Plant availability of organic phosphorus in low fertile Andosols Biodisponibilité du phosphore organique dans les andosols

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1 Scientific registration n : 1431 Symposium n : 13B Presentation : oral Plant availability of organic phosphorus in low fertile Andosols Biodisponibilité du phosphore organique dans les andosols OTANI Takashi, AE Noriharu National Institute of Agro-Environmental Sciences, Kannondai, Tsukuba, 305 Japan INTRODUCTION Andosols, developed on deposits of volcanic ash, are the major upland soils in Japan. In these soils, about 30 to 80 % of the total phosphorus (P) is present in organic forms. Soil organic P (Po) is usually quantified as its total amount released by ignition or alkaline extraction. There were various reports about the characterizing Po substrates in soils (Anderson 1961; Islam and Mandal 1977; Hong and Yamane 1981; Condron et al. 1985). However, the extent to which these Po compounds are meaningful in plant nutrition is uncertain. Various soil P tests, mainly extraction methods, are used to evaluate plant available P. Generally, only extractable inorganic P (Pi), and not Po, is considered in these methods. Bowman and Cole (1978) fractionated Po in a sandy loam soil, and pointed out that Po extracted by the Olsen method can be used in assessing Po contribution to plant P uptake. However, this method is scarcely used in Japan, and there are few attempt to quantify Po extracted with dilute acids used in the Truog and Bray II methods, which are commonly used for the evaluation of available P in Japan. Some crops [rice (Oryza sativa L.), pigeonpea (Cajanus cajan L.), and groundnut (Arachis hypogaea L.)] showed higher P uptake ability from low fertile Andosols than that from a Yellow soil, in spite of nearly the same amounts of extractable Pi by the Truog or Olsen methods in these soils (Otani and Ae, 1996). Therefore, a study about the possibility of Po as a P source for upland crops in Andosols is important. The objectives of this report were 1) to determine the amounts of extractable Pi and Po, respectively, by the soil P tests in Andosols and a Yellow soil, another major upland soil in Japan, at different fertilization levels, 2) to determine the amounts of Pi released by phosphatases from Po in the extracts, and 3) to discussed about the evaluation of Po availability in upland soils, especially in Andosols. MATERIALS AND METHODS Soils. Soils were sampled from four sites with three treatment plots (uncultivated land, abbreviated as "Uc" ; only chemical fertilizer applied, "Cf"; and organic matter applied besides chemical fertilizer, "Org". The Tsukuba soil ("T" a Haplic Andosol; Hydric 1

2 Hapludand) was collected from uncultivated land (T-Uc), and from a field with upland rice (T-Cf, T-Org) of the National Agriculture Research Center (NARC), Tsukuba, Ibaraki Pref. The Utsunomiy a soil ("U", a Cumulic Andosol; Hydric Pachic Melanudand) was collected from uncultivated land (U-Uc), and from a field with soybean-barley (U-Cf, U-Org) at Tochigi Agriculture Experimental Station, Utsunomiya, Tochigi Pref. The Naruko soil ("N", a Cumulic Non-Allophanic Andosol; Alic Pachic Melanudand) was collected from uncultivated land (N-Uc), a field with maize (N-Cf), and a field with garlic (N-Org) at Tohoku University Farm, Naruko, Miyagi Pref. The Ishigaki soil ("I", a Terrace Yellow soil; Typic Hapludult) was collected from uncultivated land (I-Uc), a pasture (I-Cf), and a green manure field (I- Org) at the Okinawa Subtropical Station of Japan International Research Center for Agricultural Sciences, Ishigaki, Okinawa Pref. The soils were classified according to the criteria adopted by the Classification Committee of Cultivated Soils (1996), and Soil Survey Staff (1992). The chemical fertilizer plots were treated with recommended amounts of P for actual crops as an N-P-K compound fertilizer. The organic matter plots were amended with 17 t ha-1 of dried cow dung in T-Org, 30 t ha-1 of cow dung manure in U-Org, N-Org, and green manure, an estimated 6 t DM ha-1 of crotalaria in I- org, per year for at least three years ( ), besides the chemical fertilizer. The soil samples were collected from the surface layer (0-20 cm). Plant debris were removed and the samples were air-dried, passed through a 2-mm sieve, andground in a mortar. Quantification of extractable Pi and Po by soil P tests. The extraction procedures according to Truog (Truog 1930), modified Bray II (soil:solution ratio, 1:20; shaking time, 60 s ; Shoji et al. 1964), Olsen (Olsen et al. 1954) and citrate extraction (Simard et al. 1991) were used to the soil samples. Each extract was filtered through a 0.20 µm membrane filter, and a part of the filtrate was digested with HNO3-HClO4 for determination of total extractable P (Pt). The method of Murphy and Riley (1962) was used to measure theorthophosphate concentration in the neutralized extract (Pi), and in the neutralized digest of the extract (Pt). The amount of Po in each extract was calculated by subtracting Pi from Pt. Determination of Pi hydrolyzed by the phosphatases from Po in the soil extracts. Three kinds of phosphatases, acid phosphatase (EC : Type I, from wheat germ), phytase (EC : crude, from wheat), and alkaline phosphatase [EC : Type III, bacterial (Eschericia coli)] (Sigma Chemical Company) were used. The phytase was dialyzed 100 times more using a macrosolute concentrator (Amicon, Inc.) for removing contaminant orthophosphate. The extracts of uncultivated Utsunomiya and Naruko soils (U-Uc, N-Uc) were used to determine Pi hydrolyzed by the enzymes from Po in the soil extracts. Each soil extract solution by the method of Truog [ten times concentrated of the usual concentration, abbreviated as "Truog (x10)], modified Bray II ("Bray II), Olsen, or citrate extraction ("Citrate") methods, each enzyme solution, and the appropriate buffer solution [acid phosphatase, phytase:2 M acetate buffer (ph 5.0); alkaline phosphatase: 0.5 M glycine/sodium hydroxide buffer (ph 9.6)] were mixed. It was necessary to add adequate amounts of sulfuric acid (reaction system of acid phosphatase or phytase, with Olsen solution), or sodium hydroxide [reaction system of alkaline phosphatase, with Truog (x10), Bray II, and Citrate solution] to keep the optimal ph. After adjusting the 2

3 total volume to 5 ml by adding distilled water, each mixed solution was allowed to react at 37 C (acid phosphatase and alkaline phosphatase) or 55 C (phytase) for 8 hours. After the reaction time, each solution was added 0.05 g of active carbon (powder), passed through a 0.20 µm membrane filter,and the amount of released orthophosphate was measured immediately by the method of Murphy and Riley (1962). RESULTS AND DISCUSSION Amounts of extractable Pi and Po by the different soil P tests Extractable Pi from the soils in uncultivated land (T-, U-, K-, I-Uc) were very low by all the extraction methods (Truog, Bray II, Olsen, and Citrate), and increased remarkably by addition of chemical fertilizer or organic matter (Table 1). The amount of extractable Po was nearly the same as Pi extracted in most Uc soils, but much higher for the Olsen and Citrate extractable Po in U-Uc and N-Uc. The amounts of extractable Po did not increase by fertilization for all the methods. The separate extraction in Pi and Po forms showed that only the amounts of Pi extracted by each method increased by fertilization, while that of Po extracted did not increase even after addition of organic matter. Therefore, the evaluation of Po as available P is scarcely needed in usual upland fields, because the P availability is mainly defined by the status of Pi. On the other hand, the ratio of extractable Po / total extractable P (Pi + Po) were higher in the uncultivated soils than in the fertilized soils. Especially, the amounts of Olsen-, and Citrate-Po in the uncultivated Andosols of U-Uc and N-Uc soils, in which total C and organic P contents were high (Otani and Ae, 1997), were much more than that in the T- Uc and I-Uc soils. Extractable Pi by the different soil P tests is usually considered to indicate the P availability in the soils, that is, the amounts of labile or easily soluble Pi accessible for plants. Therefore, extractable Po may also be worth to consider as "available" (Jfor plants, especially in uncultivated land of high humic Andosols. At least in part, the Po can be regarded as the form which has the chance to exist in a soluble form in the soil solution. Phosphatases, like acid phosphatase, phytase, and alkaline phosphatase, produced by plant roots or soil microorganisms are present in the rhizosphere soils (Jungk 1991). Therefore, the Po extracted will be plant available, in the case that the Po is mineralized in the soils by these enzymes, since plants cannot use organic P forms. Pi hydrolyzed by the enzymes from Po in the soil extracts The amounts of orthophosphate released by the phosphatases in the extracts of U-Uc and N-Uc soils are shown in Table 2. The Truog or Bray II methods extracted only small amounts of Po, and the amounts of Pi hydrolyzed by the enzymes were small. The Olsen and Citrate methods extracted much Po. Citrate-Po was hydrolyzed to % by phytase, or 12-13% by acid phosphatase, while Olsen-Po was not hydrolyzed by any of the phosphatases. This result suggests that the properties, that is, the chemical structures or molecular sizes, are quite different between the Po extracted by these methods. In the Citrate-Po, nucleotides or sugar phosphates may be hydrolyzed by acid phosphatase, and in addition, phytates seemed to be hydrolyzed partly by phytase (Nagai and Funakoshi 1962). The Olsen method was the sole alkaline extraction method (ph 8.5), and the Olsen extracts from the Andosols were more black compared to the extracts by the other methods. This may indicate that a large amounts of organic 3

4 matter in the soils was decomposed and extracted from humic polymers with the Olsen method due to alkaline hydrolyzation. In spite of high amounts, Olsen-Po could not be hydrolyzed by any of the tested enzymes. Therefore, Olsen-Po seemed to extract mainly the hardly hydrolyzable forms, like those in corporated with fulvic, and/or humic acids, or lipid P, which are the main constituents of soil organic P next to phytates (Daral 1977). Evaluation of soil Po for plant availability Only the amounts of Pi extracted by the soil P tests, which were very low in the uncultivated soils, were usually used for the evaluation of the soil P availability. However, it is expected that certain amounts of Citrate-Po (U-Uc, N-Uc; 7, and 7 mg P kg-1 soil, respectively, calculated from Table 1 and 2) were hydrolyzed by phytase. With the hypothesis of enough phytase activity in soils, the amounts of available P by the Citrate method may be twice (U-Uc), or 8 times more compared to the usual manner when considering only the amounts of Pi extracted. Therefore, the Citrate-Po may be evaluated as plant available P to a certain extent, and important especially in the high humic Andosols with low fertility. It is also important to note that only the Citrate-Po could be hydrolyzed by the phosphatases to orthophosphate. It was pointed out that Po in the rhizosphere may be mineralized by phosphatases released from plant roots (Caradus et al. 1987; Tadano et al. 1991) or microorganisms (Bishop et al. 1994). However, the Po probably must be present as the released form in the soil solution for hydrolyzation by the enzymes. For example, phytates as calcium or magnesium salts was hydrolyzed by phytase, but iron or aluminum salt with low solubility was not (Jackman and Black 1951). In our study, most of Po accumulated in Andosols was bonded with Al and Fe (Otani and Ae, 1997), and the forms may be difficult to react with soil phosphatases. However, Po seems to be the form attacked by the enzymes, if released from Al and Fe by Citrate extraction, with high ability to dissolve Al- and Fe-bound P (Otani et al. 1996). Tarafdar and Claassen (1988) pointed out that phosphatase activity is not the limiting factor in the use of Po but rather the availability of phosphatase hydrolysable P compounds. In our study, released Po from Al and Fe (Citrate-Po) was hydrolyzed by the enzyme. Therefore, the most important strategy to enhance the availability of Po in Andosols may be, how to release Po, as well as Pi, from Al- and Fe-bound forms. REFERENCES Anderson G (1961) A partial fractionation of alkali-soluble soil organic phosphorus. J Soil Sci 12: Bishop ML, Chang AC, Lee EWK (1994) Enzymatic mineralization of organic phosphorus in a volcanic soil in Chile. Soil Sci 157: Bowman RA, Cole CV (1978) Transformations of organic phosphorus substrates in soils as evaluated by NaHCO3 extraction. Soil Sci 125:49-54 Caradus JR, Snaydon RW (1987) Aspects of phosphorus nutrition of white clover population. II. Root exocellular phosphatase activity. J Plant Physiol 10: Classification Committee of Cultivated soils (1996) Classification of Cultivated Soils in Japan, 3rd Approximation (English version) Misc Publ Natl Inst Agro-Environ Sci No. 17, National Institute of Agro-Environmental Sciences, Tsukuba, Japan, pp

5 Condron LM, Goh KM, Newman RH (1985) Nature and distribution of soil phosphorus as revealed by a sequential extraction method followed by 31P nuclear magnetic resonance analysis. J Soil Sci 36: Dalal RC (1977) Soil organic phosphorus. Adv Agron 29: Hong JK, Yamane I (1981) Distribution of inositol phosphate in the molecular size fractions of humic and fulvic acid fractions. Soil Sci Plant Nutr 27: Islam A, Mandel R (1977) Amounts and mineralization of organic phosphorus compounds and derivatives in some surface soil of Bangladesh. Geoderma 17:57-68 Jackman RH, Black CA (1951) Hydrolysis of iron- aluminum, and magnesium inositol phosphates by phytase at different ph values. Soil Sci 72: Jungk A (1991) Dynamics of nutrient movement at the soil-root interface. In: Waisel Y, Eshel A, Kafkafi U (eds) Plant Root, The Hidden Half. Marcel Dekker, Inc., New York, USA, pp Murphy J, Riley JP (1962) A modified single solution method for the determination of phosphorus in natural waters. Anal Chem Acta 27:31-36 Nagai Y, Funahashi S (1962) Phytase (myoinositolhexaphosphate phosphohydrolase) from wheat bran. Agr Biol Chem 26: Olsen SR, Cole CV, Watanabe FS, Dean LA (1954) Estimation of available phosphorus in soils by extraction with sodium bicarbonate. U.S. Dept Agr Circ 939:1-19 Otani T, Ae N (1996) Phosphorus (P) uptake mechanisms of crops grown in soils with low P status. I. Screening of crops for efficient P uptake. Soil SciPlant Nutr 42: Otani T, Ae N, Tanaka H (1996) Phosphorus (P) uptake mechanisms of crops grown in soils with low P status. II. Significance of organic acids in root exudates of pigeonpea. Soil Sci Plant Nutr 42: Otani T, Ae N (1997) The status of inorganic and organic phosphorus in some soils in relation to plant availability. Soil Sci Plant Nutr 43: Shoji S, Miyake M, Takeuchi Y (1964) Comparison of methods of available soil phosphorus determination. II. Correlation between soil tests and "A"values. Res Bull Hokkaido Natl Agric Exp Stn 84: (in Japanese with English summary) Simard RR, Tran TS, Zizka J (1991) Strontium chloride-citric acid extraction evaluated as a soil-testing procedure for phosphorus. Soil Sci Soc Am J 55: Soil Survey Staff (1992) Keys to Soil Taxonomy, 5th edn. SMSS Technical Monograph, No. 19, Pocahontas Press Inc., Blacksburg, Virginia, USA, pp Tadano T, Sakai H (1991) Secretion of acid phosphatase by the roots of several crop species under phosphorus-deficient conditions. Soil Sci Plant Nutr 37: Tarafdar JC, Claassen N (1988) Organic phosphorus compounds as a phosphorus source for higher plants through the activity of phosphatases produced by plant roots and microorganisms. Biol Fertil Soils 5: Truog E (1930) Determination of readily available phosphorus of soils. J Am Soc Agron 22: Keywords : Andosol, organic phosphorus, phosphatase, phosphorus availability Mots clés : andodol, phosporeorganique, phosphatase, biodisponibilité 5

6 Table 1. The amounts of inorganic and organic P in the soils extraceted by the four soil P test methods Truog Bray II Olsen Citrate Soil Pi Po Pi Po Pi Po Pi Po -- mg P kg T a -Uc b T-Cf T-Org U-Uc U-Cf U-Org N-Uc N-Cf N-Org I-Uc I-Cf I-Org a T, Tsukuba soil; U, Utsunomiya soil; N, Naruko soil; I, Ishigaki soil b Uc, uncultivated; Cf, addition of chemical fertilizer; Org, addition of organic matter 6