Biological Determinations of Availability Indexes


Determination of Essentiality
Nitrogen Metabolism and Nitrogenous Constituents in Plants
  Nitrate Assimilation
    - Nitrate Reductase
    - Nitrite Reductase
  Ammonium Assimilation
    - Glutamine Synthetase
    - Glutamate Synthase
    - Glutamic Acid Dehydrogenase
    - Transamination
    - Amidation
  Proteins and Other Nitrogenous Compounds
Diagnosis of Nitrogen Status in Plants
  Symptoms of Deficiency and Excess
  Concentrations of Nitrogen in Plants
    - Concentrations of Nitrogen in Plant Parts
    - Ratios of Concentrations of Nitrogen to Other Nutrients in Plants
Nitrogen in Soils
  Forms of Nitrogen in Soils
    - Organic Nitrogen in Soil
    - Inorganic Nitrogen in Soil
Soil Testing for Nitrogen
  Determinations of Total Nitrogen
  Biological Determinations of Availability Indexes
    - Determination of Inorganic Nitrogen
      - Ammonium
      - Nitrate
      - Amino Sugars
Nitrogen Fertilizers
  Properties and Use of Nitrogen Fertilizers
    - Anhydrous Ammonia: 82% N
    - Aqua Ammonia: 21% N
    - Urea: 46% N
    - Ammonium Nitrate: 34% N
    - Ammonium Sulfate: 21% N
    - Nitrogen Solutions: 28–32% N
    - Ammonium Phosphates: 10–21% N
    - Other Inorganic Nitrogen Fertilizers
    - Organic Nitrogen Fertilizers: 0.2–15% N
Aerobic incubation of soil samples for 2 to 4 weeks under nearly optimum conditions of microbial decomposition of organic matter and measurements of nitrogen mineralization is an extensively employed biological procedure for the development of an availability index (96,101,112-114). Incubated samples are tested for the amounts of nitrate, ammonium, or both forms released. Since determinations are run under nearly optimum conditions, only an estimate of the potential for mineralization is provided. Results may differ from mineralization in a field in a particular year. Determinations of indexes by anaerobic incubation involve estimations of ammonium released (115). Other biological tests involve bioassays of microbial growth or pigment production (116), chlorophyll production by algae (117), and carbon dioxide production (118).

Determination of Inorganic Nitrogen

These determinations are considered to be chemical indexes of availability of nitrogen soil organic matter. The utility of chemical indexes depends on their correlation for a broad range of soils with biological criteria, such as crop yields, nitrogen accumulation in plants, and biological indexes (101). Inorganic nitrogen is determined in an extraction of soil with water or solutions of acids, bases, chelating agents, or salts at differing concentrations and temperatures (101). Severe extractants, such as moderately concentrated (4.5 to 6 M) boiling mineral acids or bases, generally give nitrogen releases that correlate well with total soil nitrogen. However, total soil nitrogen as such is not a reliable index of nitrogen availability in soils. Also, release of nitrogen by moderate extraction procedures, such as alkaline permanganate, sodium carbonate, and molar solutions of mineral acids and bases, generally are poorly correlated with biological measurements (96,101). Relatively mild extractions with cold, hot, or boiling water or solutions of cold dilute (0.01 M) acids, bases, or salts have been used with the premise that these methods determine nitrogen of which a high proportion is derived from microbial action on the soils (101). Ammonium or nitrate may be determined in the extracts (96,105,106).


The rate-controlling step in nitrogen mineralization is the conversion of organic nitrogen into ammonium. The conversion of ammonium into nitrate is a rapid step, as a result ammonium generally does not accumulate in well-drained mineral soils. Ammonium in soil, initially present in soils at sampling, is correlated weakly with nitrogen accumulation in plants (113). Temperatures in handling and storage of soil samples are important in judging the correlation between ammonium in soils and accumulation in plants (119). Waterlogging, high acidity (pH<5.0) or alkalinity (pH>8.0), or use of nitrification inhibitors can lead to mineralization that stops with the formation of ammonium and hence to accumulation beyond that occurring in well-drained, mineral soils. Determination of ammonium present in soil without any manipulation generally gives better correlations with biological processes than the correlation of ammonium that accumulates with manipulation of processes that lead to ammonium accumulation.


Nitrate is the form of nitrogen that is used most commonly by plants and that may accumulate in agricultural soils. In combination with other factors, such as soil water, nitrate concentrations in soils have been used in assessments of soil fertility since the early 1900s (113,120–122). Ozus and Hanway (123) reported that nitrogen accumulation in crops during early growth was related to nitrate content in soils. Early workers related nitrate in soils to crop yields. Nitrate in soil was shown to be a reliable evaluation of soil nitrogen that is residual from previous fertilization (124–126). Recent work has related tests for nitrate in soils to prediction of the needs of crops for nitrogen fertilization. These tests are commonly called preplant nitrate tests and are conducted in the early spring to a soil-sampling depth of 60, 90, 120 cm, or greater.

Nitrate is a soluble form of nitrogen that is subject to downward movement in soils in humid temperate climates (105). Sometimes, soil tests for nitrate in the top 15 or 30 cm of soils have not been well correlated with crop yields because of depletion of nitrate in these zones by leaching in humid regions (113). Good correlations between soil nitrate tests and crop yields have been noted with soil samples taken from 120- to 180-cm depth in the profile. Roth and Fox (125) reported nitrate concentrations that ranged from 36 to 295 kg N/ha in the 120-cm profile following the harvest of corn. Soils fertilized with nitrogen applied at economiclly optimum amounts had nitrate concentrations ranging from 41 to 138 kg N/ha. Soils with more than 169 kg nitrate-N/ha in the 120-cm profile did not show an increase in corn yields in response to nitrogen fertilization. Jokela and Randall (124) reported that nitrate concentrations in a 150-cm profile ranged from 150 to 500 kg N/ha over a range of fertilizer treatments after corn harvest in the fall but fell by 50 to 70% by the following spring.

Nitrate concentrations vary among soils and among seasons of the year for a given soil and climate (105,127). In humid temperate climatic areas, nitrate in soils is low in the cold of winter, rises in spring and through the summer with warming of soils and falls in the fall with the rains. In unfertilized fields in the winter, nitrate in topsoil (top 30 to 60 cm) is less than 5 or 10 mg N/kg (105). The concentration can rise to 40 to 60 mg nitrate-N/kg in spring and summer. Depending on the permeability of soil, the depletion of nitrate from topsoil can be rapid with fall rains. Tillage of land can bring about an increase of nitrate, as mineralization and nitrification are increased by aeration of the soil due to tillage. Generally, the more intensive the tillage, the greater the nitrate concentrations in the soil (128–130). For example, in the 120-cm-deep soil profile, following a crop of corn, the nitrate in conventionally tilled soils (100 to 120 kg N/ha) was twice that in the profile of soils cropped in a no-tillage system (129). In dry seasons, soil nitrate can be very low due to low microbiological activity, perhaps less than 10 mg N/kg, but increases as rain falls and mineralization and nitrification result in the wetted soil. In some cases, if the subsoil contains nitrate, nitrate may rise with capillary action and accumulate in dry surface soils. Absorption by plants is a principal path of removal of nitrate from soils. Removal is unique with various soils and crops (105). Perennial crops having a developed root system can absorb nitrate as soon as conditions are favorable for plant growth. Grassland soils generally are low in nitrate throughout the year. However, annual crops do not absorb much nitrate from soils until the root systems are developed.

Many soil test recommendations for correlation of soil nitrate with crop yields require soil sampling to a minimum depth of 60 cm (113). Sampling to this depth involves considerable costs, and attempts have been made to develop a test based on shallower sampling. Alvarez et al. (131) developed prediction equations that related nitrate in the top 30 cm stratum to that in the top 60 cm stratum. Recent research has shown good correlations between crop yields and concentrations of nitrate in the surface 30 cm layer of soils early in the growing season (132–135). Determination of the amount of nitrate in the upper stratum of soil early in the season has led to the development of a test called the early season nitrate test or pre-sidedress soil nitrate test (PSNT).

The basis of the PSNT is the concentration of nitrate in the surface 30 cm of soils at the time that a crop starts rapid growth, for example, when corn is 30 cm tall (133,134). The amount of nitrate in the soil at this depth at this time is an assessment of the amount of nitrogen available for crop growth for the remainder of the season and of the need for nitrogen fertilization. The critical concentration of soil nitrate for the PSNT is the concentration above which yields are not expected to increase with additional nitrogen fertilization. For corn production, Sims et al. (135) in Delaware reported that the PSNT test identified nitrogen-deficient or nitrogen-sufficient sites with about 70% success. Binford et al. (132) in Iowa determined that the critical concentration of nitrate for corn was 23 to 26 mg N/kg f o r a 3 0 cm depth. Sampling 60 cm deep improved correlations between corn grain yields and soil nitrate, but it was felt that the improvement did not justify the additional costs of deep sampling. The critical concentration for the 60 cm depth was 16 to 19 mg N/kg soil. Other research has given similar results. Meisinger et al. (136) in Maryland determined a critical nitrate concentration of 22 mg N/kg with the PSNT successfully identifying nitrogen-sufficient sites across a range of textures, drainage classes, and years. Including ammonium in the analysis slightly improved the predictive use of the test (136). Heckman et al. (137) in New Jersey reported a critical nitrate concentration at the 30 cm depth to be 22 mg N/kg for corn. Evanylo and Alley (138) in Virginia reported critical nitrate concentrations of 18 mg N/kg for corn and noted that the PSNT was applicable to soils without regard to texture or physiographic region. Also for corn, Sainz-Rozas et al. (139) in Argentina reported a critical nitrate concentration of 17 to 27 mg N/kg at the 30 cm depth. They also reported that there was no improvement in reliability if the test was done on samples to 60 cm depth or with the inclusion of ammonium in the determinations. Critical concentrations, similar in magnitude to those for corn have been reported for sweet corn (Zea mays rugosa Bonaf.) (140), lettuce (Lactuca sativa L.), celery (Apium graveolens dulce Pers.) (141), cabbage (Brassica oleracea capitata L.) (142), and tomato (Lycopersicon esculentum Mill.) (143).

If the concentration of nitrate is below the critical concentration, fertilization of the crops is necessary. However, the need to collect soil samples during the growing season has limited the usage of the PSNT. Fertilization is delayed until the results of the PSNT are obtained, and bad weather can delay applications of nitrogen.

Amino Sugars

Fractionation of soil hydrolysates has been used to determine a labile pool of organic nitrogen in soil and to relate this fraction to crop responses to nitrogen fertilizers (102,144). The results of most of these studies have shown little variation among soil types or cultivation patterns in the partitioning of hydrolyzable soil nitrogen into various nitrogenous components and the capacity of soil organic matter to form nitrate. The uniformity among soils was attributed in part to errors in analysis (145,146). Mulvaney and Khan (147) developed a diffusion method for accurately determining amino sugar nitrogen in soil hydrolysates. Mulvaney et al. (145) noted that hydrolysates (6 M HCl) of soils in which crops were nonresponsive to nitrogen fertilization had higher concentrations of amino sugars (e.g., glucosamine, galactosamine, mannosamine, muramic acid) than did hydrolysates of soils in which crops responded to nitrogen fertilization. They reported no consistent differences among the total nitrogen, the ammonium nitrogen, or the amino acid nitrogen fraction of the soil hydrolysate. The amounts of amino sugars were related to mineralization of soil organic nitrogen, since production of inorganic nitrogen upon aerobic incubation of the nonresponsive soils was much greater than that in the responsive soils (145). Concentrations of amino sugars were correlated with response to fertilizer nitrogen applied. Mulvaney et al. (145) classified soils with more than 250 mg amino sugar nitrogen per kg as being nonresponsive and those with less than 200mg amino sugar nitrogen per kg as being responsive to nitrogen fertilization. Khan et al. (146) developed a simpler test for determining amino sugar nitrogen than the processes involving soil hydrolysis. The simpler test involved soil being treated with base (2 M NaOH), followed by heating (50°C) to release ammonia, and then determining the amount of ammonia releases by volumetric methods. This method determined ammonium and amino sugar nitrogen without liberating substantial nitrogen from amino acids and none from nitrate or nitrite. Test values for soils nonresponsive to nitrogen fertilization were 237 to 435 mg N/kg and for responsive soils were 72 to 223 mg N/kg soil.

Amino sugars may constitute 5 or 6% of the humic substances in soils (148). Variations in kind and amount of amino sugars have been noted with climate and with cultivation of soils (149,150).