Short Paper Soil Sci. Plant Nuti:, 51 (3), 425-430, 2005 425

Extraction of Soil Organic Nitrogen by Organic Acids and Role in Mineralization of Nitrogen in Soil

Shingo Matsumoto, Noriharu Ae*, and Toshikazu Matsumoto**

Education and Research Center for Biological Resources, Faculty of Life and Environmental Sciences, Shimane University, Matsue, 690-1 102 Japan; *Faculty of Agriculture, Kobe University, Kobe, 657-8501 Japan;

and **Shimane Agricultural Experimental Station, Izumo, 693-0035 Japan

Received October 5, 2004; accepted in revised form February 10,2005

The solubilization of organic nitrogen (Norg) in soil was examined by using organic acids often present in root exudates. The organic acids tested were classified into two groups depending on their solubilizing ability for Norg in a soil sample. Oxalate, malonate, t a ~ tarate, citrate and malate could dissolve Norg by increasing their concentrations, while suc- cinate and glutarate could not dissolve Norg even when their concentrations were increased. The amount of organic N extracted with oxalate, malonate, tartarate, citrate and malate showed a strong correlation with the sum of the amounts of Al and Fe in the extracts. Therefore, it was assumed that the solubilizing ability may be attributed to the structure of these organic acids that can form stable chelate compounds with Al and Fe (oxalate, malonate, tartarate, citrate and malate). Furthermore, the amount of 20 m~ cit- rate-extractable Norg (CEON), which was the highest among the organic acids tested, showed a strong correlation with both that of 67 m~ phosphate buffer, which is now being widely used as an index for estimating soil N availability in Japan, and N mineralized using an incubation method in 46 soil samples belonging to different soil types. These results sug- gested that CEON might be an important constituent of mineralizable Norg in soil. There- fore, organic acids secreted by crops play a role in the solubilization of available Norg surrounding the crop rhizosphere.

Key Words: citrate, extractable organic nitrogen, mineralizable organic nitrogen, organic acids, root exudates.

Several chemical extraction methods for mineraliz- able soil organic nitrogen (Norg) have been proposed for assessing N availability. These methods are substitutes for the incubation method, which cannot be easily adopted for routine analysis because it is time-consum- ing. Extraction by various solutions has recently become increasingly important because the amount of extract- able Norg shows a good agreement with net N mineral- ization in the incubation method and N uptake by crops (Appel and Steffens 1988; Appel and Mengel 1992, 1993; Groot and Houba 1995). The difference in the amount of extractable Norg among the extractants or soil types may depend on the degree of fixing ability of the soil surface and affinity to soil particles (Appel and Mengel 1998). Therefore, findings about the binding status of extractable Norg in soil might be important for the estimation of the amount of mineralizable Norg. Miltner and Zech (1999) reported that iron (Fe) oxide and aluminum (Al) hydroxide stabilized plant-derived proteins in soil. Ae et al. (1980) showed that the miner-

alization rate of available Norg in Andosols was the low- est among the soils tested, while Andosols contained a much larger amount of Norg than other soils. Ae et al. assumed that the significantly higher levels of Al and Fe in Andosols delayed mineralization due to their strong fixation to available Norg. Ito and Ae (2000) determined the binding minerals to extractable Norg based on the relationship between Norg and metallic ions in several soil samples extracted by chemical solutions such as diluted sulfuric acid. The concentrations of Fe and A1 in the extract increased with higher levels of extractable Norg in the soil, while the concentrations of other metal- lic ions (Ca, Na, K and Mg) were not related to the Norg concentrations. These results suggest that the binding status of mineralizable Norg in soil might be closely related to the behavior of A1 and Fe in soil. It is also well known that most phosphorus (P) in soils is strongly fixed to A1 and Fe. These forms of P are not available to most crops, and P deficiency in soils is the main limiting factor for crop production in developing countries

426 S. MATSUMOTO, N. AE, and T. MATSUMOTO

(Johansen et al. 1994). On the other hand, it has been reported that the unavailable P fixed to Al and Fe is released by root exudates, including some organic acids, and the solubility of Al- or Fe-bound P in soil by using organic acids has been studied extensively (Romheld and Marschner 1983; Lipton et al. 1987; Hoffland et al. 1989; Otani and Ae 1997). However, similar studies have not been conducted for mineralizable Norg, even though it also seems to be related to the behavior of A1 and Fe in soil. In the present study, we determined whether Norg fixed in the soil surface is released by some organic acids, and we considered the role of organic acid-extractable Norg in the mineralization of soil N by comparing the amount of N mineralized using the incubation method and extraction by the 67 mM neu- tral phosphate buffer, which is the standard extraction method in Japan.

Materials and methods Characteristics of the soil samples tested.

The soil samples used which belonged to different soil types were collected at 46 sites in Shimane Prefecture, Japan. Soil texture was determined by the pipet method (Gee and Bauder 1997) after sieving through a 2-mm mesh shieve. The amounts of total organic carbon and total N were determined using an automatic NC analyz- er (Model NC-80, Sumitomo Chemicals Co., Ltd., Tokyo, Japan). The chemical characteristics of the soils are shown in Table 1.

Extraction of Norg, Al and Fe in soil with several organic acids. To test the solubilization of Norg by various organic acids, soil No. 9, 20 and 27 shown in Table 1 were utilized. A 10-g soil sample with 50ml of a 1-, 5-, 10- or 20-mM organic acid solution comprised of oxalate, malonate, malate, citrate, succi- nate, tartarate and glutarate was shaken for 1 h. The soil extracts were passed through No. 6 filter paper (Advan- tec Toyo Co., Ltd., Tokyo, Japan), and the inorganic N concentration was measured by the steam distillation method with MgO. A 20-ml portion of the extract was digested by 10 ml of 36 M H2S0, in a Kjeltec auto- digestion system (Tecator Co., Ltd., Hoganas, Sweden), and the total N concentration in the extract was mea- sured by the steam distillation method with 10 M NaOH. The amount of organic acid-extractable Norg was deter- mined by subtracting the amount of inorganic-N from that of total N. The concentrations of Al and Fe in the extracts with organic acids were determined by sequen- tial plasma spectrometry (ICPS-8 100, Shimadzu, Co., Ltd., Tokyo, Japan).

Comparison with citrate-extractable Norg, phosphate-buffer extractable Norg and N min- eralized in the incubation method. For N miner- alization, 10 g of dry soil in a 100-ml plastic bottle was

Table 1. Characteristics of the soils studied.

soil type"

-

Textural Total C Total N Mineralized N class (g kg-') (g kg-') (mg kg-I) number

1 2 3 4 5 6 7 8 9

10 1 1 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Acrisols Clay loam Acrisols Clay loam Acrisols Light clay Acrisols Light clay Acrisols Light clay Acrisols Light clay Andosols Loam Andosols Loam Andosols Loam Andosols Loam Cambisols Loam Cambisols Loam Cambisols Loam Cambisols Loam Cambisols Loam Cambisols Loam Cambisols Loam Cambisols Loam Fluvisols Loam Fluvisols Loam Fluvisols Loam Fluvisols Loam Fluvisols Loam Fluvisols Loam Fluvisols Clay loam Fluvisols Clay loam Fluvisols Loam Fluvisols Loam Fluvisols Loam Fluvisols Loam Gleysols Clay loam Gleysols Clay loam Gleysols Clay loam Gleysols Clay loam Gleysols Clay loam Gleysols Clay loam Gleysols Clay loam Gleysols Clay loam Gleysols Clay loam Gleysols Clay loam Gleysols Clay loam Gleysols Clay loam Gleysols Loam Gleysols Loam Gleysols Loam Gleysols Loam

15.7 12.2 23.1 21.5 11.2 4.3

107.1 83.2

127.2 64.0

8.8 7.3

10.9 19.2 8.8 3.6 2.8 5.7

11.6 22.3 18.8 10.5 5.6

16.5 41.9 34.2 29.4 23.7 49.5 23.0 29.6 39.7 24.1 26.3 18.2 15.0 30.0 23.3 26.3 17.5 15.3 26.6 10.3 25.7 6.5

~~ ~

1.3 1 .o 1.8 1.6 0.8 0.4 5.2 5.0 5.5 4.2 0.7 0.6 0.9 1.4 1 .o 0.3 0.2 0.6 I .o 1.8 1.6 0.9 0.5 1.3 3.5 2.1 2.9 2.0 4.5 1.9 2.8 3.8 1.8 2.6 2.0 1.2 2.7 1.7 2.3 1.5 1.6 2.2 0.8 2.2 0.6

10.1 0.8

57.8 39.6

108.2 87.2 21.0 6.7

41.7 102.6 12.6 28.0 23. I 14.7 24.5

122.2 23.5 7.4 0.7 3.2

48.7 87.5

107.5 31.9 3.5

47.3 189.7 57.1

202.3 148.8 319.6 106.1 108.5 122.2 122.5 64.4 40.3 41.0

152.3 114.1 140.7 23.1 23.5

140.4 35.0

130.9 6.6

23.8 ~~

"The soils were classified according to the recent FA0 soil clas- sification system (FA0 1988).

moistened at 60% of the maximum water-holding capacity. These bottles were covered with a permeable polyethylene film (JIS 2/100), and the soils were incu- bated at 30°C for 28 d. After incubation, the inorganic N of a soil was extracted with 2 M KCI and the content was determined by the steam distillation method with MgO and Devarda alloy.

Extraction of Soil Organic Nitrogen by Organic Acids 427

For extractable Norg, a 10-g soil sample with 50 ml of each 67 mM phosphate buffer (pH 7.0) and 20 mM cit- rate solution was shaken for 1 h. The extraction proce- dure and determination of the amount of Norg were performed in the same way as those described above.

Results and discussion Difference in Norg solubilizing ability among

the organic acids tested The ability of several organic acids to release free

Norg from its fixed form in a soil was compared (Fig. 1). The amounts of Norg extracted with glutarate and succinate did not increase when we increased their con- centrations. On the other hand, the amounts extracted

90 80

0 70 3 0 - 60 %>50 Q 40 k.5 b 30 a 20

10 0

90 80

-J 70 s [<;: E z 4 0 b 30 a 20

10 0

with oxalate, malonate, tartarate, citrate and malate increased when we increased their concentrations. In the latter organic acids at a concentration of 20mM, the order of the amount of extractable Norg in 3 kinds of soils tested corresponded to the amount of mineralized N in the incubation method (see Table 1). The amount of Norg extracted by citrate was the highest among the organic acids. The amounts of A1 and Fe in the extracts with organic acids showed a similar tendency to that of Norg extracted (Fig. l), and the sum of the amounts of A1 and Fe extracted with each oxalate, malonate, tart- arate, citrate and malate showed a strong correlation with the amount of Norg extracted in the three kinds of soils tested (Fig. 2). However other metallic ions, i.e. K,

1 10 20 1 10 20 1 10 20 1 10 20 1 10 20 1 10 20

Oxalate 7 90

80 3 70

10 0 0

alonate r 3 f 9

2 % A: 1 10 20 1 10 20 1 10 20 1 10 20 1 10 20 1 10 20

90 80 [Tartarate O A l

D F e d 6

LU I IU LU 1 10 20 1 10 20 1 10 20

Concentration of organic acid

1 10 20 1 10 20 1 10 20

Concentration of organic acid

Fig. 1. Effect of organic acids on Norg, Al and Fe release from soils. *The numeral corresponds to the soil number listed in Table 1 .

428

4 s f . 2

.g 5 0 e 1 -

S. MATSUMOTO, N. AE, and T. MATSUMOTO

- A/ 0 Tartarate

Y=0.083X+0.044 r2=0 .924* * *

0

0 Malate A Malonete A Oxalate 0 Tartarate

Y=O.O27X+O. 13 1 r*=O. 887 * * *

9*

i

0 20 40 60 80 loo

Sum of Al and Fe extracted (mM k-’)

0 Ciirate g 10 Malate g - A Malonate ”

20 2 0 ’ 0 10 20 30 40 50

Sum of Al and Fe extracted (mM k-’) l r

27

Y=O. 124X-0.208 r2=0.893 ***

0 0 10 20 30 40 50

Sum of Al and Fe extracted (mM kg-’)

Fig. 2. Relationship between the amount of Norg and the sum of the amounts of A1 and Fe extracted with oxalate, mal- onate, tartarate, citrate and malate from soils. ***p<O.OOl. *The numeral corresponds to the soil number listed in Table 1.

Ca, Mg, Na and Mn did not show any significant corre- lation with the amount of Norg extracted (data not shown). In the present study, the organic acids tested were classified into two groups depending on the solubi- lizing ability for Norg in soil. That is, oxalate, malonate, tartarate, citrate and malate could dissolve Norg if their concentrations increased, while succinate and glutarate could not dissolve Norg even when their concentrations increased. If Norg is assumed to bind to Al or Fe in soil as considered above, these different solubilizing abilities of organic acids may be ascribed to the molecular struc- ture of each acid. The structure of organic acids (oxalate, malonate, tartarate, citrate and malate) is suit- able for the formation of stable 5- or 6-membered che- late rings with Fe or A1 (Hue et al. 1986) by carboxyl- and/or hydroxyl-groups. This suggests that the organic acids may be able to release Norg by forming stable chelating rings with Fe and Al. Actually, citrate, which showed the highest stability constant value for A1 and Fe among the organic acids tested (Kidd et al. 2001;

3 6 0 - 3

4 0 -

3 5 2 0 -

Y = 0 .5074~ + 11.1 94 r * = 0.7279-

0 I..@ I I

0 50 100 150 200

Phosphate bufferextractable Norg (mg kg-’)

Fig. 3. and 67 mM phosphate buffer-extractable Norg. ***p<O.OOI.

Relationship between 20 mM citrate-extractable Norg

Ochoa-Loza et al. 2001 ), displayed the highest solubiliz- ing ability for Norg. However, the amount of Norg extracted with oxalate, malonate, tartarate and malate did not necessarily correspond to the order of the stabil- ity constant value for A1 and Fe. Therefore, further stud- ies should be carried out to determine the binding status of Al, Fe and Norg in soil.

Relationship between the amount of extract- able Norg and the amount of N mineralized in the incubation method

To confirm the roll of the organic acid-extractable Norg in the mineralization of soil Norg, we compared the extraction of citrate (20 mM), which showed the highest solubilizing ability for Norg among the organic acids tested, with that of neutral 67 mM phosphate buff- er, which is widely used as an index for estimating soil N availability in Japan (Ogawa et al. 1989; Yanai et al. 1998), using an incubation method for 46 soil samples belonging to different soil types. The amount of citrate- extractable Norg (CEON) showed a strong correlation with both that of 67 mM phosphate buffer (Fig. 3) and N mineralized using the incubation method (Fig. 4). These results suggested that CEON might be an important con- stituent of mineralizable Norg in soil. On the other hand, the amount of CEON tended to be lower than that of PEON, as the amount of mineralized N increased with the incubation method (Fig. 4). The characteristics and origin of extractable Norg in soil have been investigated by many researchers, and the major constituent is con- sidered to consist of protein-like substances derived from the debris of soil microorganisms (Nemeth et al. 1988; Nunan et al. 1998; Mengel et al. 1999; Matsu- mot0 et al. 2000). Furthermore, the extractable Norg showed a very similar amino acid composition, C to N ratio (Higuchi 1982; Senwo and Tabatabai 1998; Ogi- uchi et al. 2000) and molecular weight (Wenzl 1990;

Extraction of Soil Organic Nitrogen by Organic Acids 429

200 - 0

Y = 0.4956~ + 2.2182 r = 0.8099- -

0 PEON 0 CEON

h

' ~ 1 5 0 - x

z 0 v

eD r / 2 100 /.-/ 0 0 //-- z

Y = 0.2836~ + 2.01 r = 0.7498*

3

50 W

I 1 I

0 50 100 150 200 250 300 3501

Mineralized-N in incubation method (mg kg-')

Fig. 4. Relationship between 20 mM citrate-extractable Norg (CEON), 67 mM phosphate buffer-extractable Norg (PEON) and N-mineralized in incubation method. ***p<O.OOl.

Matsumoto et al. 2000), irrespective of the kinds of test- ed extracting solutions and soil types. Matsumoto and Ae (2004) pointed out that the characteristics of Norg extracted with mild extractants (e.g., phosphate, CaCl,, diluted acids, etc.) corresponded to homogenous com- pounds, while strong extraction methods such as acid hydrolysis could extract available Norg and also recalci- trant Norg which does not contribute to the N mineral- ization and N uptake by crops. Therefore, the difference between the amount of CEON and PEON may depend on the extractability and selectivity of the extractants, while the molarity (appropriate concentration) of citrate must be considered. Further studies on the binding mechanism of available Norg and soil particles should be conducted to elucidate the relationship between the extraction procedure of Norg with chemical solutions and the mineralization of Norg in soil.

Role of organic acid-extractable Norg in N uptake by crops

In the present study, we observed that some organic acids with stable 5- or 6-membered chelate rings could dissolve Norg in relation to mineralization in soil. It is generally recognized that these organic acids are secret- ed by some specific crops and that the unavailable P bound to A1 and Fe is taken up by the crops through the dissolution of A1 and Fe by the secretion of the organic acids (Lipton et al. 1987; Ae et al. 1990 Otani and Ae 1997). On the other hand, it has been reported that the amounts of N taken up by some crops exceeded the amount of mineralized N during cultivation (Chapin et al. 1993; Yamagata et al. 1997; Nasholm et al. 1998; Mengel et al. 1999; Okamoto et al. 2003). In these cases, release of the fixed mineralizable Norg from the soil colloid surface would be necessary as an initial step of the findings. It has been reported that net N mineraliza-

tion differs considerably among fallow soils, soils culti- vated with grass and non-fertilized soils, and soils cultivated with grass and N fertilizer (Mengel et al. 1999). These findings may also be attributed to the action of roots on the available Norg. Therefore, further studies should be conducted to determine the influence of organic acids secreted by crops on the dynamics of available Norg in the crop rhizosphere.

REFERENCES

Ae N, Arihara J, Okada K, Yoshihara T, and Johansen C 1990: Phosphorus uptake by pigeonpea and its role in cropping system of Indian subcontinent. Science, 248, 477-480

Ae N, Ohyama N, and Nishi H 1980: Evaluation of the rice pro- ductivity on some kind of main soil types of paddy field in Chugoku district by lysimeter experiment. Bull. Chugoku Natl. Agric. Exp. Stn., E17, 57-74 (in Japanese with English summary)

Appel T and Mengel K 1992: Nitrogen uptake of cereals on sandy soils as related to nitrogen fertilizer application and soil nitrogen fractions obtained by electroultrafiltration (EUF) and CaCl, extraction. Eur. J . Agron., 1, 1-9

Appel T and Mengel K 1993: Nitrogen fractions in sandy soils in relation to plant uptake and organic matter incorporation. Soil Biol. Biochem., 26, 685-691

Appel T and Mengel K 1998: Prediction of mineralizable nitro- gen in soils on the basis of an analysis of extractable organic N. Z. Pjlanzenernahr. Bodenk., 161,433-452

Appel T and Steffens D 1988: Vergleich von Elektro-Ultrafiltra- tion (EUF) und Extraktion mit 0.01 molarer CaC1,-Losung zur Bestimmung des pflanzenverfugbaren Stickstoffs im Boden. Z. Pjlanzenernahr. Bodenk., 161, 127- 130

Chapin FS, Moilanen L, and Kielland K 1993. Preferential use of organic nitrogen for growth by a non-mycorrhizal arctic sedge. Nature, 361, 150-153

FA0 1988: Soil Map of the World, Revised Legend. World Soil Resource Report 60, FAO, Rome

Gee GW and Bauder JW 1997: Particle-size analysis. In Meth- ods of Soil Analysis, Part 1, Ed. A Klute, p. 383-41 I , American Society of Agronomy, Wisconsin

Groot JJR and Houba VJG 1995: A comparison of different indices for nitrogen mineralization. Biol. Fertil. Soils, 19,

Higuchi M 1982: Characterization of soil organic nitrogen extracted with phosphate buffer. Jpn. J . Soil Sci. Plant Nutr., 63, 1-5 (in Japanese)

Hoffland E, Findenegg GR, and Nelemans 1989: Solubilization of rock phosphate by rape. Local root exudation by organic acids as a response to P-starvation. Plant Soil, 113, 161- 165

Hue NV, Graddock GR, and Adams F 1986: Effect of organic acids on aluminum toxicity in subsoil. Soil. Sci. SOC. Am. J. ,

Ito C and Ae N 2000: Inference of form of available nitrogen in soils extracted by chemical solutions. Jpn. J . Soil Sci. Plant Nutr., 71, 777-785 (in Japanese with English summary)

Johansen C, Subbarao GV, Lee KK, and Sharma KK 1994:

1-9

60, 28-34

430 S. MATSUMOTO, N. AE, and T. MATSUMOTO

Genetic manipulation of crop plants to enhance integrated nutrient management in cropping system-The case of phosphorus. In Proc. FAO-ICRISAT Expert Consultancy Workshop, Ed. C Johansen et al., p. 9-29, Patancheru, India

Kidd PS, Llgany M, Poschenrieder, C, Gunse B, and Barcelo J 2001: The role of root exudates in aluminum resistance and silicon-induced amelioration of aluminium toxicity in three varieties of maize (Zea mays L.). J. Exp. Botany, 52, 1339- 1352

Lipton DS, Blanchar RW, and Blevins DG 1987: Citrate, malate and succinate concentration in exudates from P-sufficient and P-stressed Medicago sativa L. seedlings. Plant Physiol.,

Matsumoto S and Ae N 2004: Characteristics of extractable soil organic nitrogen determined by using various chemical solu- tions and its significance for nitrogen uptake by crops. Soil Sci. Plant Nutr:, 50, 1-9

Matsumoto S, Ae N, and Yamagata M 2000: The status and ori- gin of available nitrogen in soils. Soil Sci. Plant Nutr., 46,

Mengel K, Schneider B, and Kosegarten H 1999: Nitrogen com- pounds extracted by electroultrafiltration (EUF) or CaCl, solution and their relationships to nitrogen mineralization in soils. J. Plant Nutr. Soil Sci., 162, 139- 148

Miltner A and Zech W 1999: Microbial degradation and resyn- thesis of proteins during incubation of beech leaf litter in the presence of mineral phases. Biol. Fertil. Soils, 30, 48-5 1

Nasholm T, Ekblad A, Nordin A, Giesler R, Hogberg M, and Hogberg P 1998: Boreal forest plants take up organic nitro- gen. Nature, 392,914-916

Nemeth K, Barlels H, Vogel M, and Mengel K 1988: Organic nitrogen compounds extracted from arable and forest soils by electro-ultrafiltration and recovery rates of amino acids. Biol. Fertil. Soils, 5 , 27 1-275

Nunan N, Morgan MA, and Herlihy M 1998: Ultraviolet absor- bance (280nm) of compounds release from soil during

86,315-317

139-149

chloroform fumigation as an estimate of the microbial bio- mass. Soil Biol. Biochem., 30, 1599-1603

Ochoa-Loza FJ, Artiola JF, and Maier RM 2001: Stability con- stants for the complexation of various metals with a rham- nolipid biosurfactant. J . Environ. Qual., 30, 479-485

Ogawa Y, Kato H, and Ishikawa M 1989: A simple analytical method for index of soil nitrogen availability by extracting in phosphate buffer solution. Jpn. J . Soil Sci. Plant Nutr., 60, 160-163 (in Japanese)

Ogiuchi K, Nakajima N, Ae N, and Matsumoto S 2000: Amino acid composition of soil organic nitrogen extracted with phosphate buffer solution. Jpn. J . Soil Sci. Plant Nutr., 71, 385-387 (in Japanese)

Okamoto M, Okada K, and Ae N 2003: Growth responses of cereal crops to organic nitrogen in the field. Soil Sci. Plant Nutr., 49,445-458

Otani T and Ae N 1997 The status of inorganic and organic phosphorus in some soils in relation to plant availability. Soil Sci. Plant Nutr., 43, 4 19-429

Romheld V and Marschner H 1983: Mechanisms of iron uptake by peanut plants. I. Fe 111 reduction, chelate splitting and release of phenolics. Plant Physiol., 71, 949-954

Senwo ZN and Tabatabai MA 1998: Amino acid composition of soil organic matter. Biol. Fertil. Soils, 26, 235-242

Wenzl W 1990: Extraktion und Charakterisierung von stickstoff- reichen Nichthuminsoffen einer Braunerde. VDLUFA- Schrifenreihe, 32, 337-344

Yamagata M, Nakagawa K, and Ae N 1997: Differences among crops in nitrogen uptake from rice bran using the I5N tracer technique. Jpn. J . Soil Sci. Plant Nutr., 68, 291-294 (in Japanese with English summary)

Yanai M, Uwasawa M, Konno T, and Shimizu Y 1998: Extract- ing condition of soil nitrogen with phosphate-buffered solu- tion in connection with extractable soil. Jpn. J . Soil Sci. Plant Nutr., 69, 355-364 (in Japanese with English sum- mary)