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Laboratory Manual

Soil Science/Agronomy/Horticulture 326

CONTENTS Introduction Exercise 1 2 3 4 5 6 7 8 9 10 11 12 1

Plant Response to N, P, and K Nitrogen Requirement of Different Plant Species Plant Response to Nutrient Sources and Soil Placement Soil pH, pH Buffering Capacity, and Organic Matter Content Soil Potassium Buffer Power Mineralization of Organic Nitrogen Tissue Testing Total P and K concentrations in Plant Tissue Total N in Plant Tissue Determination of Available P and K in Soil Determination of Soil pH, Lime Requirement and Soluble Salts Development of Nutrient Deficiency Symptoms in Plants Growing in Solution Culture

2 7 12 18 23 28 31 37 40 43 47 53

Laboratory Manual

Soil Science/Agronomy/Horticulture 326

INTRODUCTION
During the semester, you will complete 11 of the 12 greenhouse and laboratory exercises contained in this manual. The final exercise will be conducted as a demonstration in the greenhouse. In most instances, you will be assigned to work jointly with another student in your laboratory section. This provides the opportunity to exchange ideas and discuss results as you observe. However, every student is expected to turn in individual laboratory reports and data sheets. The 11 exercises are organized into four units with the following objectives: Unit I: To demonstrate plant responses to soil applications of essential nutrients under greenhouse conditions. You will study the response of various crops to applications of nitrogen, phosphorus and potassium and compare crop responses to different sources and methods of application of these nutrients. Exercises 1, 2 and 3 make up this unit. Unit II: To become familiar with analytical methods for determining some of the soil properties and processes that affect plant growth. Exercises 4, 5 and 6 make up this unit. Unit III: To examine plant analysis as a means of identifying nutrient disorders, verifying the adequacy of soil fertilization, and gaining a more detailed understanding of how plants respond to soil treatments. Exercises 7, 8 and 9 make up this unit . Unit IV: To introduce soil analysis as a tool for assessing the fertility status of soils and for serving as a basis for fertilizer and lime recommendation. Exercises 10 and 11 make up this unit. For each laboratory exercise, results obtained by each student or student pair are tabulated and distributed to the whole class so that all of the students can see how their results fit into the big picture. For several of the exercises, each student will do additional analysis of the group data. The educational value of these exercises depends on the reliability of the greenhouse and laboratory results of each student. Sloppy work by just a few students can destroy much of the learning value of many of the exercises. Grading Your performance in the laboratory accounts for 30% of your course grade. The following factors are taken into consideration in determining your laboratory grade: 1. 2. 3. 4. 5. Careful attention to detail in setting up and carrying out each exercise. Turning in data sheets and assigned reports for all exercises on time. Errors in data entry and calculations. Neatness of your work area in the laboratory and in the greenhouse. Regular watering of pots for each greenhouse exercise.

Subject matter covered in the laboratory will be included as a separate laboratory examination at the time the final lecture examination is given.

Laboratory Manual

Soil Science/Agronomy/Horticulture 326

EXERCISE 1
PLANT RESPONSE TO N, P, AND K
Nitrogen, phosphorus, and potassium are justifiably classified as primary nutrients, not only because plants require them in relatively large quantities, but also because these are the three nutrients that most commonly limit plant growth and crop production. Thus, unless a soil has been heavily fertilized in recent times, it is generally possible to observe responses to N, P, and K under the very intensive cropping that occurs in the greenhouse. This is less likely for secondary and micronutrients. There are distinct advantages, but also distinct limitations, for studying plant response to nutrient applications made under greenhouse conditions: Table 1-1: Advantages and Limitations of Using the Greenhouse for Studying Plant Nutrient Responses. Advantages 1. Through careful control of the environment, conditions can be such that only the factor being studied is growth-limiting. 2. Root volume is restricted to the soil in the pot in which the plants are growing. Aside from gaseous nutrients absorbed through the stomata (e.g., S as SO2 ) or falling on the leaves via atmospheric deposition (dust), all of the nutrients taken up by the plant must come from the soil in the pot. In the field, plants take up nutrients from an unknown volume of surface soil and also from the subsoil below the fertilized zone. 3. Large numbers of treatments can be tested at relatively low cost and low expenditure of time. For the same cost in money and time, greenhouse experiments enable one to study a much wider range of soils and soil amendments. Limitations 1. The environment is artificial. Plants may react differently in the field because of differences in factors such as temperature, humidity or radiation level. 2. The ratio of crop dry matter to soil volume explored by the roots is much higher in greenhouse pots than in the field. Because of this, nutrient deficiencies occur at a higher level of available nutrients than in the field. The ratio of transpiring surface to water storage in the soil is also much higher in the greenhouse, so frequent watering is required.

3. Results obtained in the greenhouse cannot be applied directly to field conditions. For example, a soil testing method for a specific nutrient may correlate well with uptake of that nutrient in the greenhouse, but the critical level for yield response must be determined from field calibration studies.

Because of these differences between field and greenhouse conditions, greenhouse studies are restricted largely to studies such as the relative plant availability of various forms of a nutrient or to screening experiments that serve to develop technologies such as soil testing methods. The full advantage of greenhouse studies cannot be realized unless special precautions are taken to minimize experimental error. The soil must be mixed thoroughly so as to be homogeneous, nutrients must be applied as uniformly as possible, and efforts must be made to obtain a uniform stand of the test crop. But all of these precautions are a waste of time unless soil moisture is controlled properly. Most potted plants grow in artificial potting medium often a

Laboratory Manual Exercise 1 (Continued)

Soil Science/Agronomy/Horticulture 326

mixture containing coarse ingredients such as sand, peat, compost, vermiculite, perlite, etc. intended to promote rapid drainage of excess water. Leaching of nutrients is extreme under these conditions so this practice is not suitable for studying plant response to nutrient addition. Growing potted plants in soil (other than sand) is generally so tricky due to problems of overwatering that it is usually not recommended to the public. We will grow potted plants in soil by carefully controlling soil water content over a narrow range that is favorable to the plant and not conducive to leaching. The proper soil moisture is that corresponding to the amount of water the pot of soil can retain against drainage due to the force of gravity. This is commonly referred to as the field moisture capacity (FMC) of the soil. The FMC of the soil used in greenhouse studies generally cannot be guessed accurately. It must be measured. The apparatus commonly employed is shown in Figure 1. To measure the FMC, you need a beaker that will hold a depth of soil equal to the depth of soil in the greenhouse pots that will be used, a glass tube, and glass wool or cotton batting. The purpose of the glass wool or cotton batting and the glass tube is to allow air to escape when the soil surface is flooded with water. The beaker is initially filled to approximately 1/3 the depth that the soil will have in the pot in the greenhouse and gently tapped on a table top or in the palm of your hand two to three times to pack the soil. The process is repeated twice more so that the final soil depth is approximately equal to that in the greenhouse pot. Water is then added quickly to the soil surface to get complete coverage of the soil surface. The amount of water added should not wet the soil to more than about 1/3 of its total depth in about 5 minutes. The beaker is then covered with plastic sheeting to prevent water evaporation. After 24 to 48 hours, the soil is examined. If it is wet to its entire depth, too much water has been added and the whole procedure needs to be repeated. If wet to approximately 80% of its depth, a soil sample weighing 20 to 50 grams is removed from the middle portion of the wetted zone. The sample is weighed to +/- 0.01 g and dried for 24 hours at 105 o C. The dried soil weight is then determined and the soils FMC is calculated. Calculation of FMC Soil moisture is always expressed on a dry-weight basis. The formula used is: % H2 O = (weight H2 O) x 100% (weight dry soil)

= (weight wet soil) ! (weight dry soil) x 100% (weight dry soil)

(weight wet soil) = % H2 O x (weight dry soil) + (weight dry soil) 100% These formulas are used to calculate the soil weights needed in steps 2 and 8 of this exercise.

Laboratory Manual Exercise 1 (Continued)

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Figure 1-1: Apparatus for estimating field moisture capacity of soils for greenhouse studies.

Laboratory Manual Exercise 1 (Continued)

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Materials The materials needed are: soil passed through a 2-mm sieve, air-dried, and mixed; pots; pot liners; balances; plastic sheets; nutrient sources (nutrient treatments are shown in a separate hand-out), seed; labels; marking pens; deionized water . Procedure 1. Select three pots and line each with a plastic bag. 2. Compute the weight of air-dry soil that is equivalent to 1500 g of oven-dry soil. This weight is ________ g. 3. Weigh the amount of air-dry soil calculated in step (2) into each pot. 4. Spread the soil from the first pot onto the sheet of plastic provided and add the nutrients for the treatment assigned to you. Thoroughly mix the treated soil and return the soil to the first pot. 5. Label the container with your name(s), lab section, and amount of the variable nutrient (N, P, or K) in mg/kg. Remarks 1. Individual pots should weigh within 10 g of each other. 2. The air-dry soil contains _____ % water (get this value from the instructor). 3. Weigh the soil to +/! 10 g.

4. The added nutrients should be uniformly distributed throughout the pot. 5-8. Pot label
Required Information Name(s) ______________ Replicate (A, B or C)_ ___ Lab Section ___________ Nutrient added_____mg/kg Example Jane D. & John Q. Pot A Section 301 150 mg K/kg

6. Repeat steps 4 and 5 for the remaining pots. The total weight is comprised of: 7. Randomly label the pots A, B, or C.
Pot, plastic bag, and label ________ g Oven dry soil Water at FMC ________ g ________ g

8. Compute the weight of the container when the soil is adjusted to its field moisture percentage (FMC) of %. Include this total weight on your pot label. 9. Remove about a cup of soil from the surface of pot A and level the remaining soil. 10. Add approximately 3/4 of the water that will be needed to bring the soil to FMC. (A specific volume of water is not required at this point as long as FMC is not exceeded.)

Total weight

________ g

9. Save the soil for step 11.

10. If all of the water were added to the soil surface (step 15), the soil at the surface could become disturbed and some of the seeds uncovered.

Laboratory Manual Exercise 1 (Continued)

Soil Science/Agronomy/Horticulture 326

Procedure 11. Place ____ seeds of corn on the soil and cover with the soil just removed. 12. Place the pot on the balance and add deionized water to adjust the container to the total weight computed in step (8). Use a graduated beaker. 13. Fold the plastic liner over the soil surface to minimize water loss by evaporation until the corn germinates and emerges. 14. Thin to 4 seedlings per pot (or other number as directed by your lab instructor).

Remarks 11. Wait until all of the water has infiltrated the soil. The instructor will tell you how many seeds to plant. 12. Hereafter, all watering will be done by weight using deionized water. Tap water contains Ca, Mg, Fe, N and other unknowns. 13. As soon as plants emerge, uncover the plastic bag from the soil surface and fold down over the outside of the pot. 14. Shake the soil from roots of the seedlings removed back into the pot; discard plants removed. The remaining 4 plants should be spaced uniformly in the pot. 15. Water loss by evapotranspiration will be low the first two weeks until there is significant leaf surface area. Notice changes in water use between cloudy days and bright, sunny days. Harvest after completing Exercise (7). 16. Save all plant parts including desiccated leaves that may have fallen off during harvest. Label the bag with the same information as on the pot label. 17. For research, the samples would be kept in the drier until weighed to avoid absorption of moisture from the atmosphere. 18. Grind to pass a 20-mm screen. Samples will be taken for Ex. (8) in step (20) and for Ex. (9) in step (21). 19. Subtract the weight of each empty bag from the weight of tissue + bag. Fill out the Data Sheet and hand it in. 20. Record the weights on the Data Sheet for Exercise (8). These samples will be ashed prior to analyzing for P & K. 21. Record the weight to +/! 1 mg. The digestion tube should be dry so that the tissue will not adhere to the neck.

15. Water the pots to the computed weight three times weekly during the first two weeks and daily thereafter.

Continue watering until harvest time! 16. At the designated time, cut the plants at the soil surface and place in the paper bag provided. Label the bag and place in crop drier. Dry at 55 o C. 17. After the plants have dried to constant weight, record the dry weight of the plants plus bag to +/! 0.01 g. 18. Grind the samples and put the ground tissue into labeled plastic bags. You will analyze the tissue for N, P and K (Ex. 8 & 9). 19. Weigh each empty paper bag and calculate the dry weight of the tissue. 20. Weigh 150 to 200 mg of each ground sample into a 50 ml beaker for P & K analysis in Exercise (8). 21. Weigh 100 to 150 mg of ground plant tissue and transfer quantitatively to a dry digestion tube for nitrogen analysis in Exercise (9).

Laboratory Manual

Soil Science/Agronomy/Horticulture 326

EXERCISE 2
NITROGEN REQUIREMENT OF DIFFERENT PLANT SPECIES
Nitrogen was established as an essential element in plant nutrition in the 19th century. Plant response to nitrogen is manifested in the production of vigorous plant growth with dark green leaf color. Nitrogen is an important constituent of the chlorophyll molecule as well as amino acids, proteins, nucleotides, nucleic acids, amines, and amides. The plow layer of most soils contains nitrogen mainly in the organic form, ranging from 0.08 to 0.4% (1,600 to 8,000 lbs per acre plow layer). Over the growing season, only 2 to 3% of this organic nitrogen is made available to crops under Wisconsin climatic conditions. Soils low in organic matter will supply very little nitrogen. Continuous cropping without replacement of nitrogen reduces a soil's ability to supply nitrogen; thus, the need for nitrogen fertilizers to supplement natural supplies. Nitrogen is taken up by plants as nitrate (NO3 -) or ammonium (NH 4 + ) ions. Most plants can utilize both forms of nitrogen in their growth processes. An imbalance of nitrogen or an excess of this nutrient in relation to P, K, and S prolongs the growing period and delays maturity. Too much nitrogen produces succulent plants, which makes them more susceptible to disease. Some plants show weakening of stems causing lodging. Nitrogen requirements vary among plant species. In this exercise, you will determine the optimum nitrogen rate for biomass production under greenhouse conditions by different plant species. In the field, the nitrogen concentration varies with stage of maturity and portion of the plant sampled. The nitrogen concentration of most plant parts decreases as the plant matures. When nitrogen is the yield-limiting factor, chlorophyll in the lower leaves breaks down and nitrogen is translocated to the upper leaves. Thus, deficiency symptoms for this element show up first on the older leaves. The range in nitrogen concentration in the leaves of several crops is shown in the accompanying table. Table 2-1. Nitrogen Concentration in the Leaves of Various Crops
Crop Vegetable Crops (Geraldson and Tyler, 1990) Celery Kale and collards Lettuce Onion Pea Pepper Potato Spinach Sweet corn Sweet potato Tomato Turnip Watermelon Cotton (Sabbe and Zelinski, 1990) Peanut Soybean (Small and Ohlrogge, 1973) Sugar cane blades (Bowen, 1990) Nitrogen range (%, dry wt. basis) 2.5 4.0 2.5 1.5 3.1 3.0 3.0 4.0 2.6 3.2 2.5 3.5 2.0 3.0 2.7 4.3 1.5 - 4.0 - 5.0 - 4.0 - 2.5 - 3.6 - 4.5 - 5.0 - 6.0 - 3.5 - 4.2 - 6.0 - 4.5 - 3.0 - 4.3 - 3.8 - 5.5 - 2.7

Laboratory Manual Exercise 2 (Continued)

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Table 2-1. Nitrogen Concentrations in the Leaves of Various Crops (continued)


Crop Small grains : Oats, wheat, barley (Westfall et al., 1990) Sugar cane blades (Bowen, 1990) Rice (Westfall et al., 1990) Sorghum (Whitney, 1970) Corn (Jones, 1990) Forage crops: Alfalfa (Kelling and Matocha, 1990) Bromegrass (Krueger and Scholl, 1970) Orchardgrass (Kresge and Younts, 1963) Tall fescue (Hallock et al., 1966) Kentucky bluegrass (Butler and Hodges, 1967) Bermudagrass (Adams et al., 1967) Johnsongrass (Spooner et al., 1971) Millet (Clapp and Chambles, 1970) Pangola grass (Harris et al., 1968) Sorghum-sudan and sudangrass (Dotzenko, et al., 1966) Annual and perennial ryegrass (Thomas et al., 1952) Creeping bentgrass Nitrogen range (%, dry wt. basis)

1.7 1.5 2.5 3.0 2.7 2.5 2.5 3.2 3.4 2.6 2.6 1.6 2.5 1.7 2.0 3.8 4.5

- 3.5 - 2.7 - 4.5 - 5.0 - 3.5 - 5.5 - 3.6 - 3.5 - 3.8 - 3.2 - 3.2 - 1.8 - 3.5 - 2.0 - 3.0 - 4.2 - 5.5

References Cited
Adams, W.E., A.W. White, R.D. McCreery, and R.N. Dawson. 1967. Agron. J. 59:247-250. Bowen, J.E. 1990. In R.L. Westerman (ed.) Soil Testing and Plant Analysis, p.454. No. 3, Soil Sci. Soc. Am. Book Series. Madison. Butler, J.D., and T.K. Hodges. 1967. J. Hortic. Sci. 2:62-63. Clapp, J.G., Jr., and D.S. Chambles. 1970. Crop Sci. 10:345-349. Dotzenko, A.D., N.E. Hamburg, G.O. Hinze, and W.H. Leonard. 1966. Colorado Agric. Exp. Sta. Tech. Bull. 87. Geraldson, C.M., and K.B. Tyler. 1990. In R.L. Westerman (ed.) Soil Testing and Plant Analysis, pp.549-562. No. 3, Soil Sci. Soc. Am. Book Series. Madison. Hallock, D.L., R.H. Brown, and R.E. Blaser. 1966. Virginia Agric. Exper. Stn. Agron. Res. Rep. 112. Harris, H.D., V.N. Schroder, and R.L. Silman. 1968. Fla. Agric. Exp. STa. Tech. Bull. 725. Jones. J.B. 1990. Plant analysis as an aid in fertilizing corn and sorghum. In R.L. Westerman (ed.) Soil Testing and Plant Analysis, pp.603-643. No. 3, Soil Sci. Soc. Am. Book Series. Madison. Kelling, K.A., and J.E. Matocha. 1990. In R.L. Westerman (ed.) Soil Testing and Plant Analysis, pp.549-562. No. 3, Soil Sci. Soc. Am. Book Series. Madison. Kresge, C.B., and S.E. Younts. 1963. Agron. J. 55:161-164. Krueger, C.R., and J.M. Scholl. 1970. Wis. Agric. Exp. Sta. Res. Rep. 69. Sabbe, W.E., and L.J. Zelinski. 1990. In R.L. Westerman (ed.) Soil Testing and Plant Analysis, pp.469-493. No. 3, Soil Sci. Soc. Am. Book Series. Madison. Small,H.G., Jr., and A.J. Ohlrogge. 1973. In L.M. Walsh and J.D. Beaton (eds.), Soil Testing and Plant Analysis, pp. 315-327. Soil Sci. Soc. Am., Inc. Madison. Spooner, A.E., W.R. Jeffrey, and H.J. Hunneycutt. 1971. pp. 12-15. In Ark. Agric. Exp. Sta. Bull.769. Thomas, B.A., A. Thompson, V.A. Oyanuga, and R.H. Armstrong. 1952. Exp. Agric. 22:10-22. Westfall, D.G., D.A. Whitney, and D.M. Brandon. 1990. In R.L. Westerman (ed.) Soil Testing and Plant Analysis, pp.495-519. No. 3, Soil Sci. Soc. Am. Book Series. Madison. Whitney, D.A. 1970. Kansas State Univ. mimeo 3a-162-1-300.

Laboratory Manual Exercise 2 (Continued) Materials

Soil Science/Agronomy/Horticulture 326

Soil passed through a 2-mm sieve and air-dried; pots with a capacity of about 2 liters, plastic bag pot liners; balances with a capacity of 2 kg or more; plastic sheets for mixing soils and amendments; ammonium nitrate; triple super phosphate; potassium chloride; gypsum (CaSO 4 * 2H2 O) and zinc sulfate; labels, marking pens, corn seeds; deionized water.

Procedure 1. Select four pots and line each with a plastic bag. 2. Compute the weight of air-dry soil that is equivalent to 1500 g of oven-dry soil. 3. Weigh into each pot. g of soil of air-dry soil

Remarks 1. The pot weights should not vary by more than +/- 10 g. 2. The air-dry soil contains % moisture. Use the moisture formula from Exercise 1. 3. This is the amount of air-dry soil calculated in step (2). Check your calculations with the lab instructor before proceeding. 4. Show your calculations and results to the lab instructor before proceeding.

4. Compute the weights of NH4 NO 3 required to give the following concentrations of N. Pot N rate, mg/kg A 0 B 75 C 150 D 300 5. Spread the soil from each pot (sequentially) onto the plastic sheet provided, and add the appropriate amount of NH4 NO 3 to give the desired concentration of N. 6. Also add to each pot, 200 mg/kg P as triple super phosphate, 300 mg/kg K as KCl, 18 mg/kg S as CaSO4 * 2H2 O, and 2 mg/kg Zn as ZnSO4 . 7. Mix the treated soil thoroughly; return the soil to the pot. 8. Remove up to one cup of soil from the surface of each pot and level the remaining soil.

5. Begin with the control treatment (no N) and proceed to the highest N rate to minimize contamination of the plastic sheet with N.

6. Use the balance provided.

7. Label the N treatments: A. 0 N; B. 75 N; C. 150 N; D. 300 N. 8. This will be used later to cover the seed. Small seeds will require less than one cup to cover them. Check with the instructor.

Laboratory Manual Exercise 2 (Continued)

Soil Science/Agronomy/Horticulture 326

Procedure 9. Add approximately 3/4 of the amount of water needed to bring the soil to FMC.

Remarks 9. Use a graduated beaker. Exact volume is not needed at this point. See step 12 for the total amount of water to add, and apply about 3/4 of that amount. 10. The number of seeds depends on expected percent germination and the eventual size of the plants. 11. Sample label:

10. After the water has infiltrated, plant the number of seeds suggested by your lab instructor; cover with soil removed in step 8. 11. Label the pot as in Exercise 1. Include the rate of N applied and the crop planted.

Pete P. & Robin H. Corn 75 mg/kg Lab 301 Planted 2/5/02

12. Place the pot on the balance and adjust the soil to its FMC (_______ %) by carefully adding enough water to attain the calculated total weight.

12. Calculation of the total weight at FMC: Pot + plastic bag + label ________ g Soil (oven-dry basis) Water at FMC Total weight ________ g ________ g ________ g

13. Close the plastic liner over the soil surface. As soon as the plants emerge, uncover the soil and fold the plastic bag over the sides of the pot. 14. Record the number of plants emerged daily until the number is constant. Then thin plants to the number designated by your lab instructor. 15. Water to FMC as often as needed to assure adequate moisture. Water loss between waterings should not exceed 30% of the total water in the soil at FMC.

13. This will minimize water loss by evaporation and prevent the surface soil from drying excessively and delaying germination.

14. Discard the thinned plants.

15. The permanent wilting point is about 1/2 FMC, but photosynthesis is slowed at about 3/4 FMC.

10

Laboratory Manual Exercise 2 (Continued)

Soil Science/Agronomy/Horticulture 326

Procedure 16. At the designated time, cut the plants in each pot at the soil surface and place in separate paper bags. Label the bags the same as the labels on the pots. 17. Weigh the dried plant tissue plus the bag.

Remarks 16. Place the samples in the box provided. The lab instructor will put them in the drier and dry them to constant weight at 60o to 70o C.

17. Do not remove the tissue from the bag at this point. Record the weight of the tissue plus the bag. 18. Do not discard the bag after grinding.

18. Grind the tissue and put the ground tissue in a labeled plastic bag. The tissue will be analyzed later. 19. Weigh the paper bag after the tissue has been removed for grinding. 20. Record the dry matter yield on the data sheet.

19. Subtract the bag weight from the weight recorded in step 18. 20. Leave your data sheet with the instructor

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Laboratory Manual

Soil Science/Agronomy/Horticulture 326

EXERCISE 3
PLANT RESPONSE TO NUTRIENT SOURCE AND PLACEMENT
When fertilizing plants, the primary consideration is the appropriate rate of application. The chemical forms of the nutrients in the fertilizer are generally of secondary importance. There are, however, some instances where the chemical form of the nutrient is of concern. For example, cranberry uses nitrogen only in the ammonium form, and the rate of nutrient release to plants from fertilizer is important when fertilizing container-grown plants or turfgrass. Fertilizer is any material that contains one or more essential plant nutrients, is applied primarily for its nutrient content, and is known to promote plant growth through an increase in nutrient supply. In the fertilizer industry, fertilizers that contain one or two essential plant nutrients are known as fertilizer materials. They may be applied to the soil as such but are often mixed with other fertilizer materials to provide a complete fertilizer -- one that contains all three of the primary plant nutrients, N, P, and K. A given nutrient may be obtained from several different fertilizer materials. These materials differ in nutrient concentration and, very often, in the chemical form in which the nutrient exists. In this exercise, the class will observe plant responses to different fertilizer materials. Examples of fertilizer materials and their distinguishing characteristics are given in Table 3-1.The guaranteed nutrient content of fertilizers is expressed as their "grade". (See Table 3-1.) Grade designations consist of three numbers separated by hyphens. An example is 10-8-16. These numbers signify that the fertilizer contains a minimum of 10% total N, 8% citrate-soluble phosphate (expressed as P2 O5 ), and 16% water-soluble potash (expressed as K2 O). The numbers are always given in the same sequence: N-P2 O5 -K2 O. A fertilizer such as ammonium nitrate contains no P or K, and its grade is 33.5-0-0. Note that the theoretical N concentration of NH4 NO3 is 35%. Fertilizers are not pure chemicals. The cost of purifying them is prohibitive, and there is no need to remove ordinary impurities from a product that will be applied to the soil. Also, there is no P, P2 O5 , K, or K2 O as such in fertilizer. Phosphorus is always accompanied by a cation (usually Ca2+ or NH4 + ) and potassium by an anion (usually Cl- or SO4 2-). Use of the terms phosphate (P2 O5 ) and potash (K2 O) is a carry-over from the early days of agricultural chemistry when elements analyzed in a total elemental analysis of soils were reported as oxides. To convert % P2 O5 to the elemental form (P), multiply % P2 O5 by 0.44; to convert K2 O to the elemental form (K), multiply by 0.83. Inorganic Fertilizer Materials Raw materials for inorganic fertilizers come directly from nature. Atmospheric nitrogen is the principal source of nitrogen fertilizers. Air is about 79% N2 by volume and contains about 36,000 tons over every acre of the earth's surface. Combining hydrogen with atmospheric nitrogen under pressure, heat, and a suitable catalyst produces anhydrous ammonia (NH3 ): N2 + 3 H2 =====> 2 NH3 The H2 is obtained from methane (CH4 ) or hydrolysis of water. When methane is used, urea is often made in the same fertilizer plant by converting the C from methane to CO 2 and reacting the CO 2 with NH3 : 2 NH3 + CO2 =====> (NH 2 )2 CO + H2 O

12

Laboratory Manual Exercise 3 (Continued)

Soil Science/Agronomy/Horticulture 326

Other nitrogen fertilizers are made by reacting NH3 with various acids such as H3 PO4 , HNO3 , and H2 SO4 . Fertilizer phosphorus comes from "phosphate rock," a calcium phosphate ore deposit that may be of either igneous or sedimentary origin and that contains the phosphate mineral apatite. Because of the low solubility of apatite, treatment of the ore with strong acids (H3 PO4 , H2 SO4 , HNO3 ) is necessary to produce soluble phosphate products. Fertilizer potassium is obtained by mining deposits that were created by the evaporation of ancient seas under arid conditions. In contrast to rock phosphate, potash ore can be used directly as potassium chloride (often called "muriate of potash"), potassium sulfate, or as potassium magnesium sulfate. Processing is usually necessary, however, to remove impurities such as common salt (NaCl). Organic materials Several organic materials, including manure and crop residues, can be added to soil to increase nutrient supply, especially nitrogen. Activated sewage sludge, dried blood, and fish contain a higher percentage of nitrogen than do most manures and crop residues. Most of the nitrogen from organic sources becomes available within the first three to four weeks following application. Thereafter, the amount of nitrogen released is very small. Some organic materials contain significant amounts of phosphorus but are typically low in potassium (Table 3-1). Applications of animal manure that are heavy enough to meet the nitrogen requirements of a crop usually provide more than enough phosphorus for that crop. Such rates of application over an extended period of time can increase available phosphorus to excessive levels. Slow Release Nitrogen Materials When water-soluble nitrogen fertilizers are applied to soil, significant amounts of nitrogen may be lost from the soil by leaching, denitrification, or ammonia volatilization. Use of slow-release nitrogen fertilizers generally reduces these losses. However, slow-release nitrogen fertilizers are too expensive for economical use in feed, forage, and fiber production. Slow-release nitrogen fertilizers are produced by altering the solubility of materials or by including compounds that require microbial activity for transforming organic N to available forms. A common method used is to coat water-soluble compounds with materials that are water-insoluble but contain cracks and/or pores. Water can enter by diffusion through these openings creating a saturated solution which is then forced out through the same openings or which builds up pressure sufficient to disrupt the coating material. The most common example of a coated nitrogen fertilizer is sulfurcoated urea (SCU). This material is urea with a coating of elemental sulfur, a binding agent, a sealant, and a microbiocide. Nitrogen release rates can be varied by controlling the thickness of the sulfur coating.

13

Laboratory Manual Exercise 3 (Continued)

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Table 3-1: Composition of some common fertilizers.


Fertilizer Fo rm ula Nutrient Conc. Nutrient Conc. % Inorganic N itrog en M aterials NH 3 82-0-0 N 82 -NH 4NO 3 33.5-0-0 N 33.5 -(NH 2) 2SO 4 21-0-0 N 21 S Ca(NO 3) 2 15.5-0-0 N 15.5 Ca NH 4H 2PO 4 11-48-0 N 11 P (NH 4) 2HPO 4 18-46-0 N 18 P KNO 3 13.5 -0-44.5 N 13.5 K CO(NH 2) 2 46-0-0 N 46 -NH 4NO 3 28-0-0 N 28 -+ CO(NH 2) 2 Ino rganic P ho spho rus M aterials Ca(H 2PO 4) 2 0-20-0 P + CaSO 4 Ca(H 2PO 4) 2 0-46-0 P NH 4H 2PO 4 11-48-0 P (NH 4) 2HPO 4 18-46-0 P Inorganic P otassiu m M aterials KCl 0-0-60 K 2SO 4 0-0-50 KNO 3 13.5 -0-44.5 K 2SO 4 + 0-0-22 2 MgSO 4 Grade % --26 24 21 20 37 ---

An hydrous am m onia Am m onium nitrate Am m onium sulfate Ca lcium nitrate Mo noa m m onium pho sph ate Diam m onium pho sph ate Potass ium nitrate Urea Ure a am m onium nitrate s oln.

No rm al sup erph osp hate Triple superp hos pha te Mo noa m m onium pho sph ate Diam m onium pho sph ate

8.7 20 21 20

Ca Ca N N

20 14 11 18

Potassium Potass ium Potass ium Potassium

chloride sulfate nitrate m agnesium sulfate

K K K K

50 42 37 18

Cl S N Mg S

46 17 13.5 11 22

Material Beef m anure 1 Chicke n m anure 1 Dairy man ure 1 Sheep m anure 1 Swine m anure 1

Organic F ertilize r M aterials Nutrient Am ount Nutrient lb/ton N 14 P N 25 P N 10 P N 28 P N 10 P % 5.4 6.0 5.0 P P P

Am ount Nutrient Am ount lb/ton lb/ton 3.9 K 9 10.9 K 10 2.2 K 8 4.2 K 20 2.8 K 8 % 2.5 0.9 0.9 K K K % 0.4 0.5 3.3

Se wa ge sludge (d ry) N Ac tivated sewa ge sludge (d ry) N Turke y dro pping com post (dry) N _________________________________
1

As excreted, without bedding. Most of the inorganic N is urea or NH 3, which can be lost upon drying. Slow -Re leas e M aterials N conc. Material % 32-37 Methylene-coated urea 34 Isobutylidenediurea 35-42

Material Sulfur-coated urea Resin-coated urea Urea formaldehyde

N conc. % 32 31

14

Laboratory Manual Exercise 3 (Continued)

Soil Science/Agronomy/Horticulture 326

There are three types of coating that can be applied to nitrogen materials: 1. Impermeable coatings with small pores through which solutions of the nutrients diffuse. 2. Impermeable coatings that must be broken by abrasive, chemical, or biological action before nitrogen can be released. 3. Semi-permeable coatings through which water diffuses and creates internal pressures sufficient to disrupt the coating. Uncoated organic fertilizer compounds of low water solubility are used as nitrogen sources for highvalue crops, turf, and ornamentals. These compounds are produced by the reaction of urea and a number of aldehydes to form compounds that are sparingly soluble in water. Two of the most common are the ureaforms (UF) and isobutylidenediurea (IBDU). Urea reacts with formaldehyde in the presence of a catalyst to form a mixture of compounds under the generic name ureaforms -- also known as methylene ureas. There are numerous potential processes for the dispersal of soluble fertilizer salts containing nitrogen, phosphorus, and potassium, etc. into asphalt, water, paraffins, oils, gels, polymers, and resins, which are referred to as matrixes. Nitrogen compounds of limited water solubility, such as ureaformaldehyde, have also been incorporated into a matrix or have been embodied in expanded vermiculite, perlite, clay, glass frits, and similar materials. Matrix materials have been found to be effective in rice production, pastures, and vegetable crops but too costly for field crop production. Influences of Nutrient Source and Method of Application Plant response to fertilizer is conditioned by the amount of nutrient applied, the method of application, and soil characteristics. Exercise 1 demonstrates nutrient rate effects. The present exercise examines the influence of nutrient source and method of application on plant response to nitrogen, phosphorus, and potassium. Variation in plant response to nitrogen in different fertilizer materials relates primarily to losses of the nutrient from soil-plant systems. The mechanisms for nitrogen loss are leaching of nitrate (NO3 -), denitrification, and volatilization of ammonia (NH3 ) from the soil surface. Leaching cannot occur in closed pots. However, nitrogen loss through denitrification and volatilization can occur. The extent of denitrification varies with the level of NO3 - in the soil solution and the aeration status of the soil. Volatilization of nitrogen is restricted to situations wherein solutions on or near the soil surface contain high concentrations of NH4 + and the pH approaches or surpasses 7.3. Plant response to different phosphatic fertilizers varies with the solubility of the phosphate, the ions associated with the phosphate, and conditions prevailing for diffusion transport of phosphate ions to plant root surfaces. Plant response to row applications of phosphatic fertilizers is significantly reduced when the water solubility of the phosphate is less than about 50%. Plant response to phosphate is also reduced when the fertilizer is placed in a soil zone that is subject to periodic drying. Plant recovery of fertilizer phosphate can often be enhanced by including ammoniacal nitrogen in the fertilizer.

15

Laboratory Manual Exercise 3 (Continued)

Soil Science/Agronomy/Horticulture 326

All common potassium fertilizers are totally soluble in water. Potassium moves in soil much more readily than phosphorus though not nearly as fast as NO3 -. Consequently, when variation in plant response to different potassium sources is observed, the most common reason is a nutritive effect of the anion associated with K in the fertilizer. Materials The materials needed are: soil passed through a 2-mm sieve, air-dried, and mixed; pots; pot liners; labels; plastic sheets, balances; fertilizer materials; marking pens; sorghum seeds; deionized water.

Procedure 1. Select four pots and line each with a plastic bag. 2. Compute the weight of moist soil that is equivalent to 1.5 kg of oven-dry soil. 3. Weigh _______ kg of air-dry soil (equivalent to 1.5 kg of oven-dry soil) into each pot. 4. Spread the soil from the Pot A onto a plastic sheet, and add N, P, K, S, and Zn as instructed. Mix thoroughly, and return the mixture to the pot. 5. Repeat for Pot B. Label pots A and B "incorporated". 6. Repeat step 4 with Pots C and D, but do not apply the nutrient that will be topdressed at this time. 7. Remove 1 cup of soil from the surface and level the remaining soil. 8. Add approximately 3/4 of the amount of water needed to bring the soil to FMC.

Remarks 1. The pot weights should not vary by more than +/- 10 g. 2. The air-dry soil contains _______ % moisture. Use the moisture formula from Exercise 1. 3. Check your calculations with the lab instructor before proceeding. 4. This is the incorporated treatment. Fertilizer materials and amounts to be used are given on a separated handout. 5. The incorporated and topdressed treatments are done in duplicate. 6. These pots receive the topdressed treatment. The topdressed nutrient will be applied after planting and final watering. 7. This will be used later to cover the seed. 8. Use a graduated beaker. Exact volume is not needed at this point. See step 11 for total amount of water to add and apply about 3/4 of that amount. 9. The number of seeds to plant depends on expected germination percentage.

9. After the water has infiltrated, plant 15 sorghum seeds or other number suggested by your lab instructor; cover with the soil removed in step 7.

16

Laboratory Manual Exercise 3 (Continued)

Soil Science/Agronomy/Horticulture 326

Procedure 10. Label the pot, including fertilizer source and placement.

Remarks 10. Sample Label


Irma B. & J.B. Quick Lab 301 Milorganite Topdressed - Pot C Planted 2/12/02

11. Place the pot on the balance and carefully add water to adjust the soil to its total weight when at a field moisture percentage of _____ %.

11. The total weight of the pot at FMC is: Pot, plastic bag, and label Soil (oven-dry basis) Water at FMC _______ g _______ g _______ g _______ g

12. Weigh the the fertilizer for the nutrient that will be topdressed, and spread uniformly over the soil surface of Pot C. Repeat for Pot D. 13. Place the four pots on the bench space assigned for your section, but do not cover the soil surface with the plastic liner.

Total

12. The topdressed nutrient is applied after watering so that it is not incorporated by watering. 13. Moisture accumulates on the under surface of the liner and drops onto the soil surface. This tends to incorporate the more soluble topdressed materials in a non-uniform pattern. 14. Thin any plants that emerge after initial thinning. 15. Also, notice any inhibition of germination as influenced by fertilizer placement. 16. Be sure to include all plant parts. Cut all plants at a uniform height.

14. After 7 days, thin to 5 uniform plants per pot. Water pots as needed. 15. When you water, notice any differences in plant height or color associated with fertilizer placement. 16. At the designated time, cut the plants in each pot at the soil surface and place in separate paper bags labeled with the same information as on the pot label. Dry to constant weight at 55o C. 17. Weigh the dry samples plus bags to +/0.01 g. 18. Grind the samples and put the ground tissue in labeled plastic bags for later chemical analysis. 19. Weigh the empty bags and calculate the net weight of the dry tissue.

17. Don't forget to weigh the empty bags after grinding! 18. The instructor will demonstrate the use of the tissue grinder. 19. Record your data on the Data Sheet and hand it to the instructor.

17

Laboratory Manual

Soil Science/Agronomy/Horticulture 326

EXERCISE 4
SOIL pH, pH BUFFERING CAPACITY AND ORGANIC MATTER CONTENT
Soil pH Buffering Capacity Soil pH buffering capacity is the ability of soil to resist a change in its pH when acid-forming or baseforming materials are added. The capacity to resist pH change is an extremely important characteristic of soil. Without this property, the pH of the soil would fluctuate widely whenever water passed through the profile or fertilizers or other materials were applied as a means of enhancing crop growth. In humid regions, soils without pH buffering ability would quickly become very acid and mineral weathering would accelerate drastically. Thus, agricultural sustainability of a soil is very strongly related to its pH buffering capacity. Soil pH buffering capacity is directly related to the number of pH-dependent cation exchange sites in the soil. Sources of pH-dependent charge include the weak acid functional groups on organic matter (primarily carboxylic and phenolic functional groups) and broken oxygen bonds at the edges of layer silicates and oxides of Fe and Al (which can exhibit partial positive or negative charges depending on whether they are protonated or not). Under acid conditions, most of the pH-dependent cation exchange sites are occupied by H+ or Al3+, which must be neutralized in order to raise the soil pH. [As the pH rises above 5, Al3+ is precipitated as Al(OH)3 ]. Consequently, there is a direct relationship between the pH buffering capacity of an acid soil and its pH-dependent cation exchange capacity. Neutralization of hydrogen on a pH-dependent site creates a negative charge on that site which increases the cation exchange capacity. The pH buffering capacity of Wisconsin soils is largely determined by organic matter content and to a lesser extent by clay content. Fine textured soils generally have higher organic matter and clay contents than well-drained sandy soils and thus have higher buffering capacities. In humid tropical regions where intensive weathering has taken place and soils are frequently very strongly acid (pH < 5), pH buffering capacity derives to a large extent from the protonation and deprotonation of amorphous sesquioxides and from the neutralization of exchangeable aluminum. It is not until the pH of these soils is adjusted to above pH 5.5 that organic functional groups begin to play a significant role in buffering soil pH. These soils may have a net positive surface charge (exhibit anion exchange capacity) when very acid and convert to negative surface change (exhibit cation exchange capacity) when the pH is raised. The pH buffering capacity of a soil is a direct indication of the amount of acidity or alkalinity that needs to be neutralized in order to bring about a specified change in pH. Thus, the higher the pH buffering capacity of an acid soil, the greater the amount of liming material that must be applied in order to raise the pH a specified amount. Organic soils have very high buffering capacities. It is generally not economical to lime them to as high a pH as is the practice for mineral soils. In this exercise, class members will measure pH, pH buffering capacity, and organic matter in different soils. Compilation of the results obtained by all students will permit examination of how the pH buffering capacities of Wisconsin soils differ and relate to organic matter content.

18

Laboratory Manual Exercise 4 (Continued)

Soil Science/Agronomy/Horticulture 326

Soil Organic Matter Soil organic matter can be measured several different ways. In the method used here, the soil sample is reacted with an excess of a strong oxidizing agent (chromic acid in H2 SO4 ), and the excess is titrated with a standard ferrous solution. Heat to speed the reaction is supplied by the heat of dilution of concentrated H2 SO4 . The procedure is not specific for carbon but determines any easily oxidized substance. However, experience has shown that the amount of material oxidized by this treatment is equivalent to oxidation of approximately 77% of the total carbon in soil organic matter. This value does not necessarily apply to soil organic matter from other climatic zones. Since soil organic matter contains approximately 58% carbon, a good approximation of the organic matter content can be obtained. Presence of reducing substances (chlorides, Feo ,Fe2+, Mn2+) in the soil leads to high results. To avoid the use of the heavy metal, Cr, in chromic acid and subsequent disposal problems and to speed operations, the UW Soil & Plant Analysis Lab estimates soil organic matter by weight loss when a sample is heated to 360 o C. The sample is first heated at 105 o C to remove moisture, then at 360 o C to burn off organic matter. At higher temperatures, there is the danger of weight loss from structural water in some soil clays and from decomposition of carbonates. Presence of CaSO4 *2H2 O (gypsum) or NaHCO3 in sub-humid soils leads to high estimates of organic matter determined by weight loss on ignition. The most modern methods of soil carbon analysis employ combustion of a soil sample at 600 to 1000 o C in a stream of O2 gas, with infrared detection of carbon as CO2 . Heat is applied either using an induction or resistance furnace. Above 900 o C, carbon from carbonate minerals (if present) is released so that organic carbon is not identical to total carbon. With these high temperature dry combustion methods, instrumentation costs are higher but no assumptions regarding completeness of combustion, loss on ignition of inorganic soil constituents, or average oxidation state of organic carbon are required. Reactions and Equations In this reaction, organic C, which has an average oxidation state of (0), is oxidized with the release of 4 e- per C atom: C(0) + 2 H2 O ===> C(IV)O2 + 4 H+ + 4 eThe Cr(VI) in the dichromate ion, Cr2 O7 2-, as chromic acid, undergoes reduction to Cr(III): Cr(VI)2 O7 2- + 14 H+ + 6 e- ===> 2 Cr(III)3+ + 7 H2 O And, overall, on a 12 e- basis: 3 C(0) + 2 Cr(VI)2 O7 2- + 16 H+ ===> 3 C(IV)O2 + 4 Cr(III)3+ + 8 H2 O The unit of interest for our purposes is mmol of electrons undergoing this oxidation/reduction reaction, i.e., mmol(e-). [Remember: one mole of electrons contains 6.02x1023 electrons (Avogadros number).] This unit, mmol( e-), is more relevant to the purpose than the molar concentration of the dichromate. After the chromic acid has reacted with organic carbon, the excess electron acceptors remaining as unreacted Cr(VI) are determined by electron titration with a standardized ferrous iron, Fe(II), solution, which is oxidized to Fe(III): 6 Fe2+ + Cr2 O7 2- + 14 H+ ===> 2 Cr3+ + 6 Fe3+ + 7 H2 O

19

Laboratory Manual Exercise 4 (Continued) Apparatus

Soil Science/Agronomy/Horticulture 326

Soil samples of different texture, pH and organic matter; scoop calibrated for 10 g of a "light colored silt loam" (2 million lbs/acre plow-layer); 20-mL pipets; plastic vials; pH meter; 500-mL conical flasks; 25-mL dispensers; 25- or 50-mL burettes; magnetic stirrer. Reagents KOH solutions: Dissolve 11.2 g KOH in about 800 mL of deionized water and dilute to one liter to give 0.2 M KOH. Dilute 25, 50, 100, and 200 mL of the 0.2 M KOH to one liter. The resulting concentrations will be 0, 0.005, 0.01, 0.02, and 0.04 M. Addition of 25 mL of these solutions to 10 g of soil will give the equivalent of 0, 12.5, 25, 50, and 100 mmol KOH per kg of soil, respectively. Solid NaF. Concentrated H2 SO4 . Standard 0.1667 M K2 Cr2 O7 : Dissolve 49.04 g of K2 Cr2 O7 in water and dilute to 1 liter. The concentration with respect to electrons transferred in reducing Cr(VI) to Cr(III) is 1.000 mole/L or 1.000 mmol/mL. Ferroin indicator: Dissolve 3.7 g of o-phenanthroline monohydrate and 1.74 g of FeSO4 * 7H2 O in 250 mL of water. Ferrous solution, 0.5 M: Dissolve 196.1 g of Fe(NH4 )2 (SO4 )2 * 6 H2 O in 800 mL of water containing 20 mL of concentrated H2 SO4 and dilute to 1 liter. The Fe2+ in this solution slowly oxidizes on exposure to air so it must be standardized against the dichromate solution daily. Procedure for pH and pH Buffering Capacity Procedure 1. Measure 10 g of the soil assigned to you into each of five plastic vials. 2. Add 25 mL of the solutions as shown below, using the appropriate dispenser: mmol KOH added Vial no. Solution per 10 g soil 1 Deionized water 0 2 3 4 5 0.005 M KOH 0.01 M KOH 0.02 M KOH 0.04 M KOH 0.125 0.250 0.500 1.000 Remarks 1. Use the calibrated scoop technique, which the lab instructor will demonstrate. 2. Be sure to keep a record of what solution was added to which vial.

20

Laboratory Manual Exercise 4 (Continued) Procedure 3. Shake the soil suspension intermittently for one hour. 4. Measure the pH of each suspension, using a glass electrode pH meter. 5. On graph paper, plot [mmol KOH added]/[kg(soil)] on the y-axis vs [soil pH] on the x-axis for each KOH addition. 6. From the resulting graph, find the buffer capacity (BC) from the slope of the line connecting the 0 KOH addition and the point representing each KOH addition. 7. Turn in the completed data sheet and graph to your lab instructor.

Soil Science/Agronomy/Horticulture 326

Remarks 3. Cap the vials and shake vigorously for 15 seconds every 10 minutes. 4. The instructor will standardize demonstrate the use of the pH meter. 5. and

A computer generated graph is acceptable.

6. Slope = [y2 ! y1 ]/[x2 ! x1 ] = mmol KOH/kg(soil) ! 0 pH in treated vial ! pH in vial 1

7. Next week, you will be asked to use the class data to plot pH buffering capacity vs soil organic matter.

Procedure for Determining Soil Organic Matter Procedure1 1. Weigh 1 to 2 g of oven-dried, mediumtextured soil to +/! 0.01 g and transfer to a 500mL conical flask. 2. Add 20 mL of 0.1667 M K2 Cr2 O7 and swirl to mix. 3. In a fume hood and under instructor supervision, add 20 mL conc. H2 SO4 ; swirl the flask gently for 1 minute. 4. Allow to stand for 30 minutes. Remarks 1. Weigh out 0.1 to 0.2 g for organic soils, 2 to 3 g for light-colored sandy soils. 2. Use a pipette and pipetting bulb. Molarity based on electrons transferred is 1.000 M or 1.000 mmol(e-)/mL. 3. Use gloves and eye protection! Do not get any H2 SO4 on your clothing. SO2 fumes will be generated from heat of dilution. 4. The reaction proceeds diminishes as the flask cools. slowly and

5. Dilute the suspension with about 200 mL of water. 6. Add 0.2 g of NaF and 10 drops of ophenanthroline indicator.

5. Exact volume is not important; dilution produces a clearer endpoint in a turbid solution. 6. The NaF complexes Fe3+ produced when Fe2+ is oxidized. Fe3+ interferes with the endpoint. The o-phenanthroline is a redox indicator, not a pH indicator.

21

Laboratory Manual Exercise 4 (Continued)

Soil Science/Agronomy/Horticulture 326

Procedure1 7. Place a magnet in the flask and the flask on the magnetic stirrer. Titrate with 0.5 M ferrous solution to a burgundy endpoint. The color of the solution at the beginning may be anywhere from orange-yellow to dark green, depending on the amount of organic matter in the sample. As titration proceeds, the color of the solution shifts to green, then turbid gray near the endpoint. The color changes abruptly to wine-red at the endpoint. 8. Run a reagent blank (same procedure except no soil used). 9. Calculate: Oxidized soil organic matter, mmol( e-)/g.2 Percent carbon.3 Percent soil organic matter.4

Remarks 7. If less than 4 mL of ferrous solution is used, the procedure should be repeated with a smaller sample as there is danger that the oxidation of the organic matter was not complete.

8. This determines any oxidizable material introduced in the reagents and glassware. 9. See calculations below.

10. Rinse all glassware and place in the dishpan provided.


1

10. Turn in the data sheet to your instructor.

Based on a variation of the method of Walkley and Black. Soil Sci. 37:29 (1934) as reported by D.W. Nelson and L.E. Sommers, 1996. Methods of Soil Analysis, Part 3. Chemical Methods. SSSA/ASA. Madison, WI.
2

Oxidized soil organic matter, mmol( e-)/g : mmol(e-)/g = (mL Fe2+ for blank !mL Fe2+ for sample) x (Conc. of Fe2+ solution, mmol(e-) /mL) Soil sample weight, g

Percent carbon: % C = (Oxidized soil organic matter, mmol(e-)/g ) x [0.012 g C / 4 mmol( e-)] x (1/0.77) x 100% where it is assumed that 4 mmol of e- are required to oxidize 1 mmol (12 mg or 0.012 g) of soil organic carbon and that the oxidation using the heat of dilution of concentrated sulfuric acid is 77% complete.

Percent soil organic matter (% SOM) % SOM = % C x (1/0.58) where it is assumed that the soil organic matter is 58% C by weight.

22

Laboratory Manual

Soil Science/Agronomy/Horticulture 326

EXERCISE 5
SOIL POTASSIUM BUFFER POWER
The plant availability of nutrients in soil directly relates to the quantities of the nutrients that come in contact with root surfaces. The quantities of nutrients transported to root surfaces depend on the concentrations of the nutrient ions in the soil solution and the ability of the soil to replenish the ions when plant absorption occurs. The labile forms of a nutrient include those in the soil solution and those in equilibrium with the dissolved forms. These forms vary with the nutrient and soil being considered. Labile forms of potassium that are potentially available to plants include dissolved, exchangeable, and some nonexchangeable. The K immediately available for plant nutrition is that present in the soil solution at the root surface. Replenishment of solution K from the solid phase is of great importance as the concentration of K in the soil solution is low. Of the solid-phase forms, exchangeable K is the most readily available because it is in rapid equilibrium with the soil solution. Soils that contain little exchangeable K depend on transformations from nonexchangeable forms to replenish the exchangeable and solution phases upon depletion. This nonexchangeable labile K pool is the K associated with the soil micaceous mineral, illite, or the K fixed in vermiculite. The equilibria existing between solution, exchangeable, and nonexchangeable K forms is reversible, but attainment of equilibrium with nonexchangeable K forms is relatively slow. In terms of plant nutrition, the rate of transfer between the various labile phases is of prime importance. The importance of replenishing dissolved K is illustrated by the following example: The concentration of K in the soil solution varies over an approximate range of 1 to 10 mg/L. At field moisture capacity (assuming FMC = 25% by weight), a silt loam soil weighing 2,000,000 lbs/acre plow layer contains 500,000 lbs of water per acre plow layer. This is equal to about 227,000 kg (or liters) of water. If the concentration of K is 10 mg/L, the soil solution in an acre plow layer will contain 2,270,000 mg of K or 2.27 kg of K. This is equal to 5 lbs of K. Since a 5 ton/acre alfalfa crop removes about 300 lbs of K per acre, it is apparent the amount of K in solution at any given time is less than 2% of the crops requirement. The rest must be released from solid-phase forms over the growing season. Mathematical models of plant nutrient uptake require equations describing the diffusion of nutrients from the bulk soil to the plant root. These equations include a term called the buffer power for a given nutrient. Nutrient buffer power is defined as the change in concentration of total labile nutrient per unit change in concentration of the nutrient in solution. Concentrations are expressed on a unit volume of soil basis (e.g., g/m3 soil or mg/cm3 soil). A high buffer power indicates a high ratio of labile solidphase nutrient to that in the soil solution whereas a buffer power of 1 indicates that all of the labile nutrient is in the soil solution (e.g., nitrate-N). The buffer power varies with the nutrient considered, the adsorption capacity of the soil and the degree of saturation of the nutrient adsorbing sites. For a given soil, phosphate will usually show the highest buffer power, calcium, magnesium, potassium, ammonium and sodium intermediate, and sulfate, chloride and nitrate low buffer powers. The purpose of this exercise is to obtain estimates of K buffer power of a soil that has received various K additions. You will be using the K concentration in a 4 mM Sr(NO3 )2 extract as an estimate of the K concentration in the soil solution (extracting the actual soil solution is difficult). The K extracted with 1 M NH4 OAc or Bray P-1 solution will be used as estimates of total labile K. Later, you will plot class data of [labile K/cm3 (soil)]/[dissolved K/cm3 (soil)] for soil samples with different K additions to see how buffer power varies with varying concentrations of total labile K.

23

Laboratory Manual Exercise 5 (Continued)

Soil Science/Agronomy/Horticulture 326

Materials Soil extraction flasks (50-mL conical flasks); filter tubes; filter paper; soil samples with different K additions; flame photometer; balance; scoop calibrated for 10 g of light-colored silt loam; dispenser bottles; and the following reagents: 4 mM Sr(NO3 )2 solution: Dissolve 0.846 g Sr(NO3 )2 in about 800 mL of deionized water and dilute to 1 liter. Bray P-1 extracting solution (0.03 M NH4 F+ 0.025 M HCl): Dissolve 1.11 g of NH4 F in about 900 mL of water; add 2.1 mL of concentrated HCl and dilute to 1 liter. NH4 OAc extracting solution (1 M): Add 57 mL glacial acetic acid to about 600 mL of deionized water. Add slowly, with mixing, 267 mL of concentrated NH4 OH. Cool. Adjust to pH 7.7 +/! 0.2 with HOAc or NH4 OH, and dilute to 1 liter. Standard K stock solution (315 mg/L K): Dissolve 1.0895 g of oven-dried (105 o C) KH2 PO4 in about 900 mL of deionized water. Add 5 mL of concentrated H2 SO4 as a preservative and dilute to 1 liter. This solution contains 250 mg/L P and is also used for the standard stock P solution in Exercise 11. Working K standards: Dilute the volumes of 315 mg/L K shown in the table below to 100 mL with 4 mM Sr(NO3 )2 , water, or 1 M NH4 OAc for Sr(NO 3 )2 -extractable K, Bray P-1 extractable K, or NH4 OAcextractable K, respectively. Sr(NO3 )2 -K K stock solution Conc. of K per 100 mL standard in dilute standard mL mg/L 0 1 2 4 6 8 0 3.15 6.3 12.6 18.9 25.2 Bray P-1 and NH4 OAc-K K stock solution Conc. of K per 100 mL standard in dilute standard mL mg/L 0 2 4 8 12 16 0 6.3 12.6 25.2 37.8 50.4

24

Laboratory Manual Exercise 5 (Continued)

Soil Science/Agronomy/Horticulture 326

Procedure for Determining Potassium Buffer Power Procedure 1. Label six 50-mL soil extraction flasks with numbers 1 to 6. 2. Weigh 10 g of the assigned soil and transfer to soil extraction flask [1]. 3. Weigh 2 g of the assigned soil (twice) and transfer to soil extraction flasks [2] & [3]. 4. To the 10-g sample in flask [1] and to empty flask [4], add 25 mL of 4 mM Sr(NO3 )2 solution. 5. Add 20 mL of the Bray P-1 extractant to the 2-g sample in soil extraction flask [2] and to empty soil extraction flask [5]. 6. Add 20 mL of 1 M NH4 OAc extracting solution to the 2-g sample in soil extraction flask [3] and to empty soil extraction flask [6]. Remarks 1. Keep track of what sample went in what flask. 2-4. Weigh to +/! 0.01 g. Use a funnel to transfer the soil to the extraction flask. Include a reagent blank (no soil) to account for K contamination in the reagents, glassware, filter paper, your finger tips (especially smokers), etc. Use the appropriate dispensers for the different extractants..

5. This solution is used for available K in Wisconsin and Michigan soil testing labs.

6. This solution is used to measure available K in many other states.

7. Place the soil extraction flasks on the orbital 7. Exchange reactions proceed quickly, but shaker and shake for 30 minutes. this amount of time is needed to ensure that equilibrium is reached. 8. Filter each suspension and reagent blank through Whatman No. 2 filter paper. 9. Set the flame photometer to read 0 "% T" with the extracting solution. (Use water to set 0 % T with the Bray-1 extracts.) 10. Set the flame photometer to read 100% T with 25.2 mg/L K in the Sr(NO3 )2 solution. It may be necessary to repeat steps 9 and 10. . 8. Use the funnel tubes provided. Collect 7 to 10 mL of filtrate. 9. The lab instructor will demonstrate how to use the flame photometer.

10. Standards must be made up in the same matrix as the samples. Different matrices have different viscosities and electrolyte compositions that affect K flame analysis.

25

Laboratory Manual Exercise 5 (Continued)

Soil Science/Agronomy/Horticulture 326

Procedure 11. Aspirate filtrates from the Sr(NO3 )2 flasks (1) and (4) and record % T to +/- 0.5. 12. Repeat steps 8-10 using K standards in water and your Bray P-1 extracts (2) and (5). 13. Repeat steps 8-10 using K standards in NH4 OAc and your NH4 OAc extracts (3) and (6). 14. Using the appropriate standard curve, convert % T to mg/L K in solution. 15. Record your data on the data sheet and calculate K buffer power estimates1 for your soil sample using K extracted by NH4 OAc and Bray P-1 as estimates of labile K and the K concentration in Sr(NO3 )2 as the estimate of K concentration in the soil solution. 16. Rinse all glassware and place in the dishpan provided.
1

Remarks 11-13. Aspirate both the sample and the reagent blank. If the sample reading exceeds 100 % T, dilute the sample with the appropriate extraction solution and re-run. Take the dilution factor into account in your calculations.

14. Note that the x and y axes may not be at the same scale on the three standard curves. 15. Equations for estimating the buffer power based on this one sample are given below and on the Data Sheet. Later you will plot class data to see how buffer power varies with K additions for the soil that you used.

16. The lab assistants will wash the glassware later.

This approximation assumes that the plot of Labile K vs Dissolved K is linear and passes through the origin. The class data for this soil that you will plot later will show how the buffer power (slope of the plotted line at a given point) varies with the amount of labile K in the soil. Calculations are on the next page.

26

Laboratory Manual Exercise 5 (Continued)

Soil Science/Agronomy/Horticulture 326

Calculations The units for K buffer power are: weight of labile K per unit volume of soil weight of dissolved K per unit volume of soil

Labile nutrient per unit volume of soil = labile K extracted volume of soil used Dissolved K per unit volume of soil = weight of K per unit volume of Sr(NO 3 )2 x volume of solution volume of soil The volume of solution per unit volume of soil is the volumetric fraction of water in the soil. If we want to determine the buffer power at FMC, we must convert FMC from % by weight, FMC w , to % by volume, FMC v . This requires multiplying FMCw by soil bulk specific gravity, DB (soil)/D(water). FMCv = FMCw x DB (soil) = weight of water x weight of soil/volume of soil D(water) weight of soil weight of water/volume of water where the numerical value for D(water) is assumed to be 1 for metric units of g/cm3 or Mg/m3 . Therefore: Dissolved K per unit volume of soil at FMCv = weight of K per unit volume of Sr(NO 3 )2 x FMCv . Calculation of Dissolved K, g/m3 at FMCv KSr(NO 3)2 in sample, mg/L = Measured Ksr(NO 3)2 in sample, mg/L x DF Corrected dissolved K, mg/L = KSr(NO 3)2 in sample, mg/L ! KSr(NO 3)2 in blank, mg/L Corrected dissolved K, mg/1000 cm3 = Corrected dissolved K, mg/L x 1 L/1000 cm3 Dissolved K, g/m3 (solution) = Corrected dissolved K, mg/1000 cm3 x 106 cm3 /m3 x 1 g/1000 mg (Note that dissolved K, g/m3 (solution) is numerically equal to corrected dissolved K, mg/L) Dissolved K, g/m3 (soil) = Dissolved K, g/m3 (solution) x FMCv % 100% Calculation of Labile K, g/m3 The following equations apply to Labile K determined for either NH4 OAc or Bray extractants: Corrected Labile K, mg/L = (K concentration in sample, mg/L ! K concentration in blank) x DF Labile K, g/m3 (soil) = Corrected Labile K, mg/L x 20 cm3 (solution) x DB (soil), Mg/m3 (soil) 2 g(soil) x 1000 mg/g x 1000 cm3 /L x 1 Mg/106 g Using the numerical values without including all of the units: Labile K, g/m3 (soil) = Corrected Labile K, mg/L x DB (soil) x10 Buffer Power Approximation at FMCv Buffer power approximation = [Labile K, g/m3 (soil)] [Dissolved K, g/m3 (soil)]

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EXERCISE 6
MINERALIZATION OF ORGANIC NITROGEN
The rate of mineralization of organic N is controlled by the nature of the organic material and by the suitability of the soil environment for microbiological growth. Finely divided organic material with high energy content and favorable ratios of carbon to nitrogen, phosphorus, and sulfur is decomposed much more readily than soil humus or coarse residues with high lignin content and high ratios of carbon to nitrogen, phosphorus, or sulfur. As for growth conditions, the requirements for optimum growth of microorganisms are similar to those of higher plants: soil pH near neutrality, soil moisture slightly below field capacity, and an adequate supply of all essential mineral nutrients. In this exercise, each pair of students will be assigned the same silt loam soil but the organic N source will vary. After two weeks of incubation, the soils will be analyzed for ammonium and nitrate nitrogen. Examination of the class results will reveal some of the factors regulating mineralization of organic N. Materials Silt loam soil; dried and ground organic materials with different C:N ratios; quartz sand; glass vials; polyethylene sheets, rubber bands; 5-g calibrated scoop; balances; wash bottles; dispensers; burette, Kjeldahl flasks, Kjeldahl steam distillation apparatus; and the following reagents: 2 M KCl: Add 148 g KCl to about 800 mL of deionized water in a 1-liter volumetric flask. Dissolve the salt and dilute to volume. Devarda's alloy (50% Cu, 45% Al, 5% Zn): Ball-mill the alloy to pass a 100-mesh screen and at least 75% through a 300-mesh screen. Store in a stoppered bottle. MgO: Heat "heavy" MgO in a muffle furnace at 600 to 700 o C for 2 hr. Cool in a desiccator over KOH pellets, and store in a tightly stoppered bottle. Boric acid indicator solution: Dissolve 40 g H3 BO3 in about 900 mL of deionized water. Add 25 mL of mixed indicator consisting of 0.33 g bromcresol green and 0.165 g methyl red dissolved in 500 mL of ethanol. Mix and dilute to volume. THAM, 0.050 M: Dissolve 6.057 g of tris-hydroxyamino methane (THAM), oven-dried at 105 C, in deionized water and dilute to 1 liter. Standard H2 SO4 (0.0357 M): Dilute 2 mL of concentrated H2 SO4 to 1 liter with deionized water. Mix well and standardize against 0.050 M tris-hydroxyamino methane (THAM). Calculate the amount of water that needs to be added to dilute the H2 SO4 to exactly 0.0357 M. Add the required amount of water and check the resulting concentration by titrating with the THAM solution. One mL of 0.0357 M H2 SO4 contains 0.0174 mmol of H+ which will neutralize 1 mg of NH3 -N.

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Soil Science/Agronomy/Horticulture 326

Procedure

Remarks

1. Measure one 5-g scoop of the assigned soil 1. The sand helps to provide better aeration and two 5-g scoops of quartz sand into each of promoting faster mineralization and two glass vials. discouraging denitrification of NO3 -. 2. Add 0.100 g of the assigned organic material to one of the vials and mix. 2. Weigh to +/! 0.001 g. This application is equivalent to 20 tons per acre (assuming 2 millions lbs of soil per acre). 3. Use a pipette.

3.

Add 3 mL of deionized water to each vial.

4. Cover the vials with squares of polyethylene sheeting and fasten in place with rubber bands.

4. Polyethylene is permeable to gases but not to water.

5. Label the vials and place in the incubator set at 35 +/! 2 o C. 6. After two weeks, remove the samples from the incubator. 7. Scoop 5 g of the original soil into a third vial.

5. Mineralization will be relatively rapid at this temperature. 6. You can find them in the incubator with all of the others if you labeled them properly. 7. This is a check on the amount of inorganic N in the unincubated soil. The incubated sample with no amendment applied is used to measure the amount of inorganic N mineralized from soil organic matter. 8. Mineralized N will be present as NH4 + and NO3 -. The high concentration of K+ in the KCl will displace exchangeable NH4 + into solution. 9. All of the inorganic N should be in solution. Do not try to transfer the soil to the distillation flask. Sand causes "bumping" during distillation. 10. Devarda's alloy is a reducing agent, converting NO3 - to NH4 + ; MgO raises the pH high enough to convert NH4 + to NH3 without hydrolyzing organic N. If the MgO is allowed to dissolve in the solution before connecting the flask, some NH3 may be lost.

8. Add 10 to 15 mL of 2 M KCl to each vial; shake the vial until all of the soil and sand is suspended; then decant the solution into a 100mL Kjeldahl distillation flask. 9. Repeat step 8 twice more.

10. Add one scoop of Devardas alloy (0.2 g), a drop of surfactant and, just before connecting the flask to the distillation unit, add one scoop of MgO (0.2 g).

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Laboratory Manual Exercise 6 (Continued)

Soil Science/Agronomy/Horticulture 326

Procedure 11. Place 5 mL of boric acid indicator solution into a 50-mL conical flask and position the flask under the condenser of the steam distillation apparatus. 12. Carefully connect the distillation flask to the apparatus and introduce steam. 13. Collect about 15 mL of distillate; then shut off the steam. 14. Remove the distillation flask.

Remarks 11. Boric acid traps NH3 : NH3 + H3 BO3 ----> NH4 B(OH)4 .

12. This is a delicate glass apparatus. The lab instructor will demonstrate how to use it. 13. Most of the NH3 comes over in the first 5 mL. 14. Place the distillation flask in the sink to cool.

15. Titrate the distillate to a faint pink color with the standard H2 SO4 solution. 16. Calculate the amount of N in each soil sample and the amount of N mineralized. Give the data to the instructor. 17. Rinse all glassware and place it in the dishpan provided.

15. Be sure to read the burette before and after titrating. 16. See calculations below and on the data sheet.

17. Use tap water. Glassware will be cleaned further by a lab assistant.

Chemical Reactions Involved NH3 + H3 BO3 + H2 O ===> NH4 B(OH)4 NH4 B(OH)4 + H+ ===> NH4 + + H3 BO3 + H2 O Calculations 1 mmol of H2 SO4 contains 2 mmol of H+ , therefore: (0.0357 mmol H2 SO4 /mL) x (2 mmol H+ /mL H2 SO4 ) = 0.0714 mmol H+ /mL H2 SO4 0.0714 mmol H+ /mL H2 SO4 reacts with 0.0714 mmol of NH4 B(OH)4 containing 0.0714 mmol of N (14 mg N/mmol N) x 0.0714 mmol N/mL H2 SO4 = 1.00 mg N/mL H2 SO4 N mineralized, mg = (N extracted from incubated soil, mg) ! (N extracted from original soil, mg) N mineralized from added org. N = N mineralized (org. N added) ! N mineralized (not added) N mineralized, mg/kg(soil) = N mineralized, mg x 1000 g(soil)/kg(soil) Soil weight, g(soil)

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EXERCISE 7
PLANT TISSUE TESTING
Tissue testing refers to a rapid, semi-quantitative analysis of plant tissue in the field. It is used primarily for trouble-shooting purposes but can also be used to: Call attention to the need for laboratory tests. Supplement soil testing to determine whether the fertilizer recommendation was adequate -- or excessive. Verify deficiency symptoms. Survey large areas quickly. Follow the uptake of nutrients in field research plots.

Plant analysis, on the other hand, is a quantitative analysis of one or more elements in plant tissue. It is carried out in a laboratory and is used where more precise results are required. Experience is required for reliable results with most tissue testing kits. This is so because the composition of plant tissue varies with age and portion of plant sampled. It will be affected also by weather and other factors. The N, P, and K contents of plant sap are higher early in the growing season than toward maturity. With inadequate nutrition, plants go through a period of stress about the time of flowering or early seed formation. Nutrient demands are high at this time, and the soil may not be able to supply them as fast as required. The use of starter fertilizer may provide a supply of readily available nutrients early in the season but may be inadequate later. Also, early in spring plants utilize nutrients accumulated in the soil over winter. Testing in the spring may not reflect accurately a sufficient supply of nutrients later. In the tissue testing kit used in this exercise, plant sap is analyzed for NO3 --N, H2 PO4 -, and K+ . Even plants growing under N stress will have some NO3 - accumulating overnight. This is assimilated readily into organic N in daylight. Consequently, tissue testing should not be done early in the morning. In this exercise, test strips will be used to estimate the concentrations of nitrate-N and potassium in cell sap. Cardy nitrate-nitrogen and potassium meters will also be used. The Cardy meters utilize ion-specific electrodes sensitive to nitrate and potassium ions (similar to the glass electrode used to measure hydrogen ion activity). Results from the two methods will be compared. Phosphorus in plant sap will be measured as H2 PO4 - with freshly-prepared reagents and filter paper strips. Reagents for Tissue Test Kits A complete list of the reagents employed in an early tissue testing kit and the procedures for preparing the test papers is given by Morgan and Wickstrom (1956)1 : Nitrate powder: Mix 10 g MnSO4 * H2 O, 2 g finely-powdered Zn, 4 g sulfanilic acid, and 2 g alphanaphthylamine with 25 g BaSO4 . Add 75 g citric acid and another 75 g of BaSO 4 . Thoroughly mix all ingredients. Grind any coarse materials to a fine powder before adding to the mixture. (Test strips containing these or similar ingredients are now available.2 ) P-K reagent no. 1, concentrated: (0.0032 M (NH4 )6 Mo7 O24 * 4 H2 O in 3.75 M HCl): Dissolve 4 g of ammonium molybdate in 137 mL of deionized water. Slowly, with stirring, add 63 mL conc. HCl.

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Laboratory Manual Exercise 7 (Continued)

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P-K reagent no. 1, diluted: Dilute 10 mL of concentrated P-K reagent no. 1 with 40 mL of deionized water. P reagent no. 2: Add approximately 2 g of stannous oxalate powder to 30 mL of deionized water. Shake before using. Check the chemicals. Last year's kit is useless unless the chemicals have been stored in a refrigerator. There are simple tests to check for deterioration: Nitrate strips: In good condition, the strips should be white. They turn gray as the chemicals deteriorate. Phosphate chemicals: Since saliva contains phosphate enzymes, wet an area on the test paper with your tongue. Run the P test on that area and another area where no saliva has been placed. This should give the contrast between dark blue reaction to no reaction if the chemicals are good. Potassium strips: Tips should be a bright orange color. When washed with the acid P-K reagent, the orange tip should be a pale yellow. The tips are brownish orange and brownish yellow, respectively, if the strips are too old.

The nitrate and potassium test strips are generally good for one season. Unopened test containers stored in a refrigerator and opened containers kept in a cool place away from moisture condensation may be good for more than one season. The test strips should be kept in a box separate from the liquid chemicals. The phosphorus solutions can deteriorate rapidly depending on temperature and contamination. Procedure for Using the Plant Tissue Test Kits Procedure Nitrate 1. Cut one corn plant just above soil level. Remarks Nitrate 1. Nitrate concentration is usually highest at the base of the stem because that which is not assimilated in the roots is converted to organic forms in the upper portion of the plant. 2. Be sure to use a clean pair of pliers and squeeze out enough sap to wet the tip of the test strip. 3. Nitrate reacts with the chemicals coating the tip of the test strip to give a pink to purple color.

2. With a pair of pliers, squeeze plant sap from the cut end of the stem onto the white tip of a test strip. 3. After ten seconds, observe the color at the tip of the strip and compare it with the color scale on the kit. 4. Use the nitrate table in the following section to interpret the results.

4. Record the results on your data sheet.

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Laboratory Manual Exercise 7 (Continued)

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Procedure Phosphorus 1. With a pair of pliers, squeeze plant sap from the cut end of the stem onto the test paper supplied for the P test. 2. Apply a single drop of P-K reagent no. 1 to the wetted area. 3. Next apply one drop of stannous oxalate. Phosphorus

Remarks

1. Squeeze out enough sap to wet an area equivalent at least to the size of a dime.

2. The molybdate in this reagent forms a complex with the phosphate. 3. Stannous oxalate reduces the phosphomolybdate complex to a blue colored compound.

4. From the relative intensity of the blue color that develops, estimate the relative level of P in the plant from the phosphorus table on the next page.

4. Record the results on your data sheet.

Potassium 1. With a pair of pliers, squeeze sap from the cut end of the stem onto the orange tip of the potassium test strip.

Potassium 1. Be sure to thoroughly wet the orange tip of the test strip. The K in the sap reacts with the sodium cobalti-nitrite in the test strip to form an orange compound of low solubility. 2. The acid solution dissolves the sodium cobalti-nitrite but not the orange potassium cobalti-nitrite. 3. The intensity of the orange color reflects the concentration of K in the sap.

2. After 60 seconds, dip the tip of the test strip into a small test tube containing P-K reagent no. 1 or 0.7% HNO3 . 3. After soaking for 60 seconds, remove the strip and compare the color of the test zone with the color scale on the kit. 4. Interpret the reading using the potassium table on the next page.

4. Record the results on your Data Sheet.

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Laboratory Manual Exercise 7 (Continued)

Soil Science/Agronomy/Horticulture 326

Interpretation of Tissue Test Kit Results Nitrate Estimated concentrations of nitrate associated with the color of the tip of the nitrate test strip to which plant sap has been applied are given in the table below: Approximate concentration N NO3 - - - - mg/kg - - - 0-1 0-5 2-7 10 - 30 15 - 25 60 - 100 55 - 110 250 - 500

Color White Light pink/purple Pinkish purple Deep purple

Interpretation Very low Low Medium High

Phosphorus The sufficiency of P in the test plant is estimated from the color developed on the test paper to which plant sap was applied. The interpretation is given in the table below: Color Colorless or very light blue Light blue Fair blue Deep blue Interpretation Very low Low Medium High

Potassium The sufficiency of K in the test plant is estimated from the color of the tip of the test strip to which plant sap was applied. The interpretation is given in the table below: Color Pale yellow Yellowish orange Medium orange Deep orange Interpretation Very low Low Medium High Approx. K Conc. mg/kg 0 - 250 250 - 450 700 -1000 > 1000

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Laboratory Manual Exercise 7 (Continued)

Soil Science/Agronomy/Horticulture 326

Cardy Nitrate and Potassium Meters Calibration (To be done by instructor): Calibrate the nitrate meter with standard solutions containing 150 and 2000 mg/kg NO3 - (34 and 452 mg/kg N). Follow the instructions provided with the meter. Convert values displayed as NO3 - to NO3 - - N by multiplying displayed value by 0.226. Calibrate the potassium meter using standard solutions containing 150 and 2000 mg/kg K. Follow the instructions provided with the meter.

Nitrate Determination with the Cardy Nitrate Meter Procedure 1. Cut one corn plant just above the soil. Remarks 1. Save all plant parts for dry matter analysis. all plants must be cut at the same height. 2. Use tweezers to handle the strips so as not contaminate them with perspiration from your fingertips. 3. Saturate an area approximately 1 cm x 1 cm with sap. 4. The meter should stabilize in 30 to 45 seconds. 5. mg/kg NO3 --N = mg/kg NO3 - x 0.226 Blot gently so as not to scratch the sensor.

2. Place the sampling strip provided on the sensor of the Cardy Nitrate Meter.

3. Squeeze sap from the cut end with pliers onto the sampling strip. 4. Close the cover and wait for the reading to stabilize. 5. Convert the mg/kg NO3 - reading from the meter to mg/kg NO3 --N. 6. Rinse the sensor with deionized water and blot dry with a paper towel. 7. Repeat steps 2 -5 twice.

6.

7. The procedure should be repeated to obtain a representative average measurement. In the field, the procedure would be repeated on 10 to 20 plants from different parts of the field. 8. Record the average readings on your data sheet.

8.

Calculate the average of the three readings.

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Laboratory Manual Exercise 7 (Continued)

Soil Science/Agronomy/Horticulture 326

Potassium Determination with the Cardy Potassium Meter Procedure 1. Cut one corn plant just above the soil. Remarks 1. Save all plant parts for dry matter analysis. all plants must be cut at the same height. 2. Use tweezers to handle the strips so as not contaminate them with perspiration from your fingertips. 3. Saturate an area approximately 1 cm x 1 cm with sap. 4. The meter should stabilize in 30 to 45 seconds. 5. Blot gently so as not to scratch the sensor.

2. Place the sampling strip provided on the sensor of the Cardy Potassium Meter.

3. Squeeze sap from the cut end with pliers onto the sampling strip. 4. Close the cover and wait for the reading to stabilize. 5. Rinse the sensor with deionized water and blot dry with a paper towel. 6. Repeat steps 2 -5 twice.

6. The procedure should be repeated to obtain a representative average measurement. In the field, the procedure would be repeated on 10 to 20 plants from different parts of the field. 8. Record the average readings on your data sheet.

8. Calculate the average of the three readings.

Morgan, N.D., and G.A. Wickstrom. 1956. Give your plants a blood test: Guide to quick tissue tests. Better Crops with Plant Food Magazine. American Potash Inst.
2

EM Science, Gibbstown, NJ 08027.

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EXERCISE 8
TOTAL P AND K CONCENTRATIONS IN PLANT TISSUE
Dry combustion of plant tissue in a muffle furnace drives off the organic C, H, and O as CO2 and H2 O, leaving behind carbonates, oxides, phosphates, borates, and sulfates of the cations present in the tissue. Dry combustion also volatilizes N, Cl, and some Mo and cannot be used for their analysis. Some P, B, and S will volatilize as well if precaution is not taken before combustion to ensure that there is an excess of cations over anions in the combustion vessel. If the tissue is deficient in cations, strontium acetate can be added to ensure an excess of cations without interfering with the elements normally tested for in the tissue. The inorganic compounds formed during dry combustion, with the exception of SiO2 , are readily dissolved in acid solutions. Once dissolved, the concentrations of the constituent elements can be determined by appropriate methods. The results of the analyses are expressed as percentage or mg/kg of each element in the tissue. These concentrations, when multiplied by the dry weight of the plants, give the amount of the element absorbed (total uptake). By comparing uptake from fertilized and unfertilized soils, it is possible to estimate the amount of added nutrient recovered by the crop. Materials Muffle furnace, 50-mL beakers, acid-washed filter paper, funnels, 3- and 25-mL pipettes, spectrophotometer tubes, spectrophotometer, flame photometer. 2 M HCl: Dilute 170 mL concentrated HCl to 1 liter. HNO3 - vanadomolybdate reagent: Dissolve 0.62 g of NH4 VO3 in 300 mL of hot water; cool and add 125 mL of concentrated HNO 3 . Dissolve 12 g of (NH4 )6 Mo4 O24 @4 H2 O in 400 mL of water, add to the vanadate solution, and dilute to 1 liter. Standard P solution (250 mg/L): Dissolve 1.0984 g of oven-dried (105 C) KH 2 PO4 in about 900 mL of deionized water. Add 5 mL of concentrated HNO3 as a preservative and dilute to 1 liter. (This is the same as the 315 mg/L K solution used in exercise 5.) Dilute standard P solutions: Pipette 0, 10, 20, 40, 60, and 100 mL of the 250 mg/L P standard solution into 500-mL volumetric flasks. Add 50 mL of 2 M HCl and dilute to 500 mL. The P concentrations of these standard solutions are 0, 5, 10, 20, 30, and 50 mg/L. NH4 OAc, 4 M: Add 228 mL glacial acetic acid (HOAc) to about 400 mL of deionized water. Slowly, with mixing, add 267 mL of concentrated NH4 OH. Cool. Adjust the pH to 7.0 +/! 0.2 with HOAc or NH4 OH, and dilute to 1 liter. NH4 OAc, 1 M: Transfer 250 mL of 4 M NH4 OAc to a 1-liter volumetric flask, and dilute to volume with water. Standard K solution, 5,000 mg/L: Dissolve 9.535 g of oven-dried (105 C) KCl in water and dilute to 1 liter.

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Laboratory Manual Exercise 8 (Continued)

Soil Science/Agronomy/Horticulture 326

Standard Na solution, 2,000 mg/L: Dissolve 5.084 g of oven-dried (105 C) NaCl in water and dilute to 1 liter. Concentrated K, Na standard solution; 2,000 mg/L K + 40 mg/L Na: Pipette 100 mL of 5,000 mg/L K solution and 5 mL of 2,000 mg/L Na solution into a 500-mL volumetric flask. Dilute to volume, and add a crystal of thymol as a preservative. Store in a refrigerator. Dilute K and Na standard solutions: Pipette 0, 5, 10, 15, 20, and 25 mL of 2,000 mg/L K + 40 mg/L Na solution into 500-mL volumetric flasks. Add 42 mL of 4 M NH4 OAc and 18 mL of 2 M HCl to each flask, and dilute to 500 mL.

Procedure 1. Weigh 150 to 200 mg of ground plant tissue and transfer to a 50-ml beaker. 2. Ash at 500 C for two hours.

Remarks 1. Record weights to the nearest mg.

2. Ashing at a higher temperature could result in volatilization of some K, P, S, B, Mo, Cl, and Na, (or even melt the beakers). 3. The beakers could crack if removed from the furnace while hot, and convection currents could disturb the ash. 4. Some effervescence may be seen as carbonates in the ash react with the acid. 5. The sample is diluted to get the concentrations of P and K into the desired concentration ranges for analysis.. 6. A yellow P-vanadomolybdate complex is formed. The intensity of the color is proportional to the concentration of P. 7. The color takes 10 minutes to develop fully and is stable for 2 to 3 days. 8. Fingerprints or other smudges in the optical path result in false, high P readings.

3. Cool the samples to room temperature.

4. Add 3 ml of 2 M HCl to dissolve the ash.

5. Add 25 ml of deionized water, mix and filter.

6. Pipette 3 ml of the filtrate into a spectrophotometer tube. Add 3 ml of water and 3 ml of the vanadomolybdate reagent, and mix. 7. Let stand for at least 10 minutes.

8. Wipe the bottom half of the spectrophotometer tube with lens tissue.

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Laboratory Manual Exercise 8 (Continued)

Soil Science/Agronomy/Horticulture 326

Procedure 9. Read the absorbance (red scale on spectrophotometer) of the sample at 440 nm. Convert the absorbance reading to mg/L P in solution using the standard P curve.

Remarks 9. Absorbance equals [2 ! log (% T)]. It is used in preference to % T (% of incident light transmitted through the sample) for plotting the standard curve because it gives a near linear relationship with concentration. 10. See the Calculations section below. 11. The NH4 OAc serves as a "radiation buffer" in the flame photometric determination of K, helps to prevent salt crystals from clogging the atomizer, and eliminates differences in matrix properties which might affect the flow rate through the atomizer. 12. Convert this reading to mg/L K in solution using the standard K curve.

10. Calculate % P in the tissue. 11. Pipette 3 ml of the filtrate (step 5) into a 50ml beaker or flask. Add 3 ml of deionized water and 3 ml of 1M NH4 OAc, and mix.

12. Aspirate this solution into the flame photometer, and read the % Transmission on the appropriate scale. 13. Calculate % K in the sample. .

13. See the Calculations section below. Leave your data sheet with the instructor.

Calculations %P in tissue: = [P(extract ! blank), mg/L] x 28 mL x [9mL/3mL] x [1 L/1000 mL] x 100% [tissue wt, mg] = 0.084 L x [P(extract ! blank), mg/L] x 100% [tissue wt, mg]

% K in tissue: = [K(extract ! blank), mg/L] x 28mL x [9mL/3mL] x [1 L/1000 mL] x 100% [tissue wt, mg] = 0.084 L x [K(extract ! blank), mg/L x 100% [tissue wt, mg]

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EXERCISE 9
TOTAL NITROGEN IN PLANT TISSUE
The procedure described below measures the organic nitrogen in plant tissue. It does not measure NO 3 -N quantitatively. However, in most instances NO3 --N is an insignificant fraction of the total N in plant tissue. When NO3 - is expected to be significant, addition of salicylic acid during tissue digestion results in conversion of NO3 - to NH4 + , which is measured. The majority of the N detected by this method is found in proteins. Proteins contain about 16% N. Hence, the so-called crude protein content of plant material is commonly estimated by multiplying % N by 6.25 (1 ) 0.16). In the following procedure, hot, concentrated H2 SO4 is used to oxidize the plant tissue and release the organic N in the form of NH 4 + . Selenium and copper are added as catalysts to Na2 SO4 in a "digestion mix". The Na2 SO4 raises the boiling point of the H2 SO4 . The net result is relatively rapid and complete degradation of the plant tissue. Even so, the digestion process requires 2 to 3 hours and is, therefore, completed by lab assistants prior to lab time. Materials Micro-Kjeldahl digestion apparatus, 100-mL digestion flasks, analytical balance, steam distillation apparatus, 50-mL conical flasks, burette, and the following reagents: Digestion mix: Mix thoroughly 5 g Se metal (Toxic!) and 32 g anhydrous CuSO4 with 1,000 g of anhydrous Na2 SO4 . Mixed indicator: Dissolve 0.130 g of bromcresol green and 0.065 g of methyl red in 100 mL of ethanol. Boric acid, 4%: Dissolve 40 g of H3 BO3 in about 900 mL of deionized water, add 25 mL of mixed indicator solution containing 0.33 g bromcresol green and 0.165 g methyl red dissolved in 500 mL of ethanol, and dilute to 1 liter with deionized water. Sulfuric acid, concentrated: If nitrates are to be included with "total N," dissolve 75 g of salicylic acid in one 2-liter bottle of H2 SO4 . NaOH, 15 M: Dissolve 600 g of NaOH in deionized water and dilute, when cool, to 1 liter. (Technical grade NaOH is satisfactory.) THAM, 0.050 M: Dissolve 6.057 g of tris-hydroxyamino methane (THAM), oven-dried at 105 C, in deionized water and dilute to 1 liter. Standard H2 SO4 (0.0357 M): Dilute 2 mL of concentrated H2 SO4 to 1 liter with deionized water. Mix well and standardize against 0.050 M tris-hydroxyamino methane (THAM). Calculate the amount of water that must be added to dilute the H2 SO4 to exactly 0.0357 M. Add the required amount of water, and check the resulting concentration by titrating with the THAM solution. One mL of 0.0357 M H2 SO4 contains 0.0174 mmol of H+ which will neutralize 1 mg of NH3 -N.

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Laboratory Manual Exercise 9 (Continued)

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Procedure for Determining Total Nitrogen in Plant Tissue Procedure 1. Weigh 100 to 150 mg of ground plant tissue and transfer quantitatively to a dry digestion tube. Remarks 1. Record the weight to +/! 1 mg. The digestion tube should be dry so that the tissue will not adhere to the neck. 2. Use the scoop provided. 3. Do this in a fume hood and use gloves and eye protection! Do not get any H2 SO4 on your clothing. 4. This will take 30 to 60 minutes.

2. Add 2 g of digestion mix. 3. Add 5.0 mL of concentrated H2 SO4 and allow to react for 30 minutes at room temperature.

4. Place on the digestion apparatus, and apply low heat until the initial reaction has subsided.

5. Increase the temperature to just below the boiling point of the H2 SO4 . Digest for 30 minutes after the solution becomes clear. 6. Disconnect the heaters, and allow the flasks to cool until they can be held in the hand.

5. The solution will be colored because of the Cu in the digestion mix, but it should not be turbid. 6. If the flasks cool down too far, the Na 2 SO4 will solidify, and getting it to re-dissolve may be a problem. 7. In Chem 103 you were told "Never add water to H2 SO4 ; always add H2 SO4 to water!" In the Kjeldahl procedure, there is no suitable alternative to adding water to the H2 SO4 , so be careful! Point the neck of the flask into the hood.

7. Slowly and carefully add 15 to 25 mL of deionized water with a wash bottle and allow to cool.

8. Add 5 mL of boric acid indicator solution to a 50-mL conical flask, and place the flask under the condenser of the steam distillation apparatus. 9. Slowly add 15 mL of 15 M NaOH down the side (inside) of the Kjeldahl flask, and immediately connect the flask to the distillation apparatus.

8. The boric acid traps the NH3 in the distillate as NH4 B(OH)4 .

9. The object is to get the NaOH to sink to the bottom of the flask without mixing with the H2 SO4 before the flask is connected to the still. The 15 M NaOH is denser than the diluted acid.

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Laboratory Manual Exercise 9 (Continued)

Soil Science/Agronomy/Horticulture 326

Procedure 10. Turn on the steam and distill until 15 mL of distillate have been collected.

Remarks 10. Heat will be generated when the steam mixes the NaOH and H2 SO4 . If too much H2 SO4 was added in step 3 and insufficient NaOH in step 9 to neutralize the acid, the N will remain as NH4 + instead of distilling over as NH3 . 11. One mL of this acid = 1 mg of N because the H+ concentration is 1/14 M, and the millimolar weight of N is 14 mg/mmol. (mmol x mg/mmol = mg) 12. This determines any contribution to the N measurement from the reagents used. 13. See Calculations below.

11. Titrate the distillate with 0.0357 M H2 SO4 to a pink endpoint.

12. Repeat steps 2 through 11 without soil added (a blank). 13. Calculate % N in the tissue sample, and turn in your data sheet to the instructor.

Chemical Reactions Involved NH3 + H3 BO3 + H2 O ===> NH4 B(OH)4 NH4 B(OH)4 + H+ ===> NH4 + + H3 BO3 + H2 O Calculations 1 mmol of H2 SO4 contains 2 mmol of H+ , therefore: (0.0357 mmol H2 SO4 /mL) x (2 mmol H+ /mL H2 SO4 ) = 0.0714 mmol H+ /mL H2 SO4 H+ (sample ! blank), mmol = mL H2 SO4 (sample ! blank) x 0.0714 mmol H+ /mL H2 SO4 1 mmol H+ reacts with 1 mmol of NH4 B(OH)4 which contains 1 mmol of N, therefore: N in sample, mmol = H+ (sample ! blank), mmol N in sample, mg = N in sample, mmol x 14 mg N/mmol N (The H+ concentration of 0.0714 mmol/mL H2 SO4 is used because 1 mL will react with 0.0714 mmol of N; therefore, 1 mL of H2 SO4 is the equivalent of 1 mg of N.) 0.0714 mmol N/mL H2 SO4 x 14 mg N/mmol N = 1.000 mg N/mL H2 SO4 % N in tissue = [mg N in sample] x 100% [mg of tissue] .

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Laboratory Manual

Soil Science/Agronomy/Horticulture 326

EXERCISE 10
DETERMINATION OF AVAILABLE P AND K IN SOIL

Available phosphorus exists in soils as a constituent of both organic and inorganic compounds. As organic P undergoes mineralization, the released P begins to equilibrate with the inorganic fraction. Consequently, organic P has not been found to correlate very well with plant uptake of P and is largely ignored in testing for plant-available P in soil. The rate of P uptake by plants appears to be a function of the solution P concentration at the root surface. Initially, this concentration will be that of the bulk soil solution. As uptake progresses, the concentration near the root drops, and P will have to diffuse from increasingly greater distances. If uptake of P is rapid, as in greenhouse cropping experiments and under certain optimum field conditions, the release of P from the solid phase to solution will likely become the rate-limiting step. Since the solution P concentration is a function of surface P, a test for surface P should also correlate well with plant uptake. Although a direct determination of surface P by equilibration with radioactive P is not feasible for routine analysis, various chemical extractants have been found to give high correlations with surface P. Most notable among these have been the Bray P-1 extractant (0.03 M NH4 F in 0.025 M HCl), the Mehlich extractant (0.125 M H2 SO4 + 0.05 M HCl) and the Olsen extractant (0.5 M NaHCO3 ). With the Bray extractant, the F- displaces P bound to Al and some Fe surfaces, but it does not displace Ca-bound P to any great extent. The weak acidity of the extractant may dissolve some Cabound P; but if the soil is highly calcareous, the acid will be neutralized rapidly, liberated Ca2+ will precipitate the F- as CaF2 and little P will be extracted. With the NaHCO3 extractant, the HCO 3 - ion maintains a pH of about 8.5 and, apparently, OH- displaces Fe- and Al-bound surface P and CO3 2- displaces Ca-bound P. This extractant is used by many western states that have soils containing high amounts of free CaCO3 . High concentrations of CaCO3 neutralize the HCl in the Bray extractant and P extracted initially is reprecipitated as calcium phosphate. Various acid extractants such as 0.3 M HCl, have also been used with success on acid soils. However, any calcium phosphates present will be attacked by these reagents so tests on soils containing calcium phosphate will indicate erroneously high P availabilities. The acid extractants will not give satisfactory results on alkaline soils (which usually contain calcium phosphates) nor on acid soils to which rock phosphate has been applied. Because of the many complex factors involved in P availability to plants, any soil test for P must be calibrated differently for different soils. In the field, the problem is made even more difficult by variations in the available P supply in the subsoil. The extractant used in this exercise will be Bray P-1, the most widely-used P test in the U.S.

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Laboratory Manual Exercise 10 (Continued)

Soil Science/Agronomy/Horticulture 326

Available potassium. The extractant used most commonly for measuring available soil K is 1 M NH4 OAc, which determines exchangeable plus water-soluble K. Other neutral salts may be used, but the NH4 + ion, being the same size as K+ , can displace some K at the edges of weathered micas that is inaccessible to larger hydrated ions but is available to plants. This extractant does not measure moderately available or "reserve" K, which in some soils may contribute appreciably to the available K supply to the crop. Attempts have been made to measure non-exchangeable available K but none has been generally accepted for use in soil testing laboratories. The NH4 OAc procedure extracts some K that is not immediately available to plants. That is, when plants can no longer grow in a soil depleted of K, there will still be some K extractable with NH4 OAc. The Bray P-1 extractant has a total cation charge concentration of 0.055 M compared with 1 M for NH4 OAc, but the amount of K extracted with this reagent is about 85 to 90% of that extracted with NH4 OAc. More important, however, is the fact that the amounts of K measured by the two extractants are very closely correlated. Interpretation of soil tests for available K is complicated by the fact that drying the soil sample tends to release K to exchangeable form in low-K soils and convert exchangeable K to a non-exchangeable form in high-K soils. Potassium tests on undried soils usually correlate better with plant uptake than tests on dried soils but handling undried soils in a routine testing laboratory is very difficult and is rarely used. Reagents Extracting solution (P-A) (0.03 M NH4 F in 0.025 M HCl): Dissolve 1.11 g of NH4 F in about 900 mL of water. Add 2.1 mL of concentrated HCl and dilute to 1 liter. Ammonium molybdate soln. (P-B) [0.87 M HCl, 0.0033 M (NH4 )6 Mo7 O24 * 4 H2 O, 0.08 M H3 BO3 ]: Dissolve 3.8 g of (NH4)6 Mo7 O24 * 4 H2 O in 300 mL of water at 60 C; cool. Dissolve 5.0 g H3 BO3 in 500 mL of water. Mix the two solutions, add 75 mL concentrated HCl (11.6 M), and dilute to 1 liter. Reductant solution (P-C): Prepare a stock supply of reductant powder by mixing thoroughly and grinding to a fine powder 2.5 g of 1-amino-napthol-4-sulfonic acid, 5.0 g of Na2 SO3 (sodium sulfite), and 146 g of Na2 S3 O5 (sodium metabisulfite). Dissolve 8 g of the dry powder in 50 mL of warm water. Let stand overnight if possible. A fresh reagent should be made every three weeks. (Some material may crystallize upon standing, but this does not affect the performance of the reagent.) Standard P and K solutions (250 mg/L P and 315 mg/L K): Dissolve 1.0985 g of oven-dried (105C) KH2 PO4 in about 900 mL of deionized water. Add 5 mL of concentrated HNO3 as a preservative, and dilute to 1 liter. Dilute 0, 1, 2, 4, 6, and 8 mL of this standard solution to 100 mL with water. The resulting P concentrations will be 0, 2.5, 5, 10, 15, and 20 mg/L, respectively; the K concentrations will be 0, 3.15, 6.3, 12.6, 18.9, and 25.2 mg/L. Materials 1.5-g soil scoops, 50-mL extraction flasks, 3-mL pipettes, 15-mL dispenser, Whatman no. 2 filter paper, funnel tubes, spectrophotometer tubes, oscillating shaker, spectrophotometer, flame photometer.

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Laboratory Manual Exercise 10 (Continued)

Soil Science/Agronomy/Horticulture 326

Procedure for Available Phosphorus Procedure 1. Weigh out 1.50 g of soil from each pot in Exercise 1 and transfer each soil sample to a 50-mL extraction flask labeled with the letter of the replicate from which the sample was taken. Remarks 1. Use a funnel to transfer the sample to the flask. Recall that you had three replicates in Exercise 1. Make sure you know which sample corresponds to each replicate because the soil analysis results will be related to the yield of corn tissue from each pot. 2. The blank is used to contamination in the procedure. account for

2. Add 15 mL of Bray P-1 solution to the sample and to a "blank" flask. 3. Shake for 5 minutes.

3. Equilibration is nearly complete in 5 minutes.

4. Filter the suspension through Whatman no. 2 or equivalent filter paper into a funnel tube. 5. Pipette a 3-mL aliquot of the filtrate into a clean spectrophotometer tube. 6. Add 3 mL of ammonium molybdate solution (P-B) into the same spectrophotometer tube with the pipette provided. 7. Add 5 drops of reductant solution (P-C).

4. Collect about 10 mL of filtrate.

5. Save the remaining filtrate for K analysis.

6. Ammonium molybdate forms a complex with H2 PO4 -. 7. A blue phosphomolybdate complex is formed in the presence of a reducing agent. 8. Shake the tube by hand without spilling the contents. 9. The color does not develop fully for 15 minutes and fades after 45. 10. Read absorbance for both soil and blank samples.

8. Mix the solutions.

9. Allow the solution to stand for 15 minutes, but read the color intensity before 45 min. 10. Read absorbance on the red scale of the spectrophotometer set a wavelength of 660 nm. Set a water blank at 0 absorbance. 11. Calculate P extracted in lbs/acre.

11. See Calculations at the end of this exercise and on the Data Sheet.

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Laboratory Manual Exercise 10 (Continued)

Soil Science/Agronomy/Horticulture 326

Procedure for Available Potassium Procedure 1. Use the solution left in the funnel tube for determination of K after the P aliquot has been taken. 2. Set the flame photometer to read 0 % T with water. 3. Set the flame photometer to read 100 % T with the 25.2 mg/L K solution. 4. Aspirate your filtrate from both soil and blank. 5. Use the appropriate standard curve to determine the concentration of K in the extract. 6. Calculate available K in kg/ha and in lbs/acre. Give the Data Sheet to your instructor when completed. Remarks 1. Available P and available K are determined in the same extract. 2. The instructor will demonstrate operation of the flame photometer. 3. Be sure to use the appropriate K standard solutions. 4. Read % T to +/! 0.5 % T. 5. The standard curve is plotted as % T vs K concentration in mg/L. 6. See Calculations below and on the Data Sheet.

Calculations
The calculations are done in metric units and then converted to lbs/acre ass um ing the dep th of the plow layer to be 0 .2 m (= 20 cm or 7.9 inche s). Calculation of m illigra m s of av ailable P and K per kilo gram of s oil Avail. P in soil, m g/k g soil = [P conc . (extra ct ! blank), mg/L] x 15 mL extract x 6 m L x 1L x 1000 g 1.5 g soil 3 mL 1000 mL 1 kg Avail. K in soil, mg/kg = [K co nc. (e xtrac t ! blank ), m g/L] x 15mL extract x 1 L x 1000 g 1.5 g soil 1000 mL 1 kg Calculation of kilograms of available P or K per hectare plow layer To convert the available P o r K de term ined in the lab orato ry from units o f weight pe r unit weight (m g/kg) to weight per unit volume (kg/ha plow layer), the bulk density of the soil is nee ded . Bulk den sity, D B, has units of weight per un it volum e. W hen calculating kg/ha, it is convenient to use m ega gram s pe r cub ic m eter, M g/m 3 (which is numerically equivalent to g/cm 3), as the units for D B. (1 Mg = 1000 kg; it is also called a tonne). 1 hectare = 10,000 m 2. Therefore, a hectare plow layer = 0.2 m (depth) x 10,000 m 2 (area) = 2000 m 3. The weight of this hectare plow layer, Mg/ha plow layer = D B, Mg /m 3 x 2000 m 3/ha plow layer. Available P or K in soil, kg/ha plow layer: = (Avail. P or K in soil, mg/kg) x (1kg/106 mg) x (D B, Mg /m 3) x (1000 kg/Mg) x (2000 m 3/ha plow layer) Calculation of pounds of available P or K per acre plow layer Since 1 kilogram = 2.205 pounds and 1 acre = 0.407 hectares, you can convert kilograms per hectare plow layer to pounds per acre plow layer (assuming the same plow layer depth) as follows: Available P or K in soil, lbs/acre plow layer: = (Available P or K in soil, kg/ha plow layer) x (2.205 lbs/kg) x (0.407 ha plow layer/acre plow layer)

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Soil Science/Agronomy/Horticulture 326

EXERCISE 11
DETERMINATION OF SOIL pH, LIME REQUIREMENT AND SOLUBLE SALTS
SOIL P H AND LIME REQUIREMENT The pH of a soil affects many chemical and biological properties of that soil. It affects the availability of most of the elements essential for plant growth, the activity of microorganisms, and the cation exchange capacity. Lime recommendations are designed to raise the pH of an acid soil to the optimum pH for the particular crop to be grown1 . Alfalfa, for example, grows best at pH 6.8 or above, whereas cranberries do better at a pH around 4.5. In Wisconsin, the lime requirement is calculated from the pH of the soil in water, the pH desired, the pH of the soil after reaction with SMP pH buffer2 , weight loss on ignition3 (LOI), and depth of plowing4 . Soil organic matter is estimated from LOI and, along with the SMP buffer test, estimates reserve acidity. Water pH represents active acidity. In most North-Central states, the SMP buffer test alone is used to determine the lime requirement, but Wisconsin research has given better results by including organic matter. Empirical equations used to calculate the lime requirement are shown below for pH 6.0 and pH 6.8. Equations for other pH levels are given by Kelling et al.1 LR6.0 = [1.92 x (6.0 ! pHwater) x (LOI) + 0.077(pHSMP )] x [plow depth factor] LR6.8 = [2.92 x (6.8 ! pHwater) x (LOI)] ! 0.092(pHSMP )] x [plow depth factor] The calculated lime requirements, in tons per acre, are for aglime with a neutralizing index of 60-69. The neutralizing index depends on the neutralizing value and the particle size distribution of the liming material . Soil pH measurements are affected by the soluble salt content of the soil because the addition of a cation to the soil solution will displace a small amount of H+ from the permanent charge sites (but not from pH-dependent sites): HX + K+ <=====> KX + H+, where X represents a negative charge site on a soil mineral. Consequently, the pH of a recently fertilized soil may be artificially depressed by as much as 0.5 pH unit. To circumvent this problem, some labs routinely measure soil pH in 0.01 M CaCl2 or 1 M KCl. Since a salt is added to the soil, previous fertilization will have little additional effect in lowering pH. Wisconsin soil pH values measured in 0.01 M CaCl2 or 1 M KCl typically are about 0.4 and 1.0 pH unit lower, respectively, than when measured in water. This relationship does not hold true for all soils. An increase in pH is even possible in some oxisols having a high anion exchange capacity. ____________________
1 2 3 4

Kelling, K.A., E.E. Schulte, L.G. Bundy, S.C. Combs, and J.B. Peters. 1991. UW EX Publ. A2809. Shoemak er, R.K., E.O. McLean, and P.F. Pratt. 1961. Soil Sci. Soc. Am. Proc. 25:274-277. Estimated orga nic m atter, % = 0.07 + [0.89 x LO I, %] Plow depth, inches 0 - 7.0 7.1 - 8.0 8.1 - 9.0 > 9.0 Plow dep th fac to r 1.00 1.15 1.31 1.46

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Laboratory Manual Exercise 11 (Continued)

Soil Science/Agronomy/Horticulture 326

Procedure for Determining Soil pH and Lime Requirement Materials 7.5-g soil scoop, plastic vials, 100-mL beakers, dispensers, stirring rods, glass electrode pH meter, oscillating shaker. Reagents 0.01 M CaCl2 : Dissolve 1.48 g of CaCl2 * 2 H2 O in water and dilute to 1 liter. SMP buffer: Dissolve 1.8 g p-nitrophenol and 3.0 g K2 CrO4 in about 900 mL of deionized water. Add 2.0 g of Ca(OAc)2 and 53.1 g of CaCl2 * 2 H2 O. Stir until dissolved. Add 2.5 mL of triethanolamine and mix thoroughly. After all materials have dissolved, let stand overnight; then adjust the pH to 7.7 +/! 0.02 with NaOH or HCl, using a glass electrode pH meter. Dilute to 1 liter with deionized water. Procedure 1. Measure 7.5 g of designated soil samples into each of two 40-mL plastic vails. 2. Add 10 mL of water to the first vial. Remarks 1. Use the calibrated scoops.

2. Use the dispenser. This vial is used for pH in water and SMP pH. 3. Use the dispenser. This vial is used for pH in 0.01 M CaCl2 . 4. Some time is needed for the soil pH to reach equilibrium. 5. The instructor will standardize the pH meter.

3. Add 10 mL of 0.01 M CaCl2 to the second vial. 4. Stir the samples with a glass rod; let stand 15 min. 5. Read the pH of the first two samples with a glass electrode pH meter, stirring the sample just before reading the pH. 6. Add 15 mL of SMP buffer to the first vial containing water. 7. Cap the vial and place in a horizontal position on the oscillating shaker. 8. Shake the sample for 30 minutes; then read the pH with a glass electrode pH meter.

6. Use the dispenser.

7. Shaking is much more efficient in the horizontal position. 8. This is the SMP pH. Alternatively, shake the sample for 5 min, let stand 1 hr, then shake another 5 min. 9. Fill out the first part of the Data Sheet. The equations are shown on the first page of this exercise and on the Data Sheet.

9. Calculate the lime requirements for peas (pH 6.0) and alfalfa (pH 6.8).

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Laboratory Manual Exercise 11 (Continued)

Soil Science/Agronomy/Horticulture 326

SOLUBLE SALTS All soil solutions and surface waters contain some soluble constituents as a result of geological weathering of rocks and minerals. In humid regions, these soluble constituents are carried by runoff and percolating waters to streams and oceans. In regions of high evapotranspiration and low rainfall or poor drainage, however, the soluble weathering products tend to accumulate. This accumulation is especially common on the 45% of the earth's land surface receiving less than 20 inches (500 mm) of rainfall annually. The use of irrigation water containing soluble salts further increases the salt content of soils. In the absence of leaching, water loss by evapotranspiration results in a net increase in soluble salts. Increasingly high rates of fertilizer application can lead to local salt accumulation problems, even in humid areas. Salting of highways for ice removal sometimes results in saline soils adjacent to these highways. The measurement of soluble salts has become as widely used in arid and semi-arid regions as has the pH measurement in humid areas. It serves as a rapid method for detection of toxic accumulations of salt and is a valuable aid in soil classification. Frequent analysis of soluble salts aids in determining when leaching is necessary and what the leaching requirement will be. Ground and surface water vary considerably in soluble salt content. The suitability of water for irrigation is largely dependent on its salt content. Methods for Measuring Soluble Salts Gravimetric method. The total dissolved salt content of soil extracts and waters has been determined for many years. The procedure consists of evaporating a known volume of solution and weighing the residue. The results are reported in terms of percent or ppm of salt. This method is useful but timeconsuming and dependent on the method of extraction. It may over-estimate the soluble salt content because the presence of moderately-soluble salts such as gypsum. Resistance-conductance methods. Because pure water contains few ions in solution, it is a very poor conductor of electricity. The addition of ions, however, makes water a better conductor. Materials that will not transport an electric current are said to have a high resistance. The unit of resistance is ohm, and ordinary deionized water has a resistance of about 200,000 ohms. As the salt content of a solution increases, the resistance decreases. Resistance methods were once used widely on saturated soil pastes, extracts, and solutions. Much information in the literature prior to 1950 is given in units of resistance. More recently, the results of soluble salt measurements have been expressed in units of electrical conductance (EC). The estimation of salt concentration by electrical conductance utilizes the same theory as resistance methods, since one is the reciprocal of the other: Conductance = 1/Resistance One advantage of using conductance is that it is directly proportional to salt concentration rather than inversely proportional, as are resistance units.

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Laboratory Manual Exercise 11 (Continued)

Soil Science/Agronomy/Horticulture 326

The currently accepted unit of electrical conductance is the Siemen, S, and conductivity measurements in soils are expressed as deci-siemens per meter, dS m-1 . Accepted units for electrical conductivity have changed in recent years from metric to SI measurement systems. Consequently, all but the most modern tables and instruments are marked in the older system that use the unit, mmho cm,-1 instead of the numerically equivalent SI unit, dS m -1 . (If not for the desire to maintain numerical equivalency, the preferred SI unit would have been siemens per meter, S m-1 .) The conductance of a solution is determined by measuring the current flow between two platinum electrodes connected to an alternating current voltage source when these electrodes are placed in the test solution a given fixed distance apart. Hence, the units are given as conductance per unit length (dS m-1 ). Extraction Methods Many soil:water ratios have been used for the extraction of soluble salts. However, none have been as widely accepted as the saturation extract. In most medium to fine textured soils, the moisture content of a saturated soil paste is about four times the amount present at the wilting point and twice that at field moisture capacity. By using extracts obtained from saturated soils, conductance values of soils of different texture may be compared. Obtaining a sample of the saturation extract requires a relatively large sample and vacuum extraction equipment. In the absence of vacuum extraction equipment, suspensions with extraction ratios of 1:1, 1:2, or 1:5 soil:water have been used. These are easier to work with for routine analysis than the saturation extract. The relationship between EC and crop growth is given in Table 11-1. The USDA Soil Salinity Lab classifies a soil as saline if the EC of a saturation extract exceeds 4 dS m-1 .

Table 11-1. Relationship between crop growth and salinity as measured by electrical conductivity. Electrical conductivity of the saturation extract, dS m-1 at 25 o C Non-saline Saline 0 2 4 8 16 32 Salinity Yields of very Yields of Only tolerant Only a few effects mostly sensitive crops many crops crops yield very tolerant negligible restricted restricted satisfactorily crops yield satisfactorily

Crops differ in their ability to withstand high salt concentrations. Table 11-2 groups crops according to their salt tolerance. Within any group, salt tolerance decreases from top to bottom. Spinach, for example, is less salt-tolerant than garden beet.

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Laboratory Manual Exercise 11 (Continued)

Soil Science/Agronomy/Horticulture 326

Table 11-2. Salt-tolerance of crops grouped according to ranges in the electrical conductivity of the saturation extract of saline soil corresponding to a 50% decrement of yield on saline soil from that on non-saline soil.1 High salt tolerance Med. salt tolerance Vegetable Crops EC = 10 to 12 Garden beet Kale Asparagus Spinach
2

Low salt tolerance

EC = 4 to 10 Tomato Cabbage Lettuce Potato Cucumber Forage Crops

EC = 3 to 4 Radish Celery Green beans

EC = 12 to 18 Salt grass Bermuda grass Western wheat grass Birdsfoot trefoil

EC = 4 to 12 White sweet clover Perennial ryegrass Sudan grass Alfalfa Orchard grass Brome grass Field Crops

EC = 2 to 4 White Dutch clover Meadow foxtail Alsike clover Red clover Ladino clover

EC = 10 to 16 Barley Sugar beet Canola Cotton _____________________________


1

EC = 6 to 10 Wheat Oats Rice Corn

EC = 4 Field beans

from U.S.D.A. Handbook Nol 60. 1954. Diagnosis and Improvement of Saline and Alkali Soils. EC = dS m-1 .

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Laboratory Manual Exercise 11 (Continued) Materials

Soil Science/Agronomy/Horticulture 326

10-g scoops, 100 mL beakers, dispensers, stirring rods, conductivity meter. Reagents 0.010 M KCl: Dissolve 0.7456 g of oven-dried (105 C) KCl in water and dilute to 1 liter. The electrical conductivity (EC) of this solution is 1.412 dS m-1 at 25 C. It is used to determine the cell constant of the conductivity meter. Procedure 1. Add two 10-g scoops (20 g) of designated soil to each of two 100-mL beakers. Remarks 1. One sample of the soil added to the 100-mL beakers will have had salt added; the other will not. 2. Use the burette provided. This sample is used to measure EC. 3. Some time is needed for salts to dissolve.

2. Add 40 mL of water to the soil in each of the two beakers. 3. Stir the samples with a glass rod; let stand 15 min. 4. Place the conductivity cell into the beaker of 0.010 M KCl. Read the conductivity and calculate the cell constant1. 5. Place the conductivity cell into the beaker containing the soil suspension.

4. The 0.010 M KCl has a known conductivity of 1.412 dS m-1 at 25 C. The cell constant equals the instrument reading divided by1.41. 5. Alternatively, the suspension can be poured into the conductivity cell if the cell is held upright and the air vents are closed. 6. This is the measured conductivity. 7. Multiply the measured conductivity by the cell constant, kc. 8. In medium textured soils, saturation is approximately 2 x FMC. 9. Fill out the Data Sheet and hand it to the instructor.

6. Record the conductance 7. Calculate the adjusted conductivity of the sample.2 8. Calculate the estimated soil water content at saturation 3. 9. Calculate the estimated conductivity of the saturation extract 4.
1

kc = [Conductivity of 0.010 M KCl, dS m-1 ] ) [Measured conductivity of 0.010 M KCl, dS m-1 ] = [1.41 dS m-1 ] ) [Measured conductivity of 0.010 M KCl, dS m-1 ] Adjusted conductivity of sample, dS m-1 = [Measured conductivity of sample, dS m-1 ] x kc Estimated soil water content at saturation, % = 2 x FMC % Estimated conductivity of saturation extract, dS m-1 = (Adj. cond., dS m -1 ) x 200 % ) (2 x FMC %) where 200% is the % water (dry soil basis) in a suspension with a 2:1 water:soil ratio.

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Laboratory Manual

Soil Science/Agronomy/Horticulture 326

EXERCISE 12
DEVELOPING NUTRIENT DEFICIENCY SYMPTOMS IN PLANTS GROWING IN SOLUTION CULTURE
Applications of the techniques of culturing plants in nutrient solutions range from commercial production to investigation of the mechanisms of active ion accumulation by plants and plant tissue. On the commercial scale, solution culture is frequently referred to as soil-less culture or hydroponics, and growers have developed numerous variations of the basic technique. Because of the expense involved, commercial application is normally restricted to high value crops and environments in which crops cannot be grown by conventional methods. On the research side, applications of solution culture techniques are numerous. Nutrient solutions can be rendered extremely pure (< 10 mg/kg of contaminants) and are, therefore, ideally suited for studying the essentiality of nutrients and the concentrations of elements in plants as a function of dry matter yield. By employing large solution volumes and providing for a continuous agitation of the solution, the researcher is able to maintain virtually constant ion concentrations and eliminate diffusion to root surfaces as the rate limiting step in absorption processes. Under these conditions, observed variations in ion accumulation by plants and their tissues can be ascribed to the mechanisms of ion absorption and transport by and within plant cells and tissues. Although solution techniques can be employed productively in many types of research, we must keep in mind the artificiality of the system. Results of solution culture studies cannot be extrapolated to situations where soil serves as the plant growth medium and little or no control is exerted on the plant's environment. The solution culture outlined here is based on Hoagland's solution, which over the years has undergone several modifications. The nutrient solution consists of essential major and minor elements in concentrations and ratios that prevent toxicity, maintain normal growth, and induce clear deficiency symptoms. Inclusion of Fe in chelate form proved essential for preventing Fe chlorosis, which is very difficult to control in the early stages of plant growth in solution culture. Aeration is critical, especially after the plants are two to three weeks old because oxygen diffusion through the water is too slow to support optimum root respiration in a large root system. Maintaining a well aerated system, however, tends to precipitate Fe(OH) 3 , making if difficult to maintain adequate Fe in solution. The purpose of this exercise is to introduce the technique of solution culture of plants and to allow you to view first-hand the deficiency symptoms of the essential mineral elements, N, P, K, Ca, Mg, S, Fe, Cu, Zn, B, Mo and Mn. Materials Darkened 5-liter containers, aeration system, deionized water, stock nutrient solutions (Table 12-1), 50-ml graduated cylinders , pot labels, seedlings. (The seedlings may be started in sand culture or by using germination paper. This must be planned well in advance of the time needed.)

53

Laboratory Manual Exercise 12 (Continued) Procedure 1. Select a darkened 5-liter polyethylene container. If a polyethylene container is not available, line a 5-liter pot with a clean plastic bag. 2. Add approximately 3 liters of deionized water. (Ordinary deionized water is satisfactory for !N, !P, !K, !Ca, !Mg, and !Fe treatments.) 3. Add the stock solutions listed in the column under the treatment for the element of interest as shown in Table 12-2.

Soil Science/Agronomy/Horticulture 326

Remarks 1. Light must be excluded to prevent algal growth. The plastic bag excludes contaminants which are present in most containers. 2. In this manner the stock solutions are diluted as they are added, thereby avoiding precipitation of relatively insoluble compounds such as CaHPO4 . 3. Take the pot to the stock solutions, not the solutions to the pot. The graduated cylinders must remain with their respective stock solutions; otherwise, cross-contamination will occur. 4. The lid should fit over the top of the container completely and have three 1-inch holes for seedlings and one 3/8-inch hole for the aeration tube. 5. Be careful not to damage the seedling's leaves or root system. 6. The roots should extend at least two inches below the lid and into the solution. 7. The use of more than one species will provide some indication of inter-species variation in nutrient deficiency symptoms. 8. This ensures adequate aeration for all plants in the pot. When all treatments are completed, adjust the air flow rate. 9. E.g., ! N.

4. Add deionized water to the 5-liter mark on the container and place the lid on the container.

5. Carefully remove one corn seedling from the sand culture or germination paper, and insert it through a hole in the lid. 6. Gently wrap the stem with sufficient cotton batting to hold the seedling securely. 7. Repeat steps 5 and 6 with the other plants to be used. 8. Position the lower end of the aeration tube so that it rests in the center of the bottom of the container. (See figure below.) 9. Label the container with the symbol of the omitted nutrient. 10. Add water twice weekly to bring the level of the nutrient solution to the 5-liter mark on the container. 11. Replace the nutrient solution after two weeks. 12. Note the deficiency symptoms each week as they develop.

10. Add water more frequently if necessary.

11. The nutrients in solution become depleted with time, and there is no solid phase (mineral or organic) to replenish them. 12. No report is required; however, you may be asked to describe some of the deficiency symptoms on the final examination.

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Laboratory Manual Exercise 12 (Continued)

Soil Science/Agronomy/Horticulture 326

Table 12-1. Stock nutrient solutions. Formula weight g 80.04 74.55 236.16 246.50 136.09 203.30 252.07 142.04 147.02 197.91 170.48 136.29 61.83 Nutrient content % g/L 35.0 N 52.4 K 47.6 Cl 17.0 Ca 11.9 N 9.9 Mg 13.0 S 28.7 K 22.8 P 12.0 Mg 34.9 Cl 15.9 Ca 24.6 P 32.4 Na 22.6 S 27.3 Ca 48.2 Cl 27.8 Mn 35.8 Cl 37.3 Cu 52.0 Cl 45.6 Zn 49.4 Cl 17.5 B 53.3 Mo Stock1 solution g/L 35.7 28.6 59.0 49.3 13.6 41.0 12.5 28.4 36.8 0.2715 0.0161 0.066 0.169 0.0019 Nutrient Nutrient Nutrient concentratio solution concentration mg/L mL stock/L mg/L 12,500 N 15,000 K 13,700 Cl 10,000 Ca 7,000 N 4,900 Mg 6,400 S 3,900 K 3,100 P 4,900 Mg 14,300 Cl 2,000 Ca 3,100 P 9,200 Na 6,400 S 10,000 Ca 17,800 Cl 75 Mn 97 Cl 6 Cu 8 Cl 30 Zn 32 Cl 30 B 1 Mo 5 5 5 5 5 5 5 5 5 5 5 5 5 5 62.5 N 75.0 K 16.5 Cl 50.0 Ca 35.0 N 24.5 Mg 32.0 S 19.5 K 15.5 P 24.5 Mg 71.5 Cl 10.0 Ca 15.5 P 46.0 Na 32.0 S 50.0 Ca 89.0 Cl 0.38 Mn 0.49 Cl 0.03 Cu 0.04 Cl 0.15 Zn 0.16 Cl 0.15 B 0.005 Mo

Salt NH4 NO3 KCl Ca(NO3 )2 * 4 H2 O MgSO4 * 7 H2 O KH2 PO4 MgCl2 * 6 H2 O Ca(H2 PO4 )2 * 2 H2 O Na2 SO4 * 10 H2 O CaCl2 * 2 H2 O MnCl2 * 4 H2 O CuCl2 * 2 H2 O ZnCl2 (95 %) H3 BO3

H2 MoO4 * H2 O 179.97 __________________________


1

The stock iron solution consists of 5 mM chelating agent in acid form (HEDTA, DTPA, EDDHA, or HBED), 1.35 g FeCl3 *6H2 O (5 mM), and 0.60 g NaOH (15 mM), prepared by adding the NaOH to the chelating agent in 500 mL deionized water and stirring until dissolved and then slowing adding while stirring the FeCl3 dissolved in 250 mL water. Dilute to 1 liter and store in an opaque container. (Use of EDTA or citric acid as a chelating agent for hydroponics is discouraged because of the instability of the iron chelate.) .

55

Laboratory Manual Exercise 12 (Continued)

Soil Science/Agronomy/Horticulture 326

Table 12-2. Preparation of dilute nutrient solutions from stock solutions Stock Solution NH4 NO3 KCl Ca(NO3 )2 MgSO4 KH2 PO4 MgCl2 Ca(H2 PO4 )2 Na2 SO4 CaCl2 MnCl2 CuCl2 ZnCl2 H3 BO3 H2 MoO4 Com- !N !P !K !Ca !Mg !S !Fe !Mn !Cu !Zn !B !Mo plete ------------------mL stock solution per 5 liters of dilute nutrient solutions------------------25 25 25 25 10 ------------25 25 25 25 25 10* 25 10* 25 10 ---------25 25 25 25 25 25 25 32.5 25 25 5* ------------25 25 25 25 25 25 10* 25 25 ------10 ------25 25 25 25 25 37.5 25 10* 25 10 ------------25 25 25 25 25 25 25 25 5* 10 ------25 ---25 25 25 25 25 25 25 25 10* 10 25 ---------25 25 25 25 25 25 25 25 25 10 ------------25 25 25 25 25 25 25 25 25 10 ---------------25 25 25 25 25 25 25 25 10 ------------25 ---25 25 25 25 25 25 25 10 ------------25 25 ---25 25 25 25 25 25 10 ------------25 25 25 ---25 25 25 25 25 10 ------------25 25 25 25 ----

Fe Chelate 25 25 25 25 25 25 25 25 25 25 25 25 5* * Omit when nutrient solution is changed. If these elements are omitted in the beginning, severe deficiency symptoms develop very quickly and they are not like those encountered more commonly in the field. Figure 12-1. Aeration Device for Solution Cultures.

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