Macromolecular organic phosphorus in decomposing plants and in pasture soils

0038-0717:82/020145-07103.0(3/0 Copyright 0 l9R2 Pergamon Press Ltd

Soil Biol. Biwhem. Vol. 14. pp. 145 to 151. 1982 Printed in Great Britain. All rights reserved

MACROMOLECULAR ORGANIC PHOSPHORUS IN DECOMPOSING PLANTS AND IN PASTURE SOILS 0. L. JONES and S. M. BROMF~ELD CSIRO Division of Plant industry, Canberra, A.C.T., 2601, Australia (AccepfeJ 10 Augusf 1981) Summary-The acid-insoluble organic P in hayed-off phalaris was extracted at pH 7.0 using a detergent, and a macromolecular fraction (G50 phosphorus) was separated by gel filtration. Approximate amounts of nucleic acid P in this fraction were calculated from the purine base content of the fraction. Changes in the above organic P fractions were then monitored during plant decomposition. Acid-insoluble P increased as a result of decomposition whereas changes in GSO P and nucleic acid P were less pronounced. The neutral detergent extracted only a portion of the soil organic P from unimproved and improved pasture soils. The higher total organic P content of the improved soil was reflected in the GSO P content of the extract but not in its nucleic acid content. Microbial assimilation of the GSO P isolated from undecomposed and decomposed phalaris was rapid and virtuatly complete, and so most of the organic P in this fraction could not be expected to accumulate in soil. The assimilation of that portion of the G50 P which was eluted off ion exchange cellulose was much slower for soil samples than plant samples. The unassimilated portion from the soil samples was found to be free of nucleic acid bases and warrants further chemicai examination.

INTRODUCTION P accumulates under fertilized pasture in amounts that may be equivalent to about one third of the P applied during pasture improvement (Donald and Williams, 1954). The origin and nature of much of this organic P is still obscure (Cosgrove, 1966). It appears that its accumulation is not directly related to the amount of phosphate applied to the pasture (Simpson er al., l974a), nor is it due to extensive conversion of fertilizer phosphate to organic forms during the decomposition of the tops of hayed-off pasture residues (Jones and Bromfield. 1969). These residues commonly form on improved pastures in southern Australia at the beginning of summer when soil moisture is low and hot dry winds kill the almost senescent plants by desiccation. It is possible that the organic P in freshly hayed-off pasture plants, or that subsequently formed during plant decomposition, may make a a significant direct contribution to the pool of organic P in pasture soils. This view is suggested because the amount of organic P in pasture plant residues remained relatively constant over a 6-month period of d~om~sition and leaching. The observed changes ranged from a loss of 300,; to a gain of 11% depending on the plant sample (Jones and Bromfield, 1969). The organic P in both fresh and decomposed samples of hayed-off pasture consisted of a substantial water-soluble fraction, a relatively small acid-soluble fraction and another substantial fraction that was insoluble in both water and dilute acid but soluble in alkali. It was considered likely that the acid-insoluble organic P contained more complex organic P compounds than the soluble fractions and that further examination of its was relevant to the general problem of the nature and origin of soil organic P. In this study the acid-insolubIe organic P was extracted from hayed-off plant samples without resorting to strongly alkaline solutions: a neutral Organic

extraction, based on that used by Van Soest and Wine (1967) to remove nitrogenous constituents from fodder samples, proved satisfactory. The extract was then fractionated by gel filtration and the nucleic acid bases, guanine and adenine, were determined on the macromolecular P fraction. The procedures developed for undecomposed plant material were then used to monitor quantitative and qualitative changes in the composition of the acid-insoluble P during and after microbial decomposition of the plant samples. They were also used to compare the organic P in soil samples taken at the start and after 6 yr of an experiment involving pasture improvement (Simpson et ul., 1974b) in which there had been an accumulation of organic P (Simpson er al., 1974a). Finally. various macromolecular fractions isolated from plant and soil samples were incubated with a soil inoculum to determine how readily the organic P was assimilated by microrganisms and to examine that portion which remained unassimilated.

145

MATERIALS AND METHODS Plant and soil samples

The plant material used in this study was hayed-off Phalaris aquatica. The plants were grown in the field and protected from leaching during the haying-off stage by a large plastic sheeting cover. Only the tops were used for decomposition studies, either cut up into approximately 5-8 cm pieces. or ground in a Wiley mill (<2 mm). Two sets of soil samples were selected from the soil fertility experiment mentioned previously (Simpson et al., 1974b), one from the initial soil sampling and the other after 6yr of the fertilizer and grazing treatments. The 7.5 cm deep cores were lightly ground in a mortar and pestle. and then sieved through an 0.32 mm sieve.

146

0. L. JONES and S. M. BROMFIELD

Extraction of organic P with neutrul sodium lauryl sulphate. ethylenediaminetetruacetic acid solution (SLSI EDTA)

This aqueous extractant contained SLS (37;). EDTA (1.9”~;) and was buffered at pH 7 with maleic acid (0.581,) and solid NaOH instead of the recommended phosphate buffer. It was used to extract the acid-insoluble P from plant samples and some of the organic P in soils. In the case of plants, the acid- and water-soluble P was first removed by shaking ground samples (co.64 mm) with 0.2 N H#O* (extractant ratio l/50) for 2 h at 20°C and then filtering (Whatman No. 54) and washing the residue with distilled water (50°C) until free of acid. The residue containing the acidinsoluble P was dried at 40°C. Samples of the residue in amounts varying from 0.1 to l.Og were extracted with 50ml SLS/EDTA at 1OO’C for 4 h in a 500 ml Kjeldahl flask loosely sealed to prevent evaporation. The extract was centrifuged (12,OCOg for 10 min) at 4°C to remove the bulk of the SLS which was precipitated at this temperature. At least 95”a of the acid-insoluble P was extracted from the residues by this procedure (Table I). Soils were not washed with acid before extraction and the extraction time was 6 h unless stated otherwise. Fractionation

of the organic

P in SLSIEDTA

extracts

(a) Geljltration. The first step was to separate and discard the lower molecular weight P compounds in the SLS/EDTA extract using gel filtration through Sephadex G50. The extracts were eluted through a column (70 x 4.5 cm) with distilled water and 25 ml fractions were collected. Total P in each fraction was determined and the excluded fractions that contained P were bulked for subsequent studies. The P excluded by Sephadex G50 will be referred to as “G50 P” in the remainder of this paper. (b) Ion-exchange cellulose chromatography. Samples of G50 P from various sources were fractionated at 4’C on a column of Whatman DE32 microgranular ion-exchange cellulose (12 x 2.5 cm) using a linear gradient of t&129; NH,HCO,. Twenty 10ml fractions were collected and total P determined on each. The fractions containing P were desalted on a G50 column and their U.V. absorption spectra measured. Fractions with similar spectra were bulked for further studies. Separation. identification and estimation bases in the organic P fractions

of nucleic acid

The bulked organic P fractions from the above fractionations were evaporated to dryness at 4O’C using a rotary film evaporator. They were then hydrolysed for 2 h at 100°C with concentrated HC104 (2 ml). The hydrolysate was diluted to approximately 10 ml with distilled water and centrifuged at 600 g for IO min. The residue was washed twice with 10 ml 2 N HCI at 5O’C, and the combined supernatants evaporated at 5O’C to the residual HC104 which was then diluted to 20 ml with water. This was allowed to stand for 34 days and then re-centrifuged if any further precipitate had formed.

The bases in the hydrolysates were separated by ion-exchange chromatography on a Dowex 50 column (12 x 1 cm. H + Form, lo&200 mesh). using a fractionation technique based on that of Cohn (1949). The resin column was washed first with 2 N HCI at a flow rate of 50 ml h- ’ for 5 days to reduce the level of U.V. absorbing contaminants. Standard base mixtures were then applied and eluted with N HCI (30 x 10 ml fractions) to remove the pyrimidines and guanine followed by 2 N HCI (35 x 10ml fraction) to remove adenine. The distribution of guanine and adenine in the fractions was determined by U.V. measurements at 248 and 260n~ respectively. The hydrolysate was then fractionated similarly on the standardized column, and the appropriate fractions containng the pyrimidines. guanine, and adenine bulked separately. The three fractions were then chromatographed on paper to quantitatively estimate the purine bases. guanine and adenine, and to identify the pyrmidine bases. For paper chromatography. the bulked adenine or guanine fractions from Dowex 50 were evaporated in stages to dryness at 40’C. It was found difficult to re-dissolve the small amounts of base present, particularly guanine, during the evaporation stages. To ensure complete recovery of base a few drops of lo,,, Triton X-100 were added to the evaporating flask. and only the final evaporation was taken completely to dryness. Also. when transferring solutions from larger to smaller flasks during evaporation the larger flask was heated at 7O’C to facilitate dissolution of any dessicated materials, and the flask was rinsed several times with 0.1 N HCI at 7O’C to ensure quantitative transfer of bases. The base in the dried sample was finally dissolved by dislodging all the material adhering to the sides of the evaporating flask into 3 drops of N HCI. This solution was then applied quantitatively to Whatman 3 MM chromatography paper. The samples containing adenine or guanine were purified by 2-way chromatography employing solvents described by Thomson (1960). The samples were chromatographed first by descending chromaovernight in propan-1-ol/H,O/HCl tography (130/37/33) at R.T. They were then air-dried overnight. followed by 24 h drying over moistended NaOH pellets under vacuum in a dessicator. Ascending chromatography in Hz0 at R.T. was then carried out, primarily to remove any amino acids present and contaminants in A.R. HCI which moved with the water front. For quantitative measurements the purine bases were located under U.V. light and were cut out together with an adjoining control region of equal dimensions. The bases were then eluted quantitatively from the paper with 0.1 N HCI and made up to a known volume. The control paper was treated similarly. The U.V. absorption spectra of the bases was obtained from the difference between test and control values and estimates ‘of guanine and adenine were obtained from standard curves. Since guanine and adenine generally account for 4&60% of the total base content of nucleic acids (Chargaff and Davidson, 1955) an approximate value for nucleic acid P in various preparations was obtained by doubling the P equivalent to the content of these two bases in the preparations. The-pyrimidine-containing fractions from Dowex 50 were evaporated at SY’C to the residual HCIOI.

Macromolecular

Table 1. Changes in acid-insoluble organic P and GSO P in phalaris throughout

Wt of residue (“Aof undecomposed plant material)

Treatment Undecomposed

147

phosphorus in plants and soils 16 weeks decomposition

GSO Acid-insoluble organic P organic P Gu Ad (pgP/g)* (pgP/g)* (pm/g)* (pm/g)* Gu/Ad

Approx. nucleic acid (pgP/g)*

Total P in SLS/EDTAextracted residue (pgP/g)*

100

287

183

0.94

0.88

1.07

113

2-Weeks decomposition

82

272

165

0.74

0.51

1.45

78

16

3

4-Weeks decomposition 8-Weeks decomposition 16-Weeks decomposition

78 64 51

277 431 437

156 246 173

0.66 1.01 0.67

0.50 0.73 0.47

1.32 1.38 I .42

72 108 71

5 23 22

Total P in treatments, 1470 pg/g. * Values are expressed on the basis of amounts (pg P or pm) recovered from 1 g of original undecomposed material.

which was neutralized with KOH and removed as sparingly soluble KC104 by centrifugation at 4°C (1OOOg for 10min). The pyrimidine bases, cytosine, uracil, and thymine, in the supernatant were identified on the basis of their co-chromatography with standard bases on paper chromatographs, using a range of solvents described by Thomson (1960). P estimations

Inorganic P and total P, after micro-digestion (Cosgrove, 1963), were determined on selected fractions by the method of Dickman .and Bray (1940). Total P in plant samples was determined on acid digests (Piper, 1942) using the method of Truog and Meyer (1929). Total and organic P content of soil samples were obtained by the method of Steward and Oades (1972).

EXPERIMENTAL

AND RESULTS

Changes in acid-insoluble organic P and G50 P during decomposition of phalaris Experiment 1. Changes during 16 weeks of incubation. Subsamples (10 g) of ground phalaris were mois-

tened with water (35 ml) and incubated for 2,4, 8 and 16 weeks at 2o’C in 3cm dia tubes plugged at each end with glass wool. At the end of each incubation period the residues were dried by passing air through the tubes. Samples of the undecomposed and decomposed residues were then analysed for acid-insoluble organic P, G50 P, and the purine base content of the

plant

G50 P. The P remaining in the residues after the SLS/ EDTA extraction was also determined to check the recovery of the acid-insoluble organic P (Table 1). About two thirds of the acid-insoluble organic P in the undecomposed sample was G50 P and of this about 60% was nucleic acid. During the course of incubation the acid-insoluble P remained constant for the first 4 weeks then increased markedly by the eighth week and remained so far for the next 8 weeks. The G50 P remained relatively constant except for an increase in the 8 week sample. The amount of nucleic acid in the G50 P varied during decomposition but there was a net loss after 16 weeks. From the change in the Gu/Ad ratio it is clear that a qualitative change in the nucleic acid also had occurred. Experiment 2. Changes after 6 months decomposition and leaching. Samples (3OOg) of another phalaris

sample cut into 5-8 cm segments were incubated at 20°C in 80 x 50 x 30cm stackable polythene trays under two different moisture cycles; (i) 2 weeks moist incubation, then leaching or (ii) 1 week moist incubation, 1 week air-drying, followed by leaching. These treatments are referred to as 2 M and 1 M 1 D, respectively. At each leaching the plant material was immersed in 15 1 of distilled water for 1 h and then the excess water drained off through a hole in the base of the tray. Drying was achieved by passing air at a slow rate through the trays. After 6 months of the incubation and leaching cycles the decomposed residues were air-dried, weighed, and sub-samples examined as in Expt 1. About 25% of the P in the undecomposed sample

Table 2. Changes in acid-insoluble organic P and GSO P in phalaris after 6 months decomposition

Treatment Undecomposed A(2M)t C(I1 ID)+

Wt of residue (“Aof undecomposed plant material) 100 42 52

Total P (pgP/g)*

Acid-insoluble organic P (PgP/g)*

G50 organic P (rgP/g)*

Gu (pm/g)*

Ad (pm/g)*

1226 741 655

311 428 411

227 258 245

0.97 1.08 1.13

0.89 0.85 0.87

Gu/Ad

Approx. nucleic acid (pgP/g)*

1.09 1.27 1.30

*Values are expressed on the basis of amounts (peg P or pm) recovered from 1 g of original undecomposed material. t Decomposition cycles: 2M-2 weeks moist incubation, then leaching, (repeated 13 times). 1M lD- 1 weeks moist incubation. 1 weeks drying, then leaching (repeated 13 times).

115 120 124 plant

0. L. JONES and S.

148

Table 3. Organic P and nucleic acid in SLS/EDTA

Soil and year of sampling* Unimproved 1964 Unimproved 1964 Unimproved 1964 Improved 1970 Improved 1970 Improved 1970

A B C A :

M. BR~MFIELD

extracts

Total organic P in soil k%P/g)

GSO organic P (clgP/gI

119 95 110 1.50 128 167

37.8 35.5 35.1 59.1 40.4 53.9

from unimproved and improved pasture soils

Gu/Ad 0.109 0.095 0.098 0.139 0.085 0.121

0.069 0.050 0.059 0.080 0.047 0.071

Approx. nucleic acid @P/g)

1.57 1.90 1.66 1.74 1.83 1.71

11.0 9.0 9.7 13.6 8.2 11.9

* Soils sampled before and after 6 yr of superphosphate fertilizing. pasture growth and sheep grazing.

was acid-insoluble of which about 707; was GSO P and about half of this was nucleic acid (Table 2). After 6 months. the amounts of acid-insoluble ocganic P in the residues had increased and were similar for both incubation conditions. The increases in GSO P and nucleic acid P on the other hand were small compared to the increase in acid-insoluble P. It follows that much of the increase in acid-insoluble organic P is due to P that is not excluded by Sephadex GSO. The change in the Gu/Ad ratio again indicates qualitative changes in the nucleic acid had taken place during decomposition.

Experiment 3. Extraction of soils with SLS/EDTA and separation of G50 P and purine and pyrimidine bases. Soil samples (4g) were extracted with SLS/

EDTA for 6 h and then the G50 P and nucleic acid bases were determined as for plants (Table 3). The G50 P in the extract accounted for about 30-40”,, of the soil organic P and about 20-30?,, of this was nucleic acid. The increase in total organic P during the 6 yr of pasture improvement was in general reflected in an increase in the G50 P but only 2 of the 3 soils examined showed a slight increase in the nucleic acid content of this fraction. The pyrimidine bases, uracil and thymine were readily detected on the paper chromatograms indicating that RNA and DNA respectively had been extracted from the soil.

Undecomposed phalaris

The amount of G50 P extracted from the soil by SLS/EDTA was found in another test to increase by 1.2 times when the extraction time was increased from 6 to 12 h but the amount of nucleic acid remained the same. Chromatographic

studies on plant and soil organic

Experiment 4. Examination of G50 P from soil hc ion-exchange cellulose fractionation.

P

plant and

The G50 organic P isolated from the undecomposed and decomposed phalaris (c) in Expt 2 and from soil C, 1970. in Expt 3 was fractionated on ion-exchange cellulose in an attempt to obtain a more refined separation of the organic P components. The distribution of P in the eluted fractions is shown in Fig. 1. There were two peaks in each sample. The P from plants was present largely in one well defined peak whereas for soil there were two slightly separated peaks of comparable size. The U.V.absorption spectra for fractions containing the peaks of P are presented in Fig. 2. The spectra for fractions 13 and 14 (Fig. I) from decomposed and undecomposed plant samples. respectively, gave a broad absorption peak at 260nm which was consistent with the presence of nucleic acid. Fractions between 12 and 20 also exhibited similar spectra and so fractions 12-20 for plants were bulked for purine estimation. The absorption spectra of fractions from soil had no peaks and so fractions

Decomposed phalaris

SOll

FRACTION NUMBER Fig. 1. Ion-exchange cellulose fractionation of G50 P isolated from undecomposed and decomposed phalaris and from an improved pasture soil.

149

Macromolecular phosphorus in plants and soils

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-.

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‘\

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FWCtlO” No

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13,s

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01

and soil suspension inoculum served as a control for the microbial assimilation of P. The solutions (8 ml per 50 ml conical flask) were incubated at 2OC over 4 days and the rate of microbial assimilation of P measured by daily determinations of total P in solution after centrifugation (12,OOOg per 10 min). The composition of the nutrient solution after mixing was: glucose, 0.1%; I-asparagine, 0.1%; KNO,, 0.005%; Mg S04.7H20, 0.02%; (NH&S04. 0.01%; and Ca(NO&, 0.005%. The amounts of P in the supernatant solutions are shown in Fig. 3. The bulk of the P in the G50 and ion-exchange cellulose fractions from both plant samples, and in the inorganic P controls, was rapidly assimilated within 2-3 days. The variation between the controls for (a) and (b) and those for (cj. (d) and (e) is probably due to differences in the soil inoculum since the two sets were run on different occasions. The point of interest, however, is the amount of organic P remaining in solution after the inorganic P had been virtually exhausted. After 4 days about 150,;;of the G50 P from both plant samples was still in solution whereas the organic P from the cellulose fractionation was completely assimilated (Figs 3a, 3b, cf. 3c, 3d). In contrast, a substantial part of the P in the bulked ion-exchange cellulose fractions from soil appeared to be assimilated more slowly, as about 40’;; of the P was still in solution after 4 days (Fig. 3e). Preparations of G50 P from decomposed and undecomposed plants were also incubated with water substituted for the nutrient solution. In these treatments less than 10% of the P was assimilated after 4 days.

solution

06-

_

\

‘l_

‘1..

l\

------____

‘A._.____:.-2

-_

I

‘\

10/s S/dP

I

220

240

260

280

300

.\(nm)

Fig. 2. U.V. Absorption spectra of selected fractions obtained by ion-exchange cellulose fractionation of G50 P isolated from undecomposed phalaris. ~ p; decomposed - dp: and from an improved pasture soil. phalaris. ~-S.

The recovery of G50 P applied, the amount of P in the bulked fractions and the purine estimations and approximate nucleic acid contents of the bulked samples are presented in Table 4. It is clear that much of the G50 P was retained on the cellulose and consequently the usefulness of the separation is limited. Further, it is apparent that the peak between fractions 12 and 20 for plant prep aration was a mixture of organic P compounds and nucleic acid accounted for about 65-707; of the P. The P eluted from the soil preparation on the other hand was largely non-nucleic acid P.

4-20 were bulked.

Microbial assimilation of the P fractions Experiment 5. Assimilation of organic P from p/ant and soil samples. In order to establish whether the

organic P isolated from plants and soil was resistant to microbial assimilation various preparations were inoculated with soil microorganisms. The preparations tested were the G50 P from undecomposed and decomposed phalaris (Table 2) and the bulked fractions from the ion-exchange cellulose fractionation of the G50 P from the same plant samples and from soil sample C 1970 (Table 4). Samples of each preparation containing 4pg P were mixed with a nutrient solution and inoculated with a drop of soil suspension. A treatment consisting of a mixture of inorganic phosphate (4 pg P). nutrient

Experiment 6. Examination of the soluble P remaining in cultures containing the soil organic P preparation. Nutrient solution (80 ml) containing 40 pg P

of the soil organic P fractions eluted from ionexchange cellulose were inoculated with a soil suspension and incubated in a 500 ml conical flask for 4 days at 2OC. The culture was then centrifuged at 12,000g for 10 min. and soluble P excluded by Sephadex G50 determined. This P amounted to 18 i(g. representing 457; of that initially present. Its U.V. spectrum was non specific, similar to soil fractions 10 and 13 and phalaris fraction 9. in Fig. 2. No nucleic acid bases were detected on paper chromatograms of hydrolysed samples.

Table 4. Organic P and nucleic acid in bulked fractions from ion-exchange cellulose fraction of G50 P

Material Undecomposed plant material (Expt 2) Decomposed plant material (C, Expt 2) Soil (C, 1970)

G50 organic P applied to column (rgP/g)*

P eluted from column (figP/g)*

Organic P in fractions bulked

227

157

125

0.72

0.66

1.09

86

245 54

108 27

82 27

0.48 0.034

0.38 0.024

1.26 1.42

53 3.6

Gu/Ad

Approx. nucleic acid (PgPg)

* Values for decomposed plant material are expressed on the basis of amounts (c(g P or pm) recovered from 1 g of original undecomposed plant material.

0.

150

L.

JONESand S.

M.

BROMFIELD

INCUBATIVE TIME (days)

Fig. 3. Microbial assimilation of organic P fractions isolated from plants and soil. [a) G50 fractions. undecomposed phalaris. (b) GSO fractions. decomposed phalaris. (c) Ion-exchange cellulose fractions. undecomposed phalaris. (d) Ion-exchange cellulose fractions, decomposed phalaris. (e) ion-exchange cellulose fractions, soil. M. organic P: inorganic phosphate controls.

DISCUSSION

About 2%2!$; of the total P in und~orn~s~ hayed-off phalaris plants was acid-insoluble organic P of which 64-70~~ was G50 P and SO-6Ou/oof this was nucleic acid P (Tables I and 2). The decomposition studies under extreme incubation conditions showed that there was some accumulation of acid-insoluble organic P due to microbial action. However. only a small proportion of the accumulation was due to G50 P or nucleic acid. In other words the increase was due mainly to low molecular weight organic P in the SLS/EDTA extract, possibly extracted from microbial cells. This fraction was not examined because of its gross contamination with the SLS/EDTA extractant. The fact that the G50 P content of plants remained relatively constant despite prolonged decomposition and leaching suggests that this P could be resistant to microbial assimilation. The microbial incubation studies. however, showed that when the GSO P was extracted from undecom~sed or decomposed plants it was rapidly and almost completely assimilated in the presence of added nutrients. It is seen from Tables 1 and 2 that the G50 P extracted from decomposed plants consisted of roughly equal parts of non-nucleic and nucleic acid P. It is possible that in plants much of the G50 P is present inside microbial or plant cells or as an insoluble complex and as such is resistant to further degradation. The SLQ’EDTA extraction, however. liberates this P in forms that can be readily assimilared by micoorganisms in the presence of nutrients. Another possibility is that after an initial period of decomposition and leaching of plant residues the substrates available for microbial activity become depleted and a situation, similar to that in the water controls in Expt 5. develops where the G50 P remains largely unassimilated. It is clear that at least some of the nucleic acid ‘P in senescent plants was accessible to and decomposed by microorganisms and that in the decomposed residues some of the nucleic acid was due to microbial synthesis (Tables 1 and 2).

The extent to which SLS/EDTA was able to extract organic P from soil is not known. However, the amounts of G50 P extracted from unimproved and improved pasture soils reflected the net increase in organic P due to pasture improvement (Table 3). The increase in the G50 P content of improved soils was due mainly to non-nucleic acid P rather than to nuo leic acid P. The estimates of nucleic acid extracted from soils accounted for about 6-9’10 of the total soil organic P. These values could be underestimates because of the unknown extent of extraction. They are higher than the value of ?Tl; reported for Scottish soil (Anderson, 1967). The G.50 P extracted from soils contained relatively less nucleic acid P than did the GSO P extracted from plant samples (Tables 2 and 3). The attempts to fractionate the G50 P from plants and soil on ionexchange cellulose in order to identify common fractions proved unsuccessful because of the partial retendon of this P. However, the P efuted from plant preparations was relatively enriched with nucleic acid whereas that from the soii preparation was relatively poor in nucleic acid (Table 4). The microbial assimilation studies on these preparations (Fig. 3) and the subsequent chemical examination suggest that the non-nucleic acid portion of the soil organic P exhibited some resistance to assimilation. It would appear that further studies on the non-nucleic acid P in the SLS/EDTA extracts of both soils and plant residues are warranted.

REFERENCES

ANDERBI G. (1967) Nucleic acids, derivatives and organic phosphates. In Soif Bi5chemisf~~ (A. D. McLaren and G. -H. Piterson. Eds). Marcel Dekkei. New York. CHARGAFF E. and DAVIDSON 3. N.

(1955) The Nucleic Acids, Vol. I (E. Chargaff and J. N. Davidson, Eds). Aca-

demic Press. New York. W. E. (1949) The separation of purine and pyrimidine bases and of nucleotides by ion exchange. Scirnee 109.377-378.

COHN

Macromolecular

COSGROVE D. J. (1963) The chemical nature of soil organic phosphorus. 1. Inositol phosphates. Australian Journul of Soil Research 1, 203-213. COSGROVE D. J. (1966) The chemistry and biochemistry of inositol polyphosphates. Reviews of Pure and Applied Chemistry

16, 209-224.

DICKMANS. R. and BRAYR. H. (1940) Calorimetric determination of phosphate. Industrial and Engineering Chemistrv (Anulvtical

Edition)

12, 665-668.

Fertility and productivity of a podzolic soil as influenced by subterranean clover (Trifolium subterraneum L.) and superphosphate. Australian Journal of Agricultural Research 5. 664687.

JONES 0.

L. and BROMFIELDS. M. (1969) Phosphorous changes during the leaching and decomposition of hayed-off pasture plants. Australiun Journal of Agricultural Research

20, 653-663.

Anulysis-A Munuul of Methods for the Examination of Determination of the Inoryanic Constituents

Laboratory and the of Plants.

Soil

University of Adelaide Press. SIMPSONJ. R.. BROMFIELD S. M. and JONES 0. L. (1974a)

Effects of management

III-Changes in total soil nitrogen, carbon, phosphorus and exchangeable cations. Australian Journal of Experimental

Agriculture

on soil fertility under pasture.

and Animal

Husbandry

14.487494.

SIMPSONJ. R.. BROMFIELDS. M. and MCKINNEY G. T.

(1974b) Effects of management on soil fertility under pasture. I-Influence of experimental grazing and fertilizer systems on growth, composition and nutrient status of the pasture. Australian Journal of Experimental Agriculture and Animal

DONALDC. M. and WILLIAMSC. H. (1954)

PIPER C. S. (1942) Soil and Plant

151

phospho ,rus in plants and soils

Husbandry

14, 47&478.

STEWARDJ. H. and OADESJ. M. (1972) The determination of organic phorphorus in soils. Journal of Soil Science 23, 38-50.

THOMSONR. Y. (1960) Purines and pyrimidines and their derivatives. In Chromutogruphtc and Electrophoretic Techniques, Vol. I Chromatography (1. Smith, Ed.). Heinemann, London. TRUOG E. and MEYERA. H. (1929) Improvements in the Denigks calorimetric method for phosphorus and arsenic. Industrial and Engineering Chemistry (Analpticul Edition)

1, 136-139.

VAN SOESTP. J. and WINE R. H. (1967) Use of detergents in

the analysis of fibrous feeds. IV-Determination of plant cell-wall constituents. Journal of the Association of Oficial Analytical

Chemists

50, 50-55.