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Release of organic c from growing roots of meadow fescue (Festuca pratensis L.)
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Soil Bid Biochem. Vol. 24, No. 5, pp. 421-433, 1992 Printed in Great Britain. All rights reserved
Copyright C 1992 Pergamon Press Ltd
RELEASE OF ORGANIC C FROM GROWING ROOTS OF MEADOW FESCUE (loESTUCA PRATENSIS L.) G. JOHANSSON Department
of Soil Sciences, Division of Plant Nutrition and Soil Fertility, Swedish University of Agricultural Sciences, P.O. Box 7014, S-750 07 Uppsala, Sweden [Accepted 30 November 1991)
Summery-A new method to estimate the amount of organic C released from roots during plant growth is described. The method is based on decomposing root-derived material left in the soil after plant growth and removal of all visible roots in a long-term incubation. The total amount of organic C released during plant growth is then calculated from the amount of root-derived material remaining in the soil after long-term decomposition and a stabilization factor for this material. An estimate of the total amount of organic C released from roots during plant growth allows the total rhizosphere respiration to be separated into root respiration and microbial respiration. In a previous cultivation of continuously “‘C-1abelled meadow fescue (Festuca pratensis L.) the distribution of net assimilated 14Cwas determined. Root-derived material in root-free soil from the cultivation was allowed to decompose in a long-term incubation (61 weeks). The amount of stabilized root-derived material left in the soil after the incubation, representing a certain proportion of the fresh root-derived material released during growth, was calculated. The stabilization of root-derived material was approximated through comparison with stabilization of known organic materials (glucose, grass shoots and roots). Decomposition of root-derived material resulted in more acid-stable residues than decomposition of glucose but less than shoots and roots. From the relationship, for the known organic materials, between the amount of stabilized organic material remaining after decomposition and the amount of acid-stable residue, the amount of original root-derived material was estimated. This result was combined with data from the previous determination of distribution of net assimilated ‘Y in meadow fescue on shoots, roots, soii and rhizosphere respiration. It was found that, during 7 weeks from germination, about 10% of net fixed W was released as organic material from the roots. Microbiat respiration, from decomposition of root-derived organic material during growth, was estimated to be 32% of the total rhizosphere respiration.
INTRODUCTION Below-ground plant material is an important source of soil organic matter. Usually only dead roots are considered as an input but organic material released from roots to soil during plant growth, such as root
exudates and sloughed root cells, is also produced in substantial amounts (Barber and Martin, 1976; Helal and Sauerbeck, 1989; Liljeroth et al., 1990). A zone of high microbial activity is created in the vicinity of growing roots by this input of readily available organic material. This suggests that the root-derived material is an important source of C. The “C-labelling technique has enabled distribution of the assimilated C between roots, soil and evolved “C0, to be determined (Whipps, 1990). The root-derived material is partly mineralized to CO2 by microbial decomposition during plant growth and only the remainder is Ieft to be dete~ined in the soil. Evolved 14C02, i.e. rhizosphere respiration, consists of both root respiration and microbial respiration originating from the decomposition of root-derived material. Accordingly, the release of organic material from roots to the soil can be quantified if root and microbial respiration are determined separately from each other. Root respiration has been determined either from plant growth in axenic culture (Warembourg, 1975) 427
or after temporary inhibition of microbial activity in the rhizosphere by different anti-microbial substances (Minchin and McNaughton, 1984; Helal and Sauerbeck, 1991). Determinations on plants grown in soil, like Helal and Sauerbeck’s (1991), are preferred to dete~inations on solution-cultured plants. Microorganisms present in the rhizosphere have been shown to affect root exudation (Barber and Martin, 1976; Barber and Lynch, 1977; Prikryl and Vancura, 1980). Therefore, release of organic material from plant roots cannot be determined in the absence of an active microbial population in the rhizosphere. My objective was to determine the release of organic material from growing roots of a perennial grass during the first 7 weeks from germination. Microbial respiration during plant growth could then be separated from root respiration. To attain this, an incubation experiment was performed with soil containing root-derived material from a previous cultivation of grass in a r4C-labelling growth chamber. Theory
Plant assimilated C translocated below-ground is continuously released from growing roots as root respiration and root-derived material (RDM) in the form of root exudates, mucilage, sloughed-off cells and tissue. While new RDM is continuously released
428
G. JOHANSSON
.j, t
I / t
i...j,:’
t
RUMM
were measured on the same soil sample. C RDMS
=
GDMD
-
G,,,
iI)
The degree of stabilization of each reference material, i.e. amount of C left in the soil after incubation expressed as % of original C, is calculated from the difference between C added and C mineralized to CO, during incubation. Degree of stabilization (“/) _ (C,dd”~_ Y) added
x “:y
(2)
C RDMFis calculated from the amount of stabilized root-derived C in soil after the incubation (Cc,,,,) divided by an estimated degree of stabilization of the root-derived material.
C RDMS
C RDMF
=
Table I. Definmons of abbreviations used in calculations and Fig. Abbreviation RDM RDMF RDMD RDMS RDMM R-CD, M-CO, T-CO, 10 fl f2
Description
x
loo
degree of stabilization 51
i
-_.. Root-derived material Fresh root-derived material released during plant growth Partially decomposed root-derived material remaining in soil after plant growth Stabilized root-derived material remaining after incubation Root-derived material mineralized during incubation Root respiration Microbial respiration from decomposition of root-derived maier~al during plant growth Total rhizosphere respiration (T-CO, = R-CO, + M-CO,) Time of start of plant growth period Time of end of plant growth and start of incubation period Time of end of incubation period
(3)
Organic
C released
from growing
roots
429
Incubation experiment
v
I
0
$
1’5
lb
14C in acid-stable
i0 residues
is
i0
(%)
Fig. 2. ‘+‘C-labelled C in acid-stable compounds in soil after decomposition of shoots, roots and glucose (% of total “C-labelled C) in relation to total “‘C-1abelled C remaining in soil after decomposition (% of ‘V-labelled C in original fresh organic material). To determine the total W-labelled C remaining in soil after decomposition of RDM, a linear relationship is assumed.
As the degree of stabilization of root-derived material is not known, either the degree of stabilization of one of the reference materials or an estimate derived from the relationship in Fig. 2 is used. This relationship, between acid-stable C in the residues after decomposition and the stabilized substrate-C remaining after decomposition (degree of stabilization), is determined for the reference materials. Since the acid-stable C-fraction of the CRDMs is measured, the degree of stabilization for the root-derived material can be estimated from the assumed linear relationship. Microbial rhizosphere respiration during plant growth (C, -co*) is calculated as the difference between the calculated total fresh root-derived C released from roots to soil (C,,,,) and root-derived C measured in soil after plant growth (CRDMD).
After cultivation of meadow fescue in the 14C-labelling chamber the soil contains “C-labelled roots and stabilization and RDM (CRDMD).D ecomposition OfC RDMDwere determined after long-term (61 weeks) incubation of this soil. Root-free soil was prepared by hand-picking all root parts visible at x 10 magnification. Four treatments were included: soil containing “C-labelled RDMD from two different pots after cultivation (0.37-0.61 kBq gg’ soil), and soil amended with “C-labelled meadow fescue shoots or roots (4.6 kBq mg-’ C), or amended with “C-labelled glucose (1.9 kBq mg-’ C). Shoots, roots and glucose are referred to as reference materials. Four replicates were prepared for each treatment except for glucose where two replicates were used. The soil with “C-labelled RDM, stored at - 18°C after cultivation, was thawed and dried under the light of the magnifier during the hand-picking and then ground to pass 0.6 mm. Unlabelled soil, airdried and ground (co.6 mm), was used for the treatments with the reference materials. Dried and finely-ground shoots and roots from meadow fescue and glucose solution were thoroughly hand mixed, together with 50 pg N g-’ soil as KNO,, in 20 g dry soil for each replicate. The rate of addition was 2500 pg C g-r soil for plant material and 1250 pg C g -’ soil for glucose. All soil samples were wetted to 60% of water-holding capacity (20% w/w), transferred to 50 ml glass tubes and kept at 2&22”C in the dark for 61 weeks. CO,-free moistened air was continuously passed over the soil in the tubes during the first 7 weeks of incubation and afterwards intermittently for 30 min every 4 h. Evolved CO, was trapped in 16ml 0.1 M NaOH. Fractionation
CM- co2
=
GDMF- GDMD
MATERIALS AND METHODS
Growth chamber experiment The distribution of net assimilated C in meadow fescue (Festuca pratensis L.) 7 weeks after germination was determined after growth in a closed chamber with a continuously “C-labelled CO, atmosphere (5.5 kBq mg-’ C f 7%) (Johansson, 1991). The soil was a sandy silt loam, a Typic Haplaquoll [Soil Taxonomy, Soil Survey Staff (1975)] @born and Johnsson, 1990), with 21% clay, pH(H,O) 6.3, 2.2% C and 0.21% N (Steen et al., 1984). Growth chamber and environmental conditions are described in Johansson (1991). The plants were grown in sealed pots, with shoot and root-soil compartments separated, in order to collect CO* evolved from the soil. At the end of the growth period the distribution of net assimilated t4C between shoots, roots, soil and rhizosphere respiration was determined.
The acid-stable fraction of the stabilized 14Clabelled organic material in the soils after incubation was determined by a modified proximate analytical procedure (Persson, 1968). The soil was dried and pulverized. Pre-treatment with H,SO, of increasing strength, according to the analysis, was carried out in stages. The soil was then treated with 12.5 M H,SO, at room temperature for 3 h followed by dilution to a concentration of 1.75 M HISO and boiling by refluxing for 5 h. Before and after the treatment, soil samples were taken for determination of C and “C-1abelled C. Analyses C in plant material, soil and fractions after the fractionation procedure was determined by dry combustion (Striihlein C-Mat 500). “C-labelled C was determined by liquid scintillation counting (scintillation cocktail: Permafluor V) after combustion in a sample oxidizer (Packard Tri-Carb 306) where CO, was trapped in 5 ml Carbosorb.
430
G. JOHANSSON
Table 2. Evolved CO+ (~g g ’ soil) and substrate C remaining in soil (in 5’0of original C*) and SE (n = 4 for RDM, shoots and roots, n = 2 for glucose) after incubation for 61 weeks of root-derived material (RDM) and difflerent reference materials Organic materiai
Evolved CO,-C (fin a -I soil!
RDM
30 909 1380 1190
GlUCOSe
Shoots Roots
Distribution (X of net assimilaled)
Remaining C in soil f% of original C) 72 * 27 + 45+ 52 +
I .4 0.08 1.7 0.8
*wfig C g ’ soil, as RDM in soil after plant growth. 2500 pg C g -’ soil, added as meadow fescue shoots artd roots. 125Opg C g ’ soil,added as glucose.
Total C&-C in traps was determined by titration with HCl and phenoIphtalein as indicator after precipitation of carbonates with BaCl, (Stotzky, 1965). Liquid scintilfation counting with a T&on-X cocktail (PPO, djmethyl-COOP. toluol, T&on-X) was used for determination of “CO,-C.
RESULTS
After 7 weeks of growth in the “C-labelling chamber, dry weights of shoots and roots of the meadow fescue were 11.4 and 5.7 g pot-’ for pot I and 15.1 and 9.8 g pot-’ for pot II (Johansson, 1991). At this time, 4% (3.95% for pot I and 4.14% for pot II) of the total net assimilated 14Cwas found in the root-free soil as RDMD (83 and 130 big C g’ soil for pots I and II. respectively). After incubation of the root-free soil from the two different pots for 61 weeks, 72 & 1.4% of the CRnMa in soil at the start of the incubation remained (60 and 93 pg C gg ’ soil for pots I and II, respectively) (Table 2). After 61 weeks of incubation of the reference materials, 27% of the added 14C-tabclied glucose-c remained in the soil (Table 2). Corresponding remains of ‘JC-labelled grass shoots and roots were 45 and 52%, respectively. These figures are referred to as the degree of stabilization (equation 2). Microbial respiration from decomposition of 14CIabefled RDM during plant growth in the “C-labeliing chamber was calculated (equations 3 and 4) to range from 10 to 47% of total thizosphere respiration (Table 3). The lower value refers to roots as reference material and the higher value to glucose. The corresponding values for microbial respiration, expressed as a percentage of net assimilated C, were 2 and 7% respectively {Table 4). Table 3. Microbial respiration from decomposition of RDM (% of total rhizosphere respiration) in two pots during the growth of meadow fescue m “C-fabelIed atmosphere, calculated using the decree of stabilization for different reference materials $icrobiai respiratiosn (% of total rhizosphere respiration)
Table 4. Dist~bution of assjmiiat~ “C-~a~lled f: of 7 weeks old meadow fescue grown in a “CO*-atmosph~~~. Microbial respiration is calculated by using the degrees of stabilization presented in Table 3
Reference material
Degree of stabilization (% of original C)
glucose shoots roots
27 45 52
I Plant parts and pathways Shoots CroWIs Roots Soil Rhizosphere respiration Microbial respiration Root respiration
II 50 18 11
II
Degree of stablhzation (“A) 27 45 52 ?I 45 52 -._ 53 4X i 3 28 II 4 4 15 14 7 ? 2 6 2 2 9 13 13 7 II 12
Together with results from the cultivation in the “C-labelling chamber (Johansson, 1991) the calculated microbial respiration gives a more detailed picture of the distribution of net assimilated C for meadow fescue (Table 4). The distribution was obtained for plants 7 weeks after germination. The larger part of the rhizosphere respiration is derived from root respiration. The organic rhizodeposition, i.e. RDMD in soil plus microbial respiration from decomposition of RDM (M-CO,), amounted to 6-11% of net assimilated C. When fractionating the “C-labelled organic material in the soil after 61 weeks of incubation, the acid-stable residues for the RDM was found to be considerably lower than for shoots and roots but somewhat higher than for glucose (Table 5). By assuming a linear relationship for the reference materials between stabilized 14C-labelled C remaining in soil after decomposition (degree of stabilization) (Table 2) and i4C-labelled C as acid-stable compounds after decomposition (Table 5), a degree of stabilization for the RDM can be obtained (Fig. 2). The acid-stable residues of 15% of the total stabilized residues for the RDM (Table 5) then give a degree of stabilization of 34%. In terms of stabilization. the RDM can be placed between the glucose and the grass shoots. DISCUSSION
During plant growth in the “C-labelling chamber, RDM is continuously reteased and also continuously decomposed. Thus, at the start of the incubation the RDM is already partly decomposed, as distinguished from the fresh reference materials. Provided that the time of incubation is considerably longer than the 7-week growth period, a comparison between the Table 5. Acid-stable “C-Iabeiied f m stabilized materra~ remainmg after decomposition f% of total, +SE, n = 4 for shoots. roots and soil, n = 2 for glucose)
Replicate I 43 16 IO
--
Replicate
RDM Acid-stable 14C (% of total) SE
14.7 0.513
GlllCOSe
10.44 0.075
Shoots
Roots
23.4
25.0
0.331
0.179
Organic C released from growing roots stabilization of RDM and reference materials after incubation appears justified. The residue of the RDM and the reference materials are designated as stabilized organic material after 61 weeks of incubation. This is an approximation since the decomposition will go on, although at a very slow rate (Sauerbeck, 1977). Still, it is reasonable to compare the RDM and the reference materials at the end of the incubation, since the decomposition rate was very low at that time (max. 0.7 pg CO,-C g-’ soil day-’ at an addition of 2500 pg C g- ’ soil). The soil with RDM was frozen (stored at - 18°C) and thawed before the incubation but the soil with the reference materials was not. However, al1 soil samples were dried and rewetted before the start of the incubation experiment. Freezing may have affected carbon mineralization in the soil with RDM since freezing and thawing is known to stimulate soil respiration (Jenkinson and Powlson, 1976). But, according to Soulides and Allison (1961) and Skogland et al. (1988) the effect of air-drying on the decomposition of soil organic matter was substantially higher compared to the effect of freezing.
Using the estimated degree of stabilization for the RDM (Fig. 2), the microbial respiration is calculated to be 32% of the total rhizosp~ere respiration. Thus, root respiration constitutes 68% of total rhizosphere respiration, with a range, derived from the reference materials, of 53-90%. Helal and Sauerbeck (1991) measured root respiration in cultivations of beans and maize during a short inhibition of microbial activity in the rhizosphere by anti-microbial agents. They found a ratio of 4:1 for microbial to root respiration, the opposite relationship to that estimated in my investigation with meadow fescue. One explanation may be that different plants release different amounts and quality of organic material. From determinations of microbial utilization of rootreleased material in wheat and maize, Van Veen et af. (1989) suggest that organic material from maize roots is more readily decomposable than organic material from wheat roots. Also, plant age may affect the relationship between root and microbial respiration. It is likely that the composition of the RDM is changed towards more dead root tissues as the roots grow older. Differences in quality of the RDM at different plant ages will result in different degrees of stabilization of the RDM. A change in degree of stabilization will have a larger effect on the microbial and root respiration as percentage of total rhizosphere respiration (Table 3) than on the respirations as percentage of the net plant assimilated C {Table 4). According to my results, 0.3 g of C was respired for 1 g of C incorporated into root material, i.e. C in roots and in RDM (Johansson, 1991). Compared to theoretical calculations by Lambers (1987), this estimated root respiration appears to be reasonable.
431
Based on ATP-costs for biosynthesis he calculated that for every g of C incorporated into root dry matter 0.1 g of C is respired by the roots. This must be a rni~irn~m as respiration for maintenance energy and ion transport are not included. Each of them are larger, in relation to respiration for biosynthesis, according to Lambers. Minchin and McNaughton (1984) blocked the uptake of root exudates by microorganisms in a solution culture of wheat through the addition of anti-microbial agents. After pulse-ia~liin~ with “CO,, at least 30% of the total respiration was suggested to originate from microbial activity external to the root. However, pulse-ia~lling leads to labelling of only recently fixed C. Accordingly, root death and sloughed cell parts will not be included in the determination. Comparisons between determinations in nutrient solution and in soil cultures of release of C from roots are difficult to make, Less organic material is released from roots in solution cultures compared to the release in solid growth medium (Barber and Gunn, 1974). In 16 day old barley cultivated in nutrient solution, Farrar (1985) found, that 42% of the C transiocated to the roots was respired. This respiration, being high compared to our results, may also include respiration from rhizosphere microorganisms~
Different proportions of the original material were left as stabilized residue after decomposition of the three reference materials. This is due to differences in the composition of the materials, particularly their content of lignin. In plant material, the lignin component has a long half-life in soil (Tate, 1987). Other plant compounds, e.g. cellulose and hemicellulose, are decomposed at a higher rate than lignin (Sorensen, 1963). They will be transfo~ed into secondary decomposition products during prolonged incubation. Glucose C will rather rapidly be transformed to CO,, microbial tissue and metabolites. The quality of the stabilized residue at the end of the incubation is difficult to define and accordingly difficult to compare between the different organic materials. However, an indication of the quality of the RDM can be found in the fractionation of the soils after decomposition. The content of acid-stable C in the residues after decomposition of RDM is higher than after decomposition of glucose but lower than after grass shoots and roots (Table 5). The main part of these acid-stable residues is organic matter very resistant to microbial attack (Persson, 1968). After decomposition of plant material, a considerable part of this organic matter probably consists of lignin and modified lignin. The acid-stable fraction is also derived from secondary material formed during microbial decomposition. This is apparent from the treatment with decomposition of glucose where the soil contains acid-stable residues, although there was no iignin in the glucose. Using simple substrates like
432 glucose,
G. JOHANSSON microorganisms
can synthesize
compounds
that will give rise to highly resistant substances in soil (Wagner, 1968). Stabilization of RDM
A reasonable estimate of the amount of fresh RDM released during growth can be made using the results of acid-stable residues. The degree of stabilization for the RDM is derived by assuming a linear relationship, for the three reference materials, between the acid-stable fraction and the stabilized substrate-C remaining after decomposition (degree of stabilization) (Fig. 2). There is some uncertainty with this prediction since it is based only on three determined values. However, a linear relationship was also found by Persson (1968). Also, it seems reasonable to assume that a large part of the RDM consists of fairly easily decomposable compounds since the amount of C remaining in soil after growth is low compared to the total amount of C released from the roots (Helal and Sauerbeck, 1986). After pulse-labelling of ryegrass Meharg and Killham (1988) concluded that most of the root-released C consists of recently fixed C. This implies that the release of exudates and secretions (polymeric carbohydrates and enzymes) is more important than sloughing-off of root cells which consists of more hgnified material. Release of organic C fromlit@
roots
Considerable amounts of C, ca 40% of the roottranslocated C, were released from growing roots of the meadow fescue plants and found either in soil or as CO2 (rhizosphere respiration) evolved during growth (Johansson, 1991). This confirms results from other experiments with “C-labelling of young plants of wheat (Merckx et al., 1985) and maize (Helal and Sauerbeck, 1986). At this young age (7 weeks), the perennial meadow fescue has a C distribution similar to annual cereal plants. My results suggest that microbial respiration constitutes a minor part of rhizosphere respiration. From the estimated relation between root and microbial rhizosphere respiration, about 10% of net fixed C in meadow fescue after 7 weeks’ growth was released from the roots as organic material. Higher amounts of root-derived material are proposed from other investigations, using other relations between root and microbial respiration. The methods used to determine the relationship so far are quite different. The difficulty in measuring the two processes in an intact, undisturbed plant-soil system is clear. Acknowledgements-I
thank J. Persson for support, I. Jure-
malm for technical assistance and 0. Johansson, A. Martensson, E. Witter and E. Zagal for critically reading the manuscript. REFERENCES
Barber D. A. and Gunn K. B. (1974) The effect of mechanical forces on the exudation of organic substances by the
roots of cereal pIants grown under sterile conditions. New Phytoiogist 73, 39-45. Barber D. A. and Lynch J. M. (1977) Microbial growth in the rhizosphere. soil Biology & Biochemistry 9;305-308. Barber D. A. and Martin J. K. (1976) The release of oraanic substances by cereal roots into soil. New Phycalagi~ 76, 69-80.
Farrar J. F. (1985) Fluxes of carbon in roots of barley plants. New Phytologist 99, 5769. Helal H. M. and Sauerbeck D. R. (1986) Effects of plant roots on carbon metabolism of soil microbial biomass. Zeitschrift fir 181-188.
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und Bodenkunde 149,
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navica 41, 37-46.
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Lambers H. (1987) Growth, respiration, exudation and symbiotic associations: the fate of carbon translocated to the roots. In Root D~efopment and Funetion (P. J. Gregory, J. V. Lake and D. A. Rose, Eds), pp. 125-145. Cambridge University Press, Cambridge. Liljeroth E., Van Veen J. A. and Miller H. 1. (1990) Assimilate translocation to the rhizosphere of two wheat lines and subsequent utilization by rhizosphere microorganisms at two soil nitrogen concentrations. Soil Biology & Biochemistry 22, 1015-1021.
Meharg A. A. and Killham K. (1988) A comparison of carbon flow from pre-labelled and pulse-labelled plants. Plant and Soil 112, 225-231.
Merckx R., den Hartog A. and van Veen J. A. (1985) Turnover of root-derived material and related microbial biomass formation in soils of different texture. Soil Biology & 3iochemistry 17, 565-569.
Minchin P. E. H. and McNaughton G. S. (1984) Exudation of recently fixed carbon by non-sterile roots. Journal of Experimental Botany 35, 74-82.
dbom I. and Johnsson H. (1990) Mollic gleysol at Kjettslinge. (Soil description series No. 2.) The Swedish University of Agricultural Sciences, Department of Forest Soils and Division of Soil Science and Ecochemistrv. Uppsala. Persson J. (1968) Biological testing of chemical humus analvsis. Annals of the Agricultural College _ of” Sweden 34, 81-2-17. ” Prikryl Z. and Vancura V. (1980) Root exudates of plants. IV. Wheat root exudation as dependent on growth, concentration gradient of exudates and the presence of bacteria. Plant and Soil 57, 69-83. Sauerbeck D. R. (1977) The turnover of organic matter in soils as traced by radio carbon. Journal of Nuclear Agricultural Biology 6, 3341.
Skogland T., Lomeland S. and Goksoyr J. (1988) Respiratory burst after freezing and thawing of soil: experiments with soil bacteria. Soil Biology & Biochemistry 20, 851-856.
Soil Survey Staff (1975) Soil taxonomy. A basic system of soil classification for making and interpreting soil surveys, 754 pp. USDA Handbook No. 436. U.S. Government Printing Office, Washington, DC. Sorensen H. (1963) Studies on the decomposition of ‘Ylabelled barley straw in soil. Soil Science 95, 45-51.
Organic C released from growing roots Soulides D. A. and Allison F. E. (1961) Effect of drying and freezing soils on carbon dioxide production, available mineral nutrients, aggregation and bacterial population. Soil Science 91, 291-298. Steen E., Jansson P.-E. and Persson J. (1984) Experimental site of the “Ecology of Arable Land” project. Acta Agriculturae Scandinavica 34, 153-166. Stotzky G. (1965) Microbial respiration. In Methods of Soil Analysis. Part 2. Chemical and Microbiological Properties
(C. A. Black, F. E. Clark, L. E. Ensminger, D. D. Evans and J. L. White, Eds), pp. 155&1572. American Society of Agronomy, Madison. Tate R. L. III (1987) Soil Organic Matfer. Wiley, New York.
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Van Veen J. A., Merckx R. and van de Geijn S. C. (1989) Plant- and soil-related controls of the flow of carbon from roots through the soil microbial biomass. In Ecology of Arable Land (M. Clarholm and L. Bergstrdm, Eds), pp. 43-52. Kluwer Academic, Dordrecht. Wagner G. H. (1968) Significance of microbial tissues to soil organic matter. In Isotopes and Radiation in Soil Organic Matter-Studies, pp. 197-205. IAEA, Vienna. Warembourg F. R. (1975) Application de techniques radioisotopiques a l’etude de l’activite biologique dans la rhizosphere des plantes. Revue D’Ecologie et de Biologie du Sol 12, 261-272. Whipps J. M. (1990) Carbon economy. In The Rhizosphere (J. M. Lynch, Ed.), pp. 59-97. Wiley, Chichester.
Soil Bid Biochem. Vol. 24, No. 5, pp. 421-433, 1992 Printed in Great Britain. All rights reserved
Copyright C 1992 Pergamon Press Ltd
RELEASE OF ORGANIC C FROM GROWING ROOTS OF MEADOW FESCUE (loESTUCA PRATENSIS L.) G. JOHANSSON Department
of Soil Sciences, Division of Plant Nutrition and Soil Fertility, Swedish University of Agricultural Sciences, P.O. Box 7014, S-750 07 Uppsala, Sweden [Accepted 30 November 1991)
Summery-A new method to estimate the amount of organic C released from roots during plant growth is described. The method is based on decomposing root-derived material left in the soil after plant growth and removal of all visible roots in a long-term incubation. The total amount of organic C released during plant growth is then calculated from the amount of root-derived material remaining in the soil after long-term decomposition and a stabilization factor for this material. An estimate of the total amount of organic C released from roots during plant growth allows the total rhizosphere respiration to be separated into root respiration and microbial respiration. In a previous cultivation of continuously “‘C-1abelled meadow fescue (Festuca pratensis L.) the distribution of net assimilated 14Cwas determined. Root-derived material in root-free soil from the cultivation was allowed to decompose in a long-term incubation (61 weeks). The amount of stabilized root-derived material left in the soil after the incubation, representing a certain proportion of the fresh root-derived material released during growth, was calculated. The stabilization of root-derived material was approximated through comparison with stabilization of known organic materials (glucose, grass shoots and roots). Decomposition of root-derived material resulted in more acid-stable residues than decomposition of glucose but less than shoots and roots. From the relationship, for the known organic materials, between the amount of stabilized organic material remaining after decomposition and the amount of acid-stable residue, the amount of original root-derived material was estimated. This result was combined with data from the previous determination of distribution of net assimilated ‘Y in meadow fescue on shoots, roots, soii and rhizosphere respiration. It was found that, during 7 weeks from germination, about 10% of net fixed W was released as organic material from the roots. Microbiat respiration, from decomposition of root-derived organic material during growth, was estimated to be 32% of the total rhizosphere respiration.
INTRODUCTION Below-ground plant material is an important source of soil organic matter. Usually only dead roots are considered as an input but organic material released from roots to soil during plant growth, such as root
exudates and sloughed root cells, is also produced in substantial amounts (Barber and Martin, 1976; Helal and Sauerbeck, 1989; Liljeroth et al., 1990). A zone of high microbial activity is created in the vicinity of growing roots by this input of readily available organic material. This suggests that the root-derived material is an important source of C. The “C-labelling technique has enabled distribution of the assimilated C between roots, soil and evolved “C0, to be determined (Whipps, 1990). The root-derived material is partly mineralized to CO2 by microbial decomposition during plant growth and only the remainder is Ieft to be dete~ined in the soil. Evolved 14C02, i.e. rhizosphere respiration, consists of both root respiration and microbial respiration originating from the decomposition of root-derived material. Accordingly, the release of organic material from roots to the soil can be quantified if root and microbial respiration are determined separately from each other. Root respiration has been determined either from plant growth in axenic culture (Warembourg, 1975) 427
or after temporary inhibition of microbial activity in the rhizosphere by different anti-microbial substances (Minchin and McNaughton, 1984; Helal and Sauerbeck, 1991). Determinations on plants grown in soil, like Helal and Sauerbeck’s (1991), are preferred to dete~inations on solution-cultured plants. Microorganisms present in the rhizosphere have been shown to affect root exudation (Barber and Martin, 1976; Barber and Lynch, 1977; Prikryl and Vancura, 1980). Therefore, release of organic material from plant roots cannot be determined in the absence of an active microbial population in the rhizosphere. My objective was to determine the release of organic material from growing roots of a perennial grass during the first 7 weeks from germination. Microbial respiration during plant growth could then be separated from root respiration. To attain this, an incubation experiment was performed with soil containing root-derived material from a previous cultivation of grass in a r4C-labelling growth chamber. Theory
Plant assimilated C translocated below-ground is continuously released from growing roots as root respiration and root-derived material (RDM) in the form of root exudates, mucilage, sloughed-off cells and tissue. While new RDM is continuously released
428
G. JOHANSSON
.j, t
I / t
i...j,:’
t
RUMM
were measured on the same soil sample. C RDMS
=
GDMD
-
G,,,
iI)
The degree of stabilization of each reference material, i.e. amount of C left in the soil after incubation expressed as % of original C, is calculated from the difference between C added and C mineralized to CO, during incubation. Degree of stabilization (“/) _ (C,dd”~_ Y) added
x “:y
(2)
C RDMFis calculated from the amount of stabilized root-derived C in soil after the incubation (Cc,,,,) divided by an estimated degree of stabilization of the root-derived material.
C RDMS
C RDMF
=
Table I. Definmons of abbreviations used in calculations and Fig. Abbreviation RDM RDMF RDMD RDMS RDMM R-CD, M-CO, T-CO, 10 fl f2
Description
x
loo
degree of stabilization 51
i
-_.. Root-derived material Fresh root-derived material released during plant growth Partially decomposed root-derived material remaining in soil after plant growth Stabilized root-derived material remaining after incubation Root-derived material mineralized during incubation Root respiration Microbial respiration from decomposition of root-derived maier~al during plant growth Total rhizosphere respiration (T-CO, = R-CO, + M-CO,) Time of start of plant growth period Time of end of plant growth and start of incubation period Time of end of incubation period
(3)
Organic
C released
from growing
roots
429
Incubation experiment
v
I
0
$
1’5
lb
14C in acid-stable
i0 residues
is
i0
(%)
Fig. 2. ‘+‘C-labelled C in acid-stable compounds in soil after decomposition of shoots, roots and glucose (% of total “C-labelled C) in relation to total “‘C-1abelled C remaining in soil after decomposition (% of ‘V-labelled C in original fresh organic material). To determine the total W-labelled C remaining in soil after decomposition of RDM, a linear relationship is assumed.
As the degree of stabilization of root-derived material is not known, either the degree of stabilization of one of the reference materials or an estimate derived from the relationship in Fig. 2 is used. This relationship, between acid-stable C in the residues after decomposition and the stabilized substrate-C remaining after decomposition (degree of stabilization), is determined for the reference materials. Since the acid-stable C-fraction of the CRDMs is measured, the degree of stabilization for the root-derived material can be estimated from the assumed linear relationship. Microbial rhizosphere respiration during plant growth (C, -co*) is calculated as the difference between the calculated total fresh root-derived C released from roots to soil (C,,,,) and root-derived C measured in soil after plant growth (CRDMD).
After cultivation of meadow fescue in the 14C-labelling chamber the soil contains “C-labelled roots and stabilization and RDM (CRDMD).D ecomposition OfC RDMDwere determined after long-term (61 weeks) incubation of this soil. Root-free soil was prepared by hand-picking all root parts visible at x 10 magnification. Four treatments were included: soil containing “C-labelled RDMD from two different pots after cultivation (0.37-0.61 kBq gg’ soil), and soil amended with “C-labelled meadow fescue shoots or roots (4.6 kBq mg-’ C), or amended with “C-labelled glucose (1.9 kBq mg-’ C). Shoots, roots and glucose are referred to as reference materials. Four replicates were prepared for each treatment except for glucose where two replicates were used. The soil with “C-labelled RDM, stored at - 18°C after cultivation, was thawed and dried under the light of the magnifier during the hand-picking and then ground to pass 0.6 mm. Unlabelled soil, airdried and ground (co.6 mm), was used for the treatments with the reference materials. Dried and finely-ground shoots and roots from meadow fescue and glucose solution were thoroughly hand mixed, together with 50 pg N g-’ soil as KNO,, in 20 g dry soil for each replicate. The rate of addition was 2500 pg C g-r soil for plant material and 1250 pg C g -’ soil for glucose. All soil samples were wetted to 60% of water-holding capacity (20% w/w), transferred to 50 ml glass tubes and kept at 2&22”C in the dark for 61 weeks. CO,-free moistened air was continuously passed over the soil in the tubes during the first 7 weeks of incubation and afterwards intermittently for 30 min every 4 h. Evolved CO, was trapped in 16ml 0.1 M NaOH. Fractionation
CM- co2
=
GDMF- GDMD
MATERIALS AND METHODS
Growth chamber experiment The distribution of net assimilated C in meadow fescue (Festuca pratensis L.) 7 weeks after germination was determined after growth in a closed chamber with a continuously “C-labelled CO, atmosphere (5.5 kBq mg-’ C f 7%) (Johansson, 1991). The soil was a sandy silt loam, a Typic Haplaquoll [Soil Taxonomy, Soil Survey Staff (1975)] @born and Johnsson, 1990), with 21% clay, pH(H,O) 6.3, 2.2% C and 0.21% N (Steen et al., 1984). Growth chamber and environmental conditions are described in Johansson (1991). The plants were grown in sealed pots, with shoot and root-soil compartments separated, in order to collect CO* evolved from the soil. At the end of the growth period the distribution of net assimilated t4C between shoots, roots, soil and rhizosphere respiration was determined.
The acid-stable fraction of the stabilized 14Clabelled organic material in the soils after incubation was determined by a modified proximate analytical procedure (Persson, 1968). The soil was dried and pulverized. Pre-treatment with H,SO, of increasing strength, according to the analysis, was carried out in stages. The soil was then treated with 12.5 M H,SO, at room temperature for 3 h followed by dilution to a concentration of 1.75 M HISO and boiling by refluxing for 5 h. Before and after the treatment, soil samples were taken for determination of C and “C-1abelled C. Analyses C in plant material, soil and fractions after the fractionation procedure was determined by dry combustion (Striihlein C-Mat 500). “C-labelled C was determined by liquid scintillation counting (scintillation cocktail: Permafluor V) after combustion in a sample oxidizer (Packard Tri-Carb 306) where CO, was trapped in 5 ml Carbosorb.
430
G. JOHANSSON
Table 2. Evolved CO+ (~g g ’ soil) and substrate C remaining in soil (in 5’0of original C*) and SE (n = 4 for RDM, shoots and roots, n = 2 for glucose) after incubation for 61 weeks of root-derived material (RDM) and difflerent reference materials Organic materiai
Evolved CO,-C (fin a -I soil!
RDM
30 909 1380 1190
GlUCOSe
Shoots Roots
Distribution (X of net assimilaled)
Remaining C in soil f% of original C) 72 * 27 + 45+ 52 +
I .4 0.08 1.7 0.8
*wfig C g ’ soil, as RDM in soil after plant growth. 2500 pg C g -’ soil, added as meadow fescue shoots artd roots. 125Opg C g ’ soil,added as glucose.
Total C&-C in traps was determined by titration with HCl and phenoIphtalein as indicator after precipitation of carbonates with BaCl, (Stotzky, 1965). Liquid scintilfation counting with a T&on-X cocktail (PPO, djmethyl-COOP. toluol, T&on-X) was used for determination of “CO,-C.
RESULTS
After 7 weeks of growth in the “C-labelling chamber, dry weights of shoots and roots of the meadow fescue were 11.4 and 5.7 g pot-’ for pot I and 15.1 and 9.8 g pot-’ for pot II (Johansson, 1991). At this time, 4% (3.95% for pot I and 4.14% for pot II) of the total net assimilated 14Cwas found in the root-free soil as RDMD (83 and 130 big C g’ soil for pots I and II. respectively). After incubation of the root-free soil from the two different pots for 61 weeks, 72 & 1.4% of the CRnMa in soil at the start of the incubation remained (60 and 93 pg C gg ’ soil for pots I and II, respectively) (Table 2). After 61 weeks of incubation of the reference materials, 27% of the added 14C-tabclied glucose-c remained in the soil (Table 2). Corresponding remains of ‘JC-labelled grass shoots and roots were 45 and 52%, respectively. These figures are referred to as the degree of stabilization (equation 2). Microbial respiration from decomposition of 14CIabefled RDM during plant growth in the “C-labeliing chamber was calculated (equations 3 and 4) to range from 10 to 47% of total thizosphere respiration (Table 3). The lower value refers to roots as reference material and the higher value to glucose. The corresponding values for microbial respiration, expressed as a percentage of net assimilated C, were 2 and 7% respectively {Table 4). Table 3. Microbial respiration from decomposition of RDM (% of total rhizosphere respiration) in two pots during the growth of meadow fescue m “C-fabelIed atmosphere, calculated using the decree of stabilization for different reference materials $icrobiai respiratiosn (% of total rhizosphere respiration)
Table 4. Dist~bution of assjmiiat~ “C-~a~lled f: of 7 weeks old meadow fescue grown in a “CO*-atmosph~~~. Microbial respiration is calculated by using the degrees of stabilization presented in Table 3
Reference material
Degree of stabilization (% of original C)
glucose shoots roots
27 45 52
I Plant parts and pathways Shoots CroWIs Roots Soil Rhizosphere respiration Microbial respiration Root respiration
II 50 18 11
II
Degree of stablhzation (“A) 27 45 52 ?I 45 52 -._ 53 4X i 3 28 II 4 4 15 14 7 ? 2 6 2 2 9 13 13 7 II 12
Together with results from the cultivation in the “C-labelling chamber (Johansson, 1991) the calculated microbial respiration gives a more detailed picture of the distribution of net assimilated C for meadow fescue (Table 4). The distribution was obtained for plants 7 weeks after germination. The larger part of the rhizosphere respiration is derived from root respiration. The organic rhizodeposition, i.e. RDMD in soil plus microbial respiration from decomposition of RDM (M-CO,), amounted to 6-11% of net assimilated C. When fractionating the “C-labelled organic material in the soil after 61 weeks of incubation, the acid-stable residues for the RDM was found to be considerably lower than for shoots and roots but somewhat higher than for glucose (Table 5). By assuming a linear relationship for the reference materials between stabilized 14C-labelled C remaining in soil after decomposition (degree of stabilization) (Table 2) and i4C-labelled C as acid-stable compounds after decomposition (Table 5), a degree of stabilization for the RDM can be obtained (Fig. 2). The acid-stable residues of 15% of the total stabilized residues for the RDM (Table 5) then give a degree of stabilization of 34%. In terms of stabilization. the RDM can be placed between the glucose and the grass shoots. DISCUSSION
During plant growth in the “C-labelling chamber, RDM is continuously reteased and also continuously decomposed. Thus, at the start of the incubation the RDM is already partly decomposed, as distinguished from the fresh reference materials. Provided that the time of incubation is considerably longer than the 7-week growth period, a comparison between the Table 5. Acid-stable “C-Iabeiied f m stabilized materra~ remainmg after decomposition f% of total, +SE, n = 4 for shoots. roots and soil, n = 2 for glucose)
Replicate I 43 16 IO
--
Replicate
RDM Acid-stable 14C (% of total) SE
14.7 0.513
GlllCOSe
10.44 0.075
Shoots
Roots
23.4
25.0
0.331
0.179
Organic C released from growing roots stabilization of RDM and reference materials after incubation appears justified. The residue of the RDM and the reference materials are designated as stabilized organic material after 61 weeks of incubation. This is an approximation since the decomposition will go on, although at a very slow rate (Sauerbeck, 1977). Still, it is reasonable to compare the RDM and the reference materials at the end of the incubation, since the decomposition rate was very low at that time (max. 0.7 pg CO,-C g-’ soil day-’ at an addition of 2500 pg C g- ’ soil). The soil with RDM was frozen (stored at - 18°C) and thawed before the incubation but the soil with the reference materials was not. However, al1 soil samples were dried and rewetted before the start of the incubation experiment. Freezing may have affected carbon mineralization in the soil with RDM since freezing and thawing is known to stimulate soil respiration (Jenkinson and Powlson, 1976). But, according to Soulides and Allison (1961) and Skogland et al. (1988) the effect of air-drying on the decomposition of soil organic matter was substantially higher compared to the effect of freezing.
Using the estimated degree of stabilization for the RDM (Fig. 2), the microbial respiration is calculated to be 32% of the total rhizosp~ere respiration. Thus, root respiration constitutes 68% of total rhizosphere respiration, with a range, derived from the reference materials, of 53-90%. Helal and Sauerbeck (1991) measured root respiration in cultivations of beans and maize during a short inhibition of microbial activity in the rhizosphere by anti-microbial agents. They found a ratio of 4:1 for microbial to root respiration, the opposite relationship to that estimated in my investigation with meadow fescue. One explanation may be that different plants release different amounts and quality of organic material. From determinations of microbial utilization of rootreleased material in wheat and maize, Van Veen et af. (1989) suggest that organic material from maize roots is more readily decomposable than organic material from wheat roots. Also, plant age may affect the relationship between root and microbial respiration. It is likely that the composition of the RDM is changed towards more dead root tissues as the roots grow older. Differences in quality of the RDM at different plant ages will result in different degrees of stabilization of the RDM. A change in degree of stabilization will have a larger effect on the microbial and root respiration as percentage of total rhizosphere respiration (Table 3) than on the respirations as percentage of the net plant assimilated C {Table 4). According to my results, 0.3 g of C was respired for 1 g of C incorporated into root material, i.e. C in roots and in RDM (Johansson, 1991). Compared to theoretical calculations by Lambers (1987), this estimated root respiration appears to be reasonable.
431
Based on ATP-costs for biosynthesis he calculated that for every g of C incorporated into root dry matter 0.1 g of C is respired by the roots. This must be a rni~irn~m as respiration for maintenance energy and ion transport are not included. Each of them are larger, in relation to respiration for biosynthesis, according to Lambers. Minchin and McNaughton (1984) blocked the uptake of root exudates by microorganisms in a solution culture of wheat through the addition of anti-microbial agents. After pulse-ia~liin~ with “CO,, at least 30% of the total respiration was suggested to originate from microbial activity external to the root. However, pulse-ia~lling leads to labelling of only recently fixed C. Accordingly, root death and sloughed cell parts will not be included in the determination. Comparisons between determinations in nutrient solution and in soil cultures of release of C from roots are difficult to make, Less organic material is released from roots in solution cultures compared to the release in solid growth medium (Barber and Gunn, 1974). In 16 day old barley cultivated in nutrient solution, Farrar (1985) found, that 42% of the C transiocated to the roots was respired. This respiration, being high compared to our results, may also include respiration from rhizosphere microorganisms~
Different proportions of the original material were left as stabilized residue after decomposition of the three reference materials. This is due to differences in the composition of the materials, particularly their content of lignin. In plant material, the lignin component has a long half-life in soil (Tate, 1987). Other plant compounds, e.g. cellulose and hemicellulose, are decomposed at a higher rate than lignin (Sorensen, 1963). They will be transfo~ed into secondary decomposition products during prolonged incubation. Glucose C will rather rapidly be transformed to CO,, microbial tissue and metabolites. The quality of the stabilized residue at the end of the incubation is difficult to define and accordingly difficult to compare between the different organic materials. However, an indication of the quality of the RDM can be found in the fractionation of the soils after decomposition. The content of acid-stable C in the residues after decomposition of RDM is higher than after decomposition of glucose but lower than after grass shoots and roots (Table 5). The main part of these acid-stable residues is organic matter very resistant to microbial attack (Persson, 1968). After decomposition of plant material, a considerable part of this organic matter probably consists of lignin and modified lignin. The acid-stable fraction is also derived from secondary material formed during microbial decomposition. This is apparent from the treatment with decomposition of glucose where the soil contains acid-stable residues, although there was no iignin in the glucose. Using simple substrates like
432 glucose,
G. JOHANSSON microorganisms
can synthesize
compounds
that will give rise to highly resistant substances in soil (Wagner, 1968). Stabilization of RDM
A reasonable estimate of the amount of fresh RDM released during growth can be made using the results of acid-stable residues. The degree of stabilization for the RDM is derived by assuming a linear relationship, for the three reference materials, between the acid-stable fraction and the stabilized substrate-C remaining after decomposition (degree of stabilization) (Fig. 2). There is some uncertainty with this prediction since it is based only on three determined values. However, a linear relationship was also found by Persson (1968). Also, it seems reasonable to assume that a large part of the RDM consists of fairly easily decomposable compounds since the amount of C remaining in soil after growth is low compared to the total amount of C released from the roots (Helal and Sauerbeck, 1986). After pulse-labelling of ryegrass Meharg and Killham (1988) concluded that most of the root-released C consists of recently fixed C. This implies that the release of exudates and secretions (polymeric carbohydrates and enzymes) is more important than sloughing-off of root cells which consists of more hgnified material. Release of organic C fromlit@
roots
Considerable amounts of C, ca 40% of the roottranslocated C, were released from growing roots of the meadow fescue plants and found either in soil or as CO2 (rhizosphere respiration) evolved during growth (Johansson, 1991). This confirms results from other experiments with “C-labelling of young plants of wheat (Merckx et al., 1985) and maize (Helal and Sauerbeck, 1986). At this young age (7 weeks), the perennial meadow fescue has a C distribution similar to annual cereal plants. My results suggest that microbial respiration constitutes a minor part of rhizosphere respiration. From the estimated relation between root and microbial rhizosphere respiration, about 10% of net fixed C in meadow fescue after 7 weeks’ growth was released from the roots as organic material. Higher amounts of root-derived material are proposed from other investigations, using other relations between root and microbial respiration. The methods used to determine the relationship so far are quite different. The difficulty in measuring the two processes in an intact, undisturbed plant-soil system is clear. Acknowledgements-I
thank J. Persson for support, I. Jure-
malm for technical assistance and 0. Johansson, A. Martensson, E. Witter and E. Zagal for critically reading the manuscript. REFERENCES
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