 1998) By contrast, screening of 57 3- to 4-wk-old rice cultivars showed that 13C discrimination averaged about 1.7% lower in indica types compared with tropical japonica types (Xu et al 2009) In the present studies, d13C levels differed between tropical japonica and indica rice only in greenhouse pots where the 13C discrimination was 3.8% greater in shoots of PI 312777 indica compared with Lemont (Table 1) Similar but nonsignificant trends were observed for root d13C levels in pots in both greenhouse and field environments Under environments that may be particularly stressful to one of these rice types and not the other (e.g., cool early-season conditions that are more stressful to indicas than japonicas), the d13C signatures of these two rice types could change slightly These differences would be expected to be no more than a few percent, however, and are not likely to contribute substantially to errors in d13C root analysis studies Separate monoculture standards of tropical japonica and indica cultivars could be grown if greater precision for rice d13C values is desired Overall, the d13C levels in rice in the field and greenhouse environments in the present study ranged from about 227% to 232% A d13C of 232% equates to about the greatest 13 C discrimination reported by Xu et al (2009) in a comparison of 116 accessions from seven different Oryza species in well-watered greenhouse pots The lowest 13C discrimination level in our test (d13C 227%) equates to about 12% lower than the minimum reported by Xu et al (2009) This may be attributable to our later growth stage of sampling (mature vs 3- to 4-wk-old plants), and the possibility that as plants matured in pots, they experienced additional stress due to pot-bound roots (Comstock et al 2005) This type of stress was apparently avoided in the Xu et al (2009) study Scartazza et al (1998) have reported 13C discrimination levels in potted rice plants similar to those in the present study and showed that the discrimination decreased by , 10% in 170-d-old plants compared with 20-d-old plants Corrected root d13C levels in C3 plants averaged 100% (14%) lower, and all rice cultivars were at least 58% (9.9%) lower compared with the C4 plants grown in field plots in 2007 and 2008 (Table 2) These contrasts between C3 and C4 plants are similar to those observed in the pot experiment, again confirming our C4 weeds to be ideal for d13C rice root interaction studies in field soils The d13C values for barnyardgrass and fall panicum roots were statistically indistinguishable in all of the environments/years evaluated in this study (Tables and 2) Thus, 13C discrimination methods potentially could be used to evaluate the combined/ average effects of these two common C4 weeds in mixtures with rice in the field Because of their distinctively high d13C levels and the resulting large d13C differential with rice, yellow nutsedge, and perhaps crabgrass may be especially well suited to 13C discrimination studies, and potentially could yield root mixture data that are more accurate than those of the other C4 weed species (Table 2) 594 N Weed Science 59, October–December 2011 Root d13C levels in gooseweed, redstem, AR-1995-StgB red rice, and all rice cultivars were similar, averaging , 227.9% (Table 2) These data confirmed that these three weed species are C3 plants similar to rice, and thus unsuitable for 13C discrimination studies with rice–weed root mixtures United States red rice types often share key genetic traits with indica rice (Gealy et al 2009; Londo and Schaal 2007; Vaughan et al 2001) Generally consistent trends between d13C levels of roots and shoots of key C4 weed species and rice were observed in these studies (Tables and 2) d13C levels in roots of C4 weeds (except for crabgrass and yellow nutsedge) and rice were 1.0 to 1.1 times and 0.9 to 1.0 times the respective levels in shoots Such trends between the d13C levels in these plant organs have been observed in numerous species (Badeck et al 2005; Klumpp et al 2005; Scartazza et al 1998) If monoculture root samples were unreliable or unavailable for some reason, shoot d13C values potentially could be substituted for root d13C values, or used as an internal standard check for d13C levels within the same plant Carbon Content and Mass of Roots and Shoots The shoot C fraction of C4 weed species ranged from 41 to 44% in pot studies (Table 3) and from 39 to 42% in the field survey (Table 4) The shoot C fraction of rice in pot studies (Table 3) ranged between 41 and 42%, and in the expanded field survey (Table 4) was more variable and slightly lower, ranging from 34 to 39% These rice shoot C fraction levels are similar to those reported for rice in an earlier study where the foliar C fraction of an indica subgroup, aus (0.392), was lower (P , 0.01) than for indica (0.402) or tropical japonica (0.404) groups (Dingkuhn et al 1991) In the pot studies, C fraction of root samples was generally greatest in barnyardgrass and broad-leaved signalgrass in the greenhouse and lowest in rice in the field (Table 3) The C fraction of root samples, particularly for rice, was often much lower than in shoots (Tables and 4), a phenomenon that has been attributed primarily to presence of difficult-to-remove soil residue (Gealy and Fischer 2010) In the pot and survey studies conducted in the field in 2007, C fraction of root samples of some rice cultivars (e.g., PI 312777 and CL 141) averaged as much as 80% lower than the levels in shoots (Tables and 4) Implementation of a more vigorous root cleaning/extraction process in the 2008 field study resulted in substantially greater C fraction levels in roots that often approached those in shoots (data not shown), and a trend toward higher root C fraction values that year (Table 4) This improvement was also evident in the additional rice and red rice entries sampled from field plots in 2008 compared with 2007 (Table 4; ‘‘additional entry’’ section) In most entries collected exclusively in 2008, C fractions were only to 4% less in root samples than in shoots, although in PI 312777 the C fraction was 23% less in roots than shoots By contrast, the C fraction of root samples collected exclusively in 2007 averaged 63% less than in shoots The masses of the 2007 root samples were also unusually high, averaging four times greater than those in 2008, which further indicated heavy soil contamination in 2007 Similar large discrepancies in C fractions and masses between rice root and shoot samples also were observed in the 2007 field pot study (Table 3) Because of the variation and uncertainties in C fraction and mass of root samples caused by soil contamination, we performed a mathematical calculation that corrected root Table Carbon content and mass of weed and rice samples from pots maintained in greenhouse or field environments, and the application of a mathematical correction for soil contamination in roots.a,b Species C content (C fraction 100) Growth environment Shoot Mass Root Shoot Corrected rootc,d Root Soil calculatedc % -g plant21 Barnyardgrass Bearded sprangletop Broadleaf signalgrass Fall panicum Lemont rice PI 312777 rice Greenhouse Field Greenhouse Field Greenhouse Field Greenhouse Field Greenhouse Field Greenhouse Field 40.7 f 41.5 d–f 42.5 a–d 43.5 ab 42.1 c–e 41.7 c–f 42.9 a–c 43.48 a 42.1 b–e 41.5 d–f 40.9 ef 41.2 d–f 28.9 11.5 27.2 19.0 32.0 27.3 26.9 24.2 25.1 9.8 16.1 7.8 ab c–e a–c a–e a a– a–c a–d a–d de b–e e 27.1 17.2 20.2 29.0 17.1 6.8 29.3 4.3 21.9 19.3 18.9 17.6 a a–c ab a a–c bc a c a ab ab a–c 7.8 41.2 7.9 18.4 4.7 11.4 12.2 10.9 10.7 59.2 14.7 51.7 c ab c bc c c c c c a c a 5.3 9.6 4.6 6.8 2.9 6.4 7.0 4.3 5.9 13.1 5.4 8.6 not significant at P 0.05 2.3 31.4 3.0 9.8 1.5 4.8 5.1 6.5 4.6 45.9 9.1 42.9 b a b b b b b b b a b a a Plants were grown in flooded pots in soil in the field or greenhouse during 2007 Values in columns are the estimated means according to an LSmeans test Values followed by the same letter were not different according to LSmeans (P 0.05) c Corrected root mass was calculated using Equation Soil mass was calculated using Equation d Corrected root mass Main effect means for growth environment; field 8.12 g and greenhouse 5.18 g (P 0.0042) Main effect means for species (P 0.0865) Species growth environment interaction (P 0.1012) b Table Carbon content and mass of additional weed species and rice cultivars growing in or near rice field plots, and the application of a mathematical correction for soil contamination in roots.a,b C content (C fraction 100) Speciesc Shoot Mass Root Shoot Root Corrected rootd Soil calculatedd - % g plant21 -Bearded sprangletop Amazon sprangletop Barnyardgrass Broadleaf signalgrass Fall panicum Crabgrass Yellow nutsedgec Redstem Gooseweed AR-1995-StgB awned red rice Wells long-grain rice 4593 indica rice Bengal medium-grain rice XL723 hybrid rice 41.4 41.5 39.6 39.6 40.6 38.8 39.4 43.1 45.0 37.7 37.8 37.3 37.9 37.8 bc bc c–e c–e cd de c–e ab a e e e e e Additional entries from same field locationc AR-1995-StgS awnless 36.6 0.8 red rice (2007 only) (n 4) AR-1994-8 awned 36.4 0.6 red rice (2008 only) (n 4) AR-1994-11D awned 35.3 1.0 red rice (2008 only) (n 4) LA-1995-LA3 awned 34.9 1.3 red rice (2008 only) (n 4) Lemont (2008 only) 35.7 0.6 (n 4) CL 141 (2007 only) 38.9 0.5 (n 4) PI 312777 (2008 only) 34.2 2.5 (n 4) 29.0 30.5 37.0 34.4 28.1 41.1 38.9 24.7 27.9 26.4 22.1 21.3 18.9 21.4 b–f a–e a–c a–d b–f a ab d–f b–f c–f ef ef f ef 89.4 54.0 66.3 54.4 141.8 94.6 25.8 18.4 26.2 101.6 67.9 55.6 46.3 56.7 20.2 6.2 (n 4) 35.3 2.9 (n 4) 33.8 3.1 (n 4) 33.7 1.5 (n 4) 35.7 5.3 (n 3) 7.2 2.2 (n 4) 26.4 3.7 (n 4) bc b–d b–d b–d a a–c d d d ab b–d b–d cd b–d 100.1 62.9 (n 4) 69.8 18.4 (n 4) 116.8 53.3 (n 4) 145.6 54.1 (n 4) 15.8 4.2 (n 4) 70.4 44.8 (n 4) 24.5 13.7 (n 4) 18.3 9.0 9.6 4.6 16.3 4.3 5.8 5.1 16.0 21.6 47.5 45.4 46.2 39.6 b–d d d d cd d d d cd a–d a ab ab a–c 41.9 39.7 (n 4) 12.0 5.1 (n 4) 16.6 4.2 (n 4) 20.8 5.5 (n 4) 6.7 1.8 (n 4) 70.3 42.8 (n 4) 8.4 4.9 (n 4) 10.2 5.1 8.7 3.1 10.9 4.1 5.2 2.7 6.4 13.6 14.0 8.6 8.6 10.7 a–c b–d a–d cd ab b–d b–d d b–d a a a–d a–d ab 17.5 7.8 (n 4) 11.5 5.4 (n 4) 15.6 64.1 (n 4) 19.9 5.8 (n 4) 6.4 2.1 (n 4) 11.6 7.5 (n 4) 6.2 4.9 (n 4) 8.1 3.9 0.9 1.5 5.4 0.2 0.5 2.4 9.6 8.0 33.5 17.5 37.7 28.9 bc c c c c c c c bc bc a a–c a ab 24.4 32.2 (n 4) 0.5 0.6 (n 4) 1.5 (n 4) 0.9 0.8 (n 4) 0.3 0.6 (n 4) 58.6 35.8 (n 4) 2.1 1.7 (n 4) a Plants were grown in or near flooded rice field plots in 2007 or 2008 (or both years) Values in columns are the arithmetic means Values followed by the same letter were not different according to Duncan’s multiple range test (P 0.05) The additional O sativa entries (bottom section of table) that were evaluated in yr only were not included in the statistical analysis with other data Only the means and standard deviations of subsamples were calculated c Carbon content of yellow nutsedge nutlets averaged 41.9% (data from 2008 only, and were not included in statistical analysis; values not corrected for soil contamination) The additional O sativa entries were obtained from same field location as species above, evaluated yr only, and were not included in statistical analysis Plant growth environment: rice cultivars, red rice lines, redstem, and gooseweed obtained from flooded rice fields; bearded sprangletop from flooded rice fields or area adjacent to rice field levees; all other plant species from areas adjacent to rice field levees d Corrected root mass was calculated using Equation Soil mass was calculated using Equation b Gealy and Gealy: 13 Carbon isotope discrimination in rice–weed root interactions N 595 Figure Response of carbon content to the quantity of supplemental soil present in ground root samples of Wells rice (A) and barnyardgrass (B) mass values on the basis of the observation that the expected C fractions in roots and shoots should be about equal These corrections revealed that soil contamination of root samples was substantially greater in the field pots than in the greenhouse pots (Table 3) In field pots, soil contamination in rice was typically greater than in the weed species (except barnyardgrass), a trend that appeared to be associated with greater corrected root mass (Table 3) In field pots, PI 312777 root samples contained five times as much soil mass as root mass Similar to the results in field pots, rice root samples from the field plot areas were more contaminated with soil than were the weed species (Table 4) Densely packed roots emanating from below the crown area of some rice cultivars or their fibrous nature may have facilitated the retention of soil particles by rice roots Although few differences among soilcorrected root mass values were significant, root masses of Wells rice and AR-1995-StgB red rice averaged more than four times the mass of broadleaf signalgrass and redstem (Table 3) An additional helpful procedure for future studies may be to discard all tissues from the top to cm of the crown area of rice roots below the soil surface where soil can be heavily compacted within the dense root mass This step would ensure that soil trapped in these upper roots would not be inadvertently included with standards or ordinary samples Soil Supplementing Experiment Supplementing clean monoculture rice and barnyardgrass roots with soil demonstrated that increased soil mass in root mixtures caused large, near-linear reductions in the sample C fraction (f ) levels 596 N Weed Science 59, October–December 2011 Figure Comparison of calculated and actual mass of ground Wells rice (A) and barnyardgrass (B) roots with supplemental soil added Calculated values were determined using Equation (Figures 1A and 1B) Equation shows how the corrected root mass (M1) will be reduced when the sample C fraction (f ) is reduced by soil contamination Equation shows the mass of the soil contamination Because the C fraction of soil (fs ) was ,, that of the roots (f1) or sample (f ) in our study, its effect on root mass estimates should be minimal except at high soil contamination levels in which the C fractions of the sample begin to approach the low levels for soil (Equation 4) The calculated and actual root masses of rice and barnyardgrass in sample mixtures were highly correlated (R 0.98; R 0.97), indicating a substantial benefit from the soil correction (Figures 2A and 2B) It should be noted, however, that an underestimation of the true root C fraction value (i.e., from an unusually low f1shoot value in the present study) used in Equation would result in a higher-than-expected corrected root mass (M1) value In contrast to its large effect on sample C fraction, soil contamination affected d13C levels minimally at levels below 87% (Figures 3A and 3B) With a C fraction of only 0.008335 (, 1.44% soil organic matter according to a conventional estimation procedure for this soil; Morteza Mozaffari, personal communication, University of Arkansas, 2010), this soil contributed only , 14% of the carbon to samples containing 87% soil Thus, its influence on sample d13C values will be minor except at low sample C fraction levels Corrected d13C values closely paralleled those for the Figure d13C levels in ground root tissues of Wells rice (A) and barnyardgrass (B) with supplemental soil added, as determined from laboratory analysis and calculation by correction equations Calculated values were determined using Equation Most of the symbols depicting calculated values (black diamonds) at soil levels less than 85% have been obscured significantly by the symbols depicting the corresponding sample values (black circles), and may not be visible Expected d13C values for soil levels of 88 to 99.8% were simulated by solving Equation (after rearrangement) for d13C of sample at the sample C fractions equivalent to the respective soil% values, and all other variables held constant Figure Simulation of the expected d13C (d) values in rice (A) and barnyardgrass (B) samples containing increasing soil C fractions (fs ) The soil contamination level was held constant at , 85% (g/g) by adjusting the sample C fractions to the appropriate level All other variables were held constant Expected d13C values were determined from Equation (after rearrangement) by solving for d13C of sample (d) at each sample C fraction analyzed sample mixtures containing up to 75% soil, but at 87.5% soil, they underestimated the analyzed samples by 4% in rice and overestimated them by 5% in barnyardgrass (Figures 3A and 3B) The reason for these discrepancies is uncertain, but incomplete mixing during sample preparation or errors in any of the measured or estimated variable inputs could have contributed Simulated d13C sample values at %soil levels $ 88% gradually curved toward the d13C soil value of 221.27% at 100% soil To gain insight into the relatively larger effect on d13C sample values of roots contaminated with much higher levels of soil, we simulated a scenario of increasing C fraction (organic matter) levels using Equation (rearranged to solve for the d value) The %soil level was held constant at , 85% soil (g/g), sample C fraction levels manipulated to maintain the constant soil%, and other variables held constant At increasing soil C fractions, d13C values of rice root samples increased (Figure 4A), whereas those of barnyardgrass root samples decreased (Figure 4B) The opposite trends for the responses of the two species occurred because the d13C of soil lies approximately midway between the d13C of rice and barnyardgrass roots Because the root d13C values are constants in this context, the sample d13C values for the C3 and C4 species will always converge toward the d13C value of Gealy and Gealy: the soil as the soil carbon component becomes more prominent with increasing soil C fraction levels Mathematical equations were developed to correct for the effects of soil contamination on measurements of plant mass and d13C levels in root samples d13C correction equations typically resulted in minor adjustments to the raw data from both rice and C4 weeds because the d13C level of the carbon associated with soil contamination was greater than that for rice and less than that for the C4 weeds Relative to uncorrected values, the corrected d13C values averaged 0.61% greater in the C4 plants and 0.89% lower in C3 plants, and were always within 2.6% of these values, largely because the C fraction of our low-organic-matter soil was very small compared with that of the plants Root d13C values for rice were usually affected slightly more than those for C4 weeds, probably because of the greater soil contamination in rice roots Development of Predictive Correction Equations for Rice and C4 Weed Root Mixtures with Soil Contamination Root interaction studies using 13C methods are inevitably accompanied by unpredictable variability in the d13C values 13 Carbon isotope discrimination in rice–weed root interactions N 597 Figure Simulated effect of sample C fraction on the mass of rice and C4 weed roots and soil as calculated from the two-species soil correction equation: (A) light infestation of bearded sprangletop with d13Csample (d) 225%; (B) light infestation of yellow nutsedge with d13Csample (d) 225%; (C) very heavy infestation of bearded sprangletop with d13Csample (d) 217%; (D) moderately heavy infestation of yellow nutsedge with d13Csample (d) 217% Variable values used in calculations: C fraction soil (fs ) 0.008335; C fraction rice (f1) 0.38; C fraction C4 weed (f2) 0.40; d13Csoil (ds) 221.27%; d13Crice (d1) 228%; d13Cyellow nutsedge (d2) 210%; d13C bearded sprangletop (d2) 216% Mass values were calculated using Equations 11, 23 or 24, and 25 of monoculture rice roots and C4 weed roots (usually small), and in the soil contamination of the samples (usually larger, less predictable) We derived equations to calculate corrected root masses of C3 rice (M1) and C4 weeds (M2) in the presence of soil (Ms) contamination (Equations 23 or 24, and 25) We propose these corrections to address uncertain levels of soil contamination and unequal species C fraction levels in root mixtures analyzed with 13C methods They should also be adaptable to root interactions of other C3-C4 species systems Insight can be obtained by graphing results from a range of expected input data For instance, at fixed values of sample d13C (e.g., d 225 or 217%) and other key input variables, increases in sample C fraction (i.e., decreasing soil contamination) will produce increases in the root masses of bearded sprangletop (Figures 5A and 5C) and yellow nutsedge (Figures 5B and 5D) Reducing the sample C fraction to 0.2 from typical actual C fraction levels of , 0.38 to 0.40 is accompanied by an increase in soil mass to about 50% of the total sample mass (Figures 5A–5D) However, for the same sample C fraction of 0.2, and soils containing a 103 higher C fraction, the soil mass will increase (, 40% at d sample 225%) relative to rice and yellow nutsedge root masses (data not shown) A greater root mass for C4 weeds relative to rice is also predicted as the sample d13C values approach those of rice The range of responses for the five other C4 weed species in this study fall between those of yellow nutsedge and bearded sprangletop, because these two species represented the maximum difference in d13C values among the C4 species 598 N Weed Science 59, October–December 2011 Plants in the present study were not intentionally exposed to stress However, it should be noted that stress can alter d13C levels in plants Rice discriminated progressively less against 13C with increased duration of water deficit stress (Scartazza et al 1998), and Zhao et al (2004) reported similar results in upland rice 13C discrimination in C4 Kallar grass was about 3% less under water deficits compared with well-watered conditions (Akhter et al 2003) In C4 corn, however, 13C discrimination was increased by water-deficit stress and decreased by nitrogendeficit stress (Clay et al 2005; 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S Rice Fields and Its Potential Use for Analysis of Rice? ? ?Weed Root Interactions. .. consistent trends between d13C levels of roots and shoots of key C4 weed species and rice were observed in these studies (Tables and 2) d13C levels in roots of C4 weeds (except for crabgrass and. .. in Roots and Shoots of Rice and Weeds: Pot Study The d13C levels in roots of the tropical C4 grass weeds were readily distinguished from those in rice cultivars in both the field and greenhouse