Soil Bid. Biochem. Vol. 18. No. 6, pp. 643-647, 1986 0038-0717/86 $3.00 + 0.00 Printed in Great Britain Pergamon Journals Ltd

THE NATURE AND STABILITY OF ORGANIC PHOSPHATES IN LEAF LITTER AND

SOIL ORGANIC MATTER IN NIGERIA

I. MUELLER-HARVEY* and A. WILD

Department of Soil Science, University, Reading RGl 5AQ. England

(Accepted 10 July 1986)

Summary-The organic phosphorus components of leaf litter from a secondary forest in Nigeria were characterized as phospholipids, acid soluble esters, LiOH-extractable esters and residual phosphate. During incubation of the litter at 30°C for 8 months all four organic fractions were synthesized and the content of inorganic phosphate decreased.

During cultivation of the same field site over a period of 22 months, the IPSf6 content of the soil did not change significantly, but there were significant decreases of phospholipids and of high molecular weight components. The high molecular weight components, which were not identified, represented the most important source of organic phosphorus that became available to plants, but phospholipids may also have been a source.

The results indicate that the inositol phosphates in leaf litter were in too low concentration to have provided the amounts found in the soil.

INTRODUCTION

Few similarities exist between the phosphate ester composition of plants and soil organic matter. In young plants Bieleski (1968) found that the phos- phate esters consisted of nucleic acids (55% of total organic P), phospholipids (28%) and acid-soluble phosphate esters (17%), and that the relative propor- tions were remarkably constant between tissues and with time. In soil on the other hand, Dalal(1977) and Anderson (1980) reported a range for nucleic acids of 0.2-2.4% of the total organic P content, 0.5-14.0% for phospholipids, and O&58.0% for inositol phos- phates. The reasons for this variable composition are not clear. The techniques used for extraction and identification of the soil organic phosphate are not entirely satisfactory and much more remains un- characterized. However, the reasons for the vari- ability in composition would be better understood if more were known of the origin of the esters. The possible sources are (i) accumulation from plant additions, (ii) synthesis during litter decomposition, and (iii) synthesis in the soil.

The study to be reported is an attempt to dis- tinguish between these possibilities. The results are reported in two parts (a) phosphate esters were characterized in leaf litter from a forest site in Nigeria, and the stability of the esters was measured in an incubation experiment, and (b) at the same site the soil organic phosphate esters were characterized, and their stability was measured after the site was brought into cultivation and cropped.

Litter

MATERIALS AND METHODS

The leaf litter (69.4% moisture content) was col- lected in June 1980 from the floor of a secondary

*Present address: Scottish Marine Biological Association, Dunstaffnage Marine Research Laboratory, P.O. Box 3, Oban PA34 4AD, Scotland.

forest at the International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria (7”30’N, 3”54’E, altitude 700 m). As most of the leaf fall occurs during the dry season, December to February, the litter had been lying on the ground for about 46 months and consisted of a dense mat of leaves and small twigs (for general forest flora description see Mueller-Harvey et al., 1985). The sample was stored moist at 5°C in a plastic bag for 16 months before being chopped in a kitchen grinder to less than 2 mm. The pH of the litter in water (l:lO, w:v) was 8.4.

For incubation, a sample of 21 g litter (oven-dry basis) was held in the dark at 30 f 1°C and 45% water holding capacity for 8 months in a 500 ml beaker covered with “cling film”. The water holding capacity had been determined by letting a wet sample drain overnight. The water content was kept approxi- mately constant by twice-weekly additions. The loss of weight of litter by respiration was calculated from the total weight of beaker plus contents after drying, allowing for the weights of samples previously re- moved. The litter was sampled and analyzed at the start and after incubation for 8 months. Sub-samples were used for two sets of analyses: (i) after NaOBr oxidation of the litter, inositol penta- and hexakis phosphates were separated from other phosphates on a Dowex 1 x 8 (Cl-) column (0.8 x 18 cm) using 0.25 M HCl; IP, + 6 were recovered by elution with 1 M HCl (Irving and Cosgrove, 1981). After acid digestion, phosphate was determined as the molyb- denum blue complex (TSAU, 1980). (ii) The methods of Bieleski (1968) for the fractionation of plant phosphorus compounds were used to separate phospholipids, acid soluble phosphates, LiOH ex- tracted phosphates and “residual” phosphates. Com- bined aqueous acid extracts and the LiOH-extract were treated with QAE-Sephadex as suggested by Redgwell (1980). In all fractions total phosphate was determined as the molybdenum blue complex after acid digestion.

643

644 I. MUELLER-HARVEY and A. WILD

The phospholipids were fractionated by 2-dimen- sional thin-layer chromatography (Bieleski, 1968; 1972) on 10 x 10 cm pre-coated SiF,,, plates (Merck, Darmstadt, West Germany). Ten to 80~1 of the phospholipid extract was applied at the origin, and elution in chloroform-methanol-water (65:25:4 v/v) was stopped when the solvent front had reached 7.5 cm. After drying, the plates were eluted at right angles in methyl isobutyl ketone-ethanoic acid-water (8:5: 1 v/v) until the solvent front had reached a distance of 5.5 cm. These conditions were required for good results, and are a modification of the original method. Detection of phospholipids was by the spray reagent of Dittmer and Lester as described by Kirchner (1978).

Acid soluble phosphates and LiOH-extractable phosphates were separated by 2-dimensional thin- layer chromatography on pre-coated 10 x 10 cm cel- lulose MN300 plates (Polygram; Macherey and Nagel, Dueren, West Germany). Two ~1 of 25 mM EDTA were applied at the origin (Bieleski, 1969) followed by IO-20 ~1 of the extracts. Elution in n-propanol-ammonia-water-EDTA (6:3: 1 :O.Ol, v/v/v/w) was performed twice to the top of the plate in the first direction. After drying, the plates were eluted twice at right angles in n- propyl ethanoate-methanoic acid-water (11: 5: 3. v/v) (Bieleski. 1968). For optimum results, the following steps were necessary: (a) before use the cellulose plates were eluted in 2M CH,COOH, dried and again eluted in distilled water; (b) the plates were sprayed with Kirchner’s (1978) ammonium molybdate re- agent until they were damp but not wet, and were then immediately placed in an oven at 80°C for 10min; (c) they were then held over a boiling water bath for 2 min and spots were recorded; (d) they were placed under U.V. light (365 nm) for 1 min and spots were recorded; (e) they were finally placed in a container holding a beaker of concentrated ammonia solution until the background colour had just disap- peared and spots were again recorded. The colours were stable.

Soil

The field site and the experimental conditions during the period of cultivations have been described

by Mueller-Harvey et al. (1985). The soil at the site is classified as a clayey skeletal kaolinitic iso- hyperthermic Oxic Paleustalf belonging to the Egbeda-Iwo association. It is medium to light tex- tured near the surface, with sandy clay to clay sub-soil, and has a layer of angular and sub-angular quartz gravel 15 cm below the surface. An area (40 x 40 m) was cleared manually from secondary forest. Soil sampling followed a stratified random sampling design. Samples (O-10 cm depth) were com- bined from nine sampling plots representing “low organic matter” soil (sample I) which came from a relatively sandy area of the site, and samples from another nine plots were collected similarly from a more clayey area as “high organic matter” soil (sample 2). They were collected in June 1979 just after bush clearance, and also in April 1981 after zero- tillage cultivation when sampling depth was reduced to 8 cm because of the increase in bulk density.

Organic phosphate esters in the NaOH extracts (Steward and Oades, 1972) were precipitated with (CH,COO),Ba and fractionated on an anion ex- change resin as described by McKercher and An- derson (1968). The aqueous eluate after the sample had been applied to the column was fraction I (“non adsorbed organic P”) and contained organic P which was not retained by the resin. Fraction 2 was eluted with 0.6 M HCOONH, (300 ml) and fraction 3. which contained inositol penta- and hexakis phosphates, with 1.2 M HCOONH, (200 ml). Inorganic phosphate was determined calorimetrically using a slight modification of the method of Asher (1980). Total phosphate was determined after acid digestion.

The molecular weight of the “non adsorbed or- ganic P” was estimated by passing the fraction through a column of Sephadex G-50 (1.5 x 50 cm) and eluting with 0.2 M NaOH.

Phospholipids were extracted after pretreating the soil samples (50 g, <0.5 mm) with a solution of 2.5% HCI and 2.5% HF (75 ml) followed by drying with acetone (40 ml). Hexane-acetone (50 ml; Kowalenko and McKercher, 1970) was used for the extraction of phospholipids. Acetone is known to be a poor solvent for phospholipids (Hance and Anderson, 1963; Kow- alenko and McKercher, 1970) but it enhances the extraction of phospholipids by hexane. The acetone

Table I. Phosphate concentrations in fractions of leaf btter at start of incubation and after 8 months (oven dry basis); in parenthesis. standard error from duplicate determinations

Concentration at Concentration after start 8 months” Changes

‘?iE % of

‘YiP,eF’ % of K?Pg ’ Level of

total P total P litter % significance

Phospholipids 42 (2) 3 52 (2) 4 + IO + 24 (NV Acid soluble esters

Inorganic 758 (IX) 58 372 (19) 28 - 386 -51 P =O.OI Organicb 59 5 I81 14 + 122 + 207 Total 817(14) 63 553 (IO) 42 - 264 - 32 P =O.OI

LiOH extracted P Inorganic 82 (5) 6 81 (0) 6 -I -I (NS) OrganG I I4 9 205 I6 +91 + 80 Total 196 (6) I5 286 (3) 22 + 90 + 46 P =O.OI

“Residual” P 203 (2) 16 366(l) 28 + 163 + 80 P =O.OOl

Sum of P in fractions 1258 97 1258” 96

Total P by direct analysis l301(30) 100 1314(29) 100

“Measured concentrations adjusted to give same total P i)s at start (I258 pgg-I). that is. concentrations are pgg ’ original litter. bBy difference (PO_ = P,,, - P,,,,, ).

Organic phosphates in litter and soil 645

drying step was incorporated in order to avoid a separation of phases during hexaneacetone extrac- tion.

RESULTS

Litter

Table 1 gives a percentage distribution of total, organic and inorganic P in four fractions: lipid, aqueous acid soluble, LiOH extracted and “resi- dual”. About 90% of the inorganic P was in the water-soluble fraction after acid extraction and the rest was in the LiOH extract. Some of this inorganic phosphate might be a product of hydrolysis. About 48% of the organic P was in the “residual-P” frac- tion. LiOH extracted 27% of the organic P and the acid extract contained about 15%. Phospholipids accounted for 9% of the total organic P. and inositol pentakis and hexakis phosphates for about 2% of the total organic P.

In order to compare P contents in each fraction before and after incubation, it was necessary to take account of the weight loss due to respiration. To do this, phosphate concentrations after incubation were recalculated assuming no change in total P during incubation. Table 1 shows that transfer of P occurred from the soluble inorganic to “residual” organic-P (+ 163 pg P), soluble organic P (+ 122 pg), LiOH extracted organic-P (+ 90 pg) and the lipid P fraction (+ 10 pg).

Characterization and semi-quantitative TLC- studies of the lipid extract at the start of incubation showed that phosphatidic acid (PA), phosphatidyl ethanolamine (PE), phosphatidyl glycerol (PG), phosphatidyl choline (PC) and phosphatidyl inositol (PI) contributed most to the lipid fraction (in de- creasing order). After incubation, this order was changed to: PE > PG > PA > PC > PI.

The acid soluble fraction contained two main phosphate esters with &values (relative to H,PO;) of 156 and 37 (compound 1) and 267 and 75 (com- pound 2) in the solvents propanol-ammonia- water-EDTA and propyl ethanoatemethanoic acid-water respectively. The characterization of com- pound 1 will be reported separately; compound 2 was not investigated further. Small amounts of inorganic P, compounds 1 and 2, plus a few (unidentified) P-esters, which were not extracted previously, were found in the LiOH extract. No IP,, IP,, DNA or ribonucleotide phosphates were detected in this frac- tion. Most of the organic P (72 pg P g-’ litter) from the LiOH extract was lost through adsorption on QAE-Sephadex during purification, whereas little (lOpgPg_r litter) was retained from the aqueous acid extract.

Soil

A preliminary ammonium formate fractionation (Anderson and Malcolm, 1974) showed that most of the extractable organic phosphates occurred in a “non-adsorbed fraction” and as inositol pentakis and hexakis phosphate. For examination of the inositol pentakis and hexakis phosphates the two-step frac- tionation of McKercher and Anderson (1968) was used.

The pentakis and hexakis phosphates in the two

646 I. MUELLEK-HARVEY and A. WILD

Table 3. Distributmn of phosphate in litter compared with reported values in young leaves, bacteria and fungi

Lioid P Inbrganic F'-water-soluble 5x 70 -

LiOH extractable 6 0 Soluble P-estera

water-soluble 5(15) 5 -

RNA DNA >

not 16 3G-50 detected 5-10

Other LiOH soluble organic P 9 (27) 0 Residual P I6 (48) 0

“The present study: hBieleski (1968); ‘Alexander (1977). 9~ of total P. “% of organic P.

soil samples accounted for 12- 13 ilg P g-l soil or 5-9% of the total organic P {Table 2). Soil phos- pholipid contents were also low, accounting for only l-2% of the total organic P, The “non-adsorbed” soil organic P accounted for more than 40% of the total soil organic P.

The changes in the soil content of the inositol phosphates which occurred during cultivation were not statistically significant. However, net mineral- ization of the phospholipids was significant at the 6% level (sample 1) and at the 5% level (sample 2). Mineralization losses were highly significant (5% and 1% level) for the “non-adsorbed organic P”, losses of 49 and 35% being measured in samples 1 and 2 respectively. The loss of organic P from this fraction was not accompanied by an increase of P esters in other fractions, that is, the organic phosphate in the “non-adsorbed fraction” appeared to be converted to inorganic phosphate.

DKXUSSION

The salient feature of our results is that the distri- bution and composition of the phosphate esters in leaf litter (Table 1) differed markedly from that in the underlying soil (Table 2). The composition of the phosphate esters in litter also differed from that reported in young leaves and microbial cells (Table 3). Thus, most of the organic phosphorus in young leaves and microorganisms occurs in RNA, DNA and phospholipids, with smaller amounts as hexose phosphates, and phosphoglyceric acid. In the leaf litter, however, no nucleotide phosphate was de- tectable, and only small amounts of phospholipids (3% of total P) and soluble P-esters (5%) were extractable; most of the organic P was diflicult to extract and remained unidentified.

Contents of inositol penta- and hexakis phosphates were very low in the leaf litter. None could be detected by TLC in the acid or alkaline extracts, and NaOBr analysis yielded only 10 pug IP, +6-P g-’ litter. However, these phosphates are present in soil (Table 2). The amounts were low compared with soils from New Zealand (Martin and Wicken. 1966) and the United Kingdom (Anderson, 1980), but they were of the same order of magnitude as reported in some Ghanaian soils (Appiah and Thomas, 1982; Halm et al., 1982).

Most of the organic P extracted from the soil was

not identified. It occurred in a fraction which was not retained by. an anion exchange resin, Gel chro- matography indicated that approximately 50% of this “non-adsorbed P” had nominal molecular weights in excess of 10,000. Such high amounts of P-esters in a large molecular weight fraction agree with other reports (Cosgrove. 1977; Tate, 1979). The percentage contribution of these P-esters to total organic P appears to be variable and depends on the extraction procedure used (Halstead and Anderson, 1970). In our experiment most of the decrease in organic phosphorus content during cultivation of the soil was associated with a decrease in this fraction.

It would appear that the net decrease of the phospholipid pool was too small to contribute phos- phate for crop nutrition (0.9 kg P ha-’ in 22 months; Table 2). There is, however, evidence that a substan- tial part (IO-20%) of the total soil phospholipids occurs in microbes (Kowalenko and McKercher, 1971). Given a sufficient rate of microbial turnover, the microbial biomass could be the source of up to 2 kg P ha-’ a--“. This estimate is based on microbial turnover rates reported by McGill ef al. (1981) for Canadian conditions and on a comparison of decom- position rates in a temperate and a tropical soil (Jenkinson and Ayanaba, 1977). The two assump- tions made are (i) that similar mineralization rates apply to soil and microbial phospholipids and (ii) that biomass turnover is faster in tropicai than tem- perate soils, in line with plant residue decomposition rates.

The big decrease in the amount of phosphate esters of large molecular weight during the period of culti- vation shows this fraction to be an important source of phosphate for crops, which agrees with obser- vations of Bettany et at. (1980) and Hedley et al. (19X2), although it differs from the conclusion of Dalal (1977). By contrast, soil inositol pentakis and hexakis phosphates were a very stable fraction and are not likely to be important. In comparing different fractions of soil organic phosphate as sources of phosphate for crops, it must, however, be noted that about 40% of the soil organic P was not accounted for using HCOONH, fractionation of the NaOH extract (see Table 2). Also, mineralization losses of the total organic P as determined by ignition (22-23%) were smaller than those of all the fractions combined (32-39%). A major difficulty is the limitations of present methods for identifying the organic phosphate esters.

Organic phosphates in litter and soil 647

The origin of soil organic phosphate can now be considered. During incubation of the litter, large amounts of inorganic P were converted to organic phosphate esters. Chauhan et al. (1981) also found that organic P compounds were synthesized by soil microorganisms from inorganic P when soils high in inorganic P were supplemented with C-additions and incubated. These two observations emphasize the important contribution of microorganisms to organic P formation.

It would appear that leaf litter is not the main source of soil inositol pentakis and hexakis phos- phates because analysis of the leaf litter yielded very low amounts. If our analysis of 10 pg IP, + 6 g-’ of litter is typical, it would take about 150 yr to accumu- late the 15 kg P ha-’ found in soil, assuming an annual litter fall of 10 t ha-’ a-’ and no mineral- ization of the esters. It is more likely that soil inositol phosphates are synthesized by microorganisms (Cos- grove, 1977). It seems probable that a large propor- tion of the soil phospholipids is also synthesized by microorganisms since (i) phospholipids were found to be labile in the leaf litter and soil and would therefore not remain in the soil for any length of time without replenishment, (ii) synthesis of phosphatidyl eth- anolamine and phosphatidyl choline was observed during litter incubation, and (iii) phospholipid con- tents were not related to organic matter contents (which in turn are related to vegetational inputs). The origin of the high molecular weight soil organic phosphate is not known.

Acknowledgements-We would like to thank Dr A. S. R. Juo for providing facilities for the field experiment and Mr J. A. Varley for advice on analysis. I.M-H. is grateful for financial assistance provided by the International Institute for Tropical Agriculture, Ibadan, Nigeria and the Research Board of Reading University.

REFERENCES

Alexander M. (1977) Introduction to Soil Microbiology, p. 336. Wiley, New York.

Anderson G. (1980) Assessing organic phosphorus in soils, In Role of Phosphorus in Agriculture (F. E. Khasewneh, E. C. Sample and E. J. Kamprath, Eds), pp. 41 l-431. American Society of Agronomy, Madison. _ _

Anderson G. and Malcolm R. E. (1974) The nature of alkali-soluble soil organic phosphates.’ Journal of Soil Science 25, 282-297.

Appiah M. R. and Thomas R. L. (1982) Inositol phosphate and organic phosphorus contents and phosphatase activ- ity of some Canadian and Ghanaian soils. Canadian Journal of Soil Science 62, 31-38.

Asher L. E. (1980) An automated method for the deter- mination of orthophosphate in the presence of labile polyphosphates. Soil Science Society of Agronomy Journal 44, 173-175.

Bettany J. R.. Saggar S. and Stewart J. W. B. (1980) Changes in the amounts and forms of sulfur in soil organic matter fractions after 65 years of cultivation. Soil Science Society of America Journal 44, 70-75.

Bieleski R. L. (1968) Levels of phosphate esters in Spirodela. Plant Physiology 43, 1297-l 308.

Bieleski R. L. (1969) Phosphorus compounds in trans- locating phloem. Plant Physiology 44, 497-502.

Bieleski R. L. (1972) Turnover of phospholipids in normal

and phosphorus-deficient Spirodela. Plant Physiology 49, 740-745.

Chauhan B. S., Stewart J. W. B. and Paul E. A. (1981) Effects of labile inorganic phosphate status and organic carbon additions on the microbial uptake of phosphorus in soils. Canadian Journal of Soil Science 61, 3733385.

Cosgrove D. J. (1977) Microbial transformations in the phosphorus cycle. Advances in Microbial Ecology I, 955134.

Dalal R. C. (1977) Soil organic phosphorus. Advances in Agronomy 29, 83-I 17.

Halm B. J., Ahenkorah Y. and Appiah M. R. (1982) Inositol penta- and hexaphosphate content of some cocoa soils in Ghana. Zeitschrtft fuer Pflanzenernaehrung und Bodenkunde 145, 586592.

Halstead R. ,L. and Anderson G. (1970) Chromatographic fractionation of organic phosphates from alkali, acid, and aqueous acetylacetone extracts of soils. Canadian Journal of Soil Science SO, 11 l-l 19.

Hance R. J. and Anderson G. (1963) Extraction and estimation of soil phospholipids. Soil Science 96, 9498.

Hedley M. J., Stewart J. W. B. and Chauhan B. S. (1982) Changes in inorganic and organic phosphorus fractions induced by cultivation practices and by laboratory incu- bations. Soil Science Society of America Journal 46, 97&976.

Irving G. C. J. and Cosgrove D. J. (1981) The use of hypobromite oxidation to evaluate two current methods for the estimation of inositol polyphosphates in alkaline extracts of soils. Communications in Soil Science and Plant Analysis 12, 495-509.

Jenkinson D. S. and Ayanaba A. (1977) Decomposition of carbon-14 labelled plant material under tropical condi- tions. Soil Science Society of America Journal 41, 912-915.

Kirchner J. G. (1978) Thin-layer Chromatography, 2nd edn, pp. 201-249. Wiley, New York.

Kowalenko C. G. and McKercher R. B. (1970) An exam- ination of methods for extraction of soil phospholipids. Soil Biology & Biochemistry 2, 2699273.

Kowalenko C. G. and McKercher R. B. (1971) Phos- pholipid P content of Saskatchewan soils. Soil Biology & Biochemistry 3, 243-247.

Martin J. K. and Wicken A. J. (1966) Soil organic phos- phorus. IV. Fractionation of organic phosphorus in alka- line soil extracts and the identification of inositol phos- phates. New Zealand Journal of Agricultural Research 9, 529-535.

McGill W. B., Hunt W. H., Woodmansee R. G. and Reuss J. 0. (1981) PHOENIX: A model of the dynamics of carbon and nitrogen in grassland soils. In Terresfrial Nitrogen Cycles-Processes. Ecosystem Strategies and Management Impacts (F. E. Clark and T. Rosswall. Eds). Ecological Bulletin(Stockholm) 33, 49-115.

McKercher R. B. and Anderson G. (1968) Content of inositol penta- and hexaphosphates in some Canadian soils. Journal of Soil Science 19, 47-55.

Mueller-Harvey I., Juo A. S. R. and Wild A. (1985) Soil organic C, N, S and P after forest clearance in Nigeria: mineralization rates and spatial variability. Journal of Soil Science 36, 585-591.

Redgwell R. J. (1980) Fractionation of plant extracts using ion-exchange Sephadex. Analytical Biochemistry 107, 44-50.

Steward J. H. and Oades J. M. (1972) The determination of organic phosphorus in soils. Journal of Soil Science 23, 3849.

Tate K. R. (1979) Fractionation of soil organic phosphorus in two New Zealand soils by use of sodium borate. New’ Zealand Journal of Soil Science 22, 1377142.

TSAU (1980). Tropical Soils Analysis Unit, Land Resources Division, ODA, Coley Park, Reading, U.K. Methods used by TSAU and supplied to the authors.