The Potential Role of Peon (Phosphate Buffer-Extractable Organic Nitrogen) in the Soil for Plant Nutrition and Its Implication for Organic Farming

Noriharu  Ae1, Shingo Matsumoto2, Yoh-ichi Koyama3, and Katsumasa Iijima3
1Kobe University, Biological and Environmental Science,
Rokkodai-cho, Nada, Kobe, 078-8501, Japan
2Shimane University, Education and Research Center
for Biological Resources, Kamihonjo, Matsue, Shimane, 690-1102, Japan
3Nippi Research Institute of Biomatrix,
Senju-Midoricho, Adachi, Tokyo, 120-8601, Japan, 2008-01-17

Key words: Organic matter, manure, available nitrogen, xylem sap, spinach, organic farming

Introduction

Modern agricultural production, including "organic farming", is based on Liebig's theory (1840) that crops take up only inorganic nitrogen (N) released from organic materials and/or soils. This theory contributes to chemical fertilizer development and stable and high yields in crop production. But in agricultural practices, compulsory use of organic matter is common not just for organic farming but also to achieve high production, especially among sugar beet, spinach and carrot cultivating farmers in Hokkaido, northern Japan. Long-term field experiments conducted at Rothamsted in the United Kingdom have reported the same phenomenon. Mattingley (1973) reported that potato and sugar beet absorbed N from organic sources more efficiently than barley and wheat. Coock (1977) described that an increase in yield of these two crops was not due to the improvement of soil physical condition by application of manure, but by organically-provided N that behaved in ways not easily imitated by fertilizer N (Fig. 1). In this context, there may be two types of crop species, one responding to organic N and another that does not.

Response of Crop Species to Organic Matter

Sedge plants preferentially took up amino acids more in Arctic tundra where mineralization of N was suppressed under low temperature, acid and moist conditions, compared to wheat (Chapin et al. 1993). Also boreal forest plants were reported to take up amino acid N rather than inorganic N (Nasholm et al. 1998). These reports suggest that amino acids in a soil can be better N sources for specific plants, compared to inorganic N. Can amino acids in arable soil become a sufficient N source for crops by replacing inorganic N? Nemeth et al. (1988) reported that the amount of amino acids in a soil was extremely low compared to inorganic N.

We conducted a pot experiment using rapeseed cake (C/N ratio 7.0) as organic N and ammonium sulphate containing the same amount of N as rapeseed cake. Five kinds of vegetable crops (Pimento: Capsicum L.; lettuce: Lactuca sativa L.; carrot: Daucus carota L.; chingensai: a kind of chinese cabbage, Braccica campestris L.; and spinach: Spinacia oleracea L.) were grown for 28 days. During the growth period, N status in the soil of unplanted pots was monitored for evaluating N supply to these crops. For N status in the soil, inorganic N (ammonium and nitrate N), amino acids and protein-like N contents were measured. Protein-like N was extracted with a 1/15M phosphate buffer at pH 7.0 and it was considered to be easily mineralizable and/or decomposable N for estimating inorganic N supply to a plant. Inorganic N levels in unplanted fallow soil with rapeseed cake were higher than in the control soil without any N source but lower than in the soil with ammonium sulphate during the 28 days of the growth period (Table 1). In contrast, the content of organic N such as amino acid and protein was higher in rapeseed cake-amended soil than the control and the ammonium sulphate-amended soil. Interestingly, there were very few amino acids compared to the amount of inorganic N and protein like N.

The N uptake by chingensai, spinach and carrot was higher in soil amended with rapeseed cake than in soil amended with ammonium sulphate, irrespective of lower inorganic N in the plot receiving rapeseed cake. However, pimento and leaf lettuce responded well to inorganic N levels in a soil and their growth was lower in rapeseed cake amended soil (Fig. 2). These facts imply that chingensai, carrot and spinach preferentially took up organic nitrogen such as amino acids and protein. However, the content of amino acids in the unplanted plot with rapeseed cake soil was only 0.6 mg N/kg dry soil at the most. Growth and N uptake of carrot, spinach, and chingensai increased 30 to 40% with application of rapeseed cake compared to chemical fertilizer. This increase in N uptake cannot be explained by the amount of amino acids in the soil, but could be explained by the amount of protein-like N (Matsumoto et al. 2000b).

Protein-like N: Peon

Protein-like N was developed for evaluating available or mineralizable organic N in a soil, and could be extracted with a 1/15 M phosphate buffer (Higuchi 1983). We called this protein-like N PEON (Phosphate-buffer Extractable Organic Nitrogen). Although several extraction methods (excepting this method) for available N or mineralizable N have been reported, organic N showed protein-like characteristics, similar amino acid composition and C/N ratios irrespective of extraction methods (Senwo & Tabatabai 1998; Marumoto et al. 1974; Ogiuchi et al. 2000; Higuchi 1982; Michrina et al. 1982). The amount of PEON in the soil was comparatively high, and it increased markedly when organic matter was applied (Table 1). We extracted PEON with a 1/15M phosphate buffer (pH 7.0) from 25 soils including paddy, upland and forest soils in Japan, grazing fields in Brazil and fertilized fields in Niger. These extracts were analyzed by size-exclusion HPLC (at 280nm). Interestingly, only one major peak of about 8,000 Da was detected in extracts from 25 types of soil irrespective of soil types, soil management and locations in the world (Fig. 3).

In order to know the structure and origin of PEON, a part of the major peak detected in the extract from the Andosol (see first soil sample in Fig. 4) was purified using gel chromatography. PEON was hydrolyzed to analyze the composition of amino acid, amino-sugar and neutral sugar. Amino acid enantiometric ratios provide useful information about the origin of nitrogenous materials (Kvenvolden 1975) because the principal source of D-amino acid is peptideglycans, the main structural component of bacterial cell walls. In bacterial cell walls, the D-enantiometric forms of alanine, glutamic acid and aspartic acid are prevalent, and D-alanine is the most abundant D-amino acid (Rogers 1983). In the present study, three D-amino acids were detected in PEON: D-alanine (12% D/L amino acid ratio), D-arginine (25%) and D-glutamic acid (15%) (Table 2). These results imply the possibility that PEON derives, at least in part, from bacterial cell walls. However, muramic acid in PEON was small in amount. This suggests that PEON may not be derived solely from pepitideglycans of cell wall components of soil bacteria. Although element analysis showed PEON contained 19% of ash (data not shown, probably minerals like Fe and Al) and sugar content was abundant, chemical structure is needed for characterization plot applied with a mixture of incorporated rice bran and rice straw, compared to a plot with chemical fertilizer only. But maize showed no response to the organic matter. It is necessary to re-evaluate the growth response of crop species to organic matter application.

Evaluation of N availability in a soil is defined by mineralizable organic N released from a soil. However, if PEON is accepted as the direct source of N, we are in a better position to re-consider the evaluation method for soil N fertility.

References

• Chapin III, F.S., Moilanen, L. & Kieland, K. 1993. Preferential use of organic nitrogen for growth by a non-mycorrhizal arctic sedge. Nature, 361, 150-153.
• Coock, G.W. 1977. The role of organic manures and organic matter in managing soils for higher crop yields, -A review of the experimental evidence-, In "Proceeding of the International Seminar on Soil Environment and Fertility Management in Intensive Agriculture, p.53-64, Japanese Society of Soil Science and Plant Nutrition, Tokyo.
• Higuchi, M. 1982. Characterization of soil organic nitrogen extracted with phosphate buffer. Japanese Soil Science and Plant Nutrition, 53, 433-437. (In Japanese.)
• Higuchi, M. 1983. Immobilization-remineralization of nitrogen in soil following addition of inorganic nitrogen and organic substances. In "Bulletin of National Institute of Agricultural Science, Series B", 34, 1-84. (In Japanese.)
• Kvenvolden, K.A. 1975. Advances in the geochemistry of amino acids. In Fred A. Donath (ed.), Annual Review of Earth Planet. Science., 3, 183-212.
• Liebig, L. 1840. Chemistry in its applications to agriculture and physiology. British Association for the Advancement of Science. London.
• Matsumoto, S., Ae, N. & Yamagata, M. 2000a. Extraction of mineralizable organic nitrogen from soils by a neutral phosphate buffer solution. Soil Biology and Biochemistry, 32, 1293-1299.
• Matsumoto, S., Ae, N. & Yamagata, M. 2000b. Possible direct uptake of organic nitrogen from soil by chingensai (Brassica campestris L.) and carrot (Daucus carota L.). Soil Biology and Biochemistry. 1301-1310.
• Matsumoto, S., Ae, N. & Yamagata, M. 1999. Nitrogen uptake response of vegetable crops to organic materials. Soil Scence and Plant Nutrition. 45, 269-278.
• Marumoto, T., Furukawa, K. & Yoshida, T. 1974. Contribution of microorganisms and cell wall to easily decomposable soil organic matter. Japanese Soil Science and Plant Nutrition, 45, 23-28 (In Japanese.)
• Mattingly, G.E.G. 1974. The Woburn organic manuring experiment. II. Design, crop yields and nutrient balance, 1964-1972. In "Report for 1973, Part 2". p.98-133. Rothamsted Experimental Station.
• Michrina, B.P., Fox, R.H. & Piekielek, W.P. 1982. Chemical characterization of two extracts used in the determination of available soil nitrogen, Plant and Soil, 64, 331-341.
• Michelsen, A., Schmidt, I.K., Jonasson, S., Quarmby, G. & Sleep, D. 1996. Leaf 15N abundance of subarctic plants provide fields evidence that ericoid, ectomycorrhizal and non- and arbuscular mycorrhizal species access different sources of soil nitrogen. Oecologia, 105, 53-63.
• Nasholm, T., Ekblad, A., Nordin, A., Giesler, R., Hogberg, M. & Hogberg, P. 1998. Boreal forest plants take up organic nitrogen. Nature, 392, 914-916.
• Nemeth, K., Bartels, M., Vogel, M. & Mengel, K. 1988. Organic nitrogen compounds extracted arable and forest soils by electro-ultrafiltration and recovery rates and amino acids. Biology and Fertility of Soils, 5, 271-275.
• Nishizawa, N.K. & Mori, S. 2001. Direct uptake of macro organic molecules. In "Plant Nutrient Acquisition, new perspectives" Ae, N., J. Arihara, K. Okada and A. Srinivasan. eds, pp. 421-444. Springer-Verlag, Tokyo.
• Ogiuchi, K., Nakajima, N., Ae, N. & Matsumoto, S. 2000. Amino acid composition of soil organic nitrogen extracted with phosphate buffer solution, Japanese Soil Science and Plant Nutrition, 60, 160-163. (In Japanese.)
• Okamoto, M., Okada, K., Watanabe, T. & Ae, N. 2003. Growth responses of cereal crops to organic nitrogen application in the field. Soil Science and Plant Nutritio , 49, 445-452.
• Robert, R.R., Yu Zengshou, Randy, A.D. & Kristina, A.V. 1995. Polyphenol control of nitrogen release from pine litter. Nature, 377, 227-229.
• Rogers, H.J. 1983. In "Aspect of Microbiology", Vol. 7, pp.6-25, Van Nostrand Reinhold, Workingham, UK.
• Schulze, E-D., Chapin III, F.S. & Gebauer, G. 1994. Nitrogen nutrition and isotope differences among life forms at the northern treeline of Alaska. Oecologia, 100, 406-412.
• Senwo, Z.N. & Tabatabai, M.A. 1998. Amino acid composition of soil organic matter, Biology and Fertility of Soils, 26, 235-242.
• Wenzel, W. 1990. Extraktion und Charakterisierung von stickstoffreichen Nichtuminsoffen einer Braunerde. VDLUFA-Schriftenreihe, 32, 337-344.
• Yamagata, M., Matsumoto, S. & Ae, N. 2002. Possibility of direct acquisition of organic nitrogen by crops. In "Plant Nutrient Acquisition, new perspectives" Ae, N., J. Arihara, K. Okada and A. Srinivasan eds, pp.399-420. Springer-Verlag, Tokyo.

Index of Images

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  Figure 1 Yield of Sugar Beet with Manure Application (Coock 1977).

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  Figure 2 N Uptake by Pimento, Leaf Lettuce, Carrot, Chingensai, and Spinach Supplied with Ammonium Sulphate and Rapeseed Cake and with No N Source at 28 Dap. Columns Indicate Se (N=3).

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  Figure 3 Chromatograms (at 280 NM) of 25 Soil Extracts with 1/15 M Phosphate Buffer at PH 7.0. Each Soil Extract from 25 Soil Samples Was Analyzed by Size-Exclusion HPLC. the Blue Line in Figure 3-a Shows a Chromatogram of Standard Sample of Mixture of Thyoglobulin (MW;670,000), Bovine-?-Globulin (MW;158,000), Chicken Ovalbumin (MW;44,000), Myoglobin (MW;17,000) and Vitamin B12 (MW;1,300).

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  Figure 4 Chromatograms of Size-Exclusion HPLC of Xylem Saps Collected from Spinach Grown on Solution Culture with Inorganic N and on an Andosol with Rapeseed Cake As N Input.

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  Figure 5 Western Blotting of Peon (a) and Xylem Saps (B) Collected from Spinach Grown in Solution Culture with Inorganic N As the N Source and in Soil Culture with Farmyard Manure with Sawdust As the N Source

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  Table 1 Inorganic, Amino Acid, and Protein-like N in an Andosol Applied with Ammonium Sulfate, Rapeseed Cake, and No N Source Over a Period of 28 Days

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  Table 2 Amino Acid and Sugar Contents of Peon

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