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Rapid tissue tests as indicators ofyield, plant composition, and soilfertility for corn and soybeansP. W. Syltie a , S. W. Melsted a & W. M. Walker aa University of Illinois, Urbana‐Champaign, Urbana, Illinois,61801Published online: 11 Nov 2008.

To cite this article: P. W. Syltie , S. W. Melsted & W. M. Walker (1972): Rapid tissue tests asindicators of yield, plant composition, and soil fertility for corn and soybeans, Communicationsin Soil Science and Plant Analysis, 3:1, 37-49

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COMM. IN SOIL SCIENCE AND PLANT ANALYSIS, 3(l) 37-^9 (1972)

RAPID TISSUE TESTS AS INDICATORS OF YIELD, PLANTCOMPOSITION, AND SOIL FERTILITY FOR CORN AND SOYBEANS

KEY WORDS: Simple correlation analysis, cross-nutrient relationships,critical sap concentration.

P. W. Syltie, S. W. Melsted, and W. M. Walker

University of Illinois at Urbana-ChampaignUrbana, Illinois 61801

ABSTRACT

Corn (Zea mays L.) and soybean (Glycine max (L.) Merr.) rapid tissue

test data for N, P, K, Mg, and Mn were obtained at two locations from

experimental plots receiving varying rates and combinations of N, P, and

K. Soil test, plant composition, and yield data for each plot were also

obtained. Relatively precise tissue test readings were obtained by using

color standard sheets. A simple correlation matrix was used to relate

specific pairs of variables for corn and soybean data at both locations.

The correlation analysis of five rapid tissue tests with plant

composition, fertility, and yield levels for corn and soybeans revealed

several significant positive nutrient-same nutrient, and many negative

cross-nutrient relationships. Most negative correlations could be

explained by ionic antagonism effects. The K tissue test proved to be the

most applicable for both crops. Critical sap nutrient concentrations were

established for four nutrients, in the case of K through the use of

critical plant composition values.

INTRODUCTION

Rapid tissue tests, or sap analyses, have as their objective the

determination of a nutrient stress in a plant in the region where

deficiency symptoms are not visible but where a decrease in yield, or

37Copyright ® 1972 by Marcel Dekker, Inc. NO PART of this work may be reproduced or utilized in anyform or by any means, electronic or mechanical, including xerography, photocopying, microfilm, and re-cording, or by any information storage and retrieval system, without the written permission of the publisher.

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SYLTIE, MELSTED, AND WALKER

growth, may occur. This is often referred to as "hidden hunger" in

crops. The value of rapid tissue tests lies in the detection of mild

nutrient stresses, distinguishing between disease and nutrient disorders,

and supplying this information in the field while other grox.'th factors

are being observed and evaluated.

The tests are designed, where possible, in such a way that negative

results indicate a nutrient stress in the plant. This is true for

nitrate, Mg and Mn tests where these elements essentially disappear from

the plant sap with the onset of a deficiency stress. Positive tests,

therefore, only indicate degrees of luxury consumption, while negative

tests do not reflect the severity of the deficiency. For P and K, both

immobile nutrients in soil, nutrient stress can occur in the plant before

these elements disappear from the sap. As a result, the P and K tissue

tests may indicate the degree of deficiency stress, if low, and the degree

of luxury consumption if very high.

Considerable research effort was expended in the area of rapid

chemical tissue testing during the mid-1920's to the early 1950's.

During this period a host of workers including Hof fer1 , Morgan2 , Emmert3 ,

Thornton, Conner and Fraser4, Bray6, and Melsted6, to mention a few,

developed several methods for determining a plant's nutritional status on

the basis of its sap nutrient concentration.

Rapid tissue tests are based on colorimetric methods for the estimation

of a particular nutrient level in the sap as measured against a prepared set

of color standards. Lynd and Turk7 utilized permanent plastic standards,

but more recent work, including this study, has employed color paper

standards. Precision is influenced to some extent by visual limitations

in detecting color changes.

Acceptance of tissue tests by agronomists has been rather slow,

due mainly to lack of understanding of the tests and their quantitative

38

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TISSUE TESTS AS INDICATORS FOR CORN AND SOYBEANS

precision. Perhaps another reason for slow acceptance is the scarcity

of detailed studies showing how well one or more of the rapid tissue

tests for particular plant nutrients indicates respective plant composi-

tion, soil fertility, or yield levels. Only generalized studies regarding

these relationships have been made, including those of Lynd and Turk7,

Lynd, Turk and Cook8, Shear9, Yuen and Pollard10, and Melsted6.

The objective of this study was to utilize N, P, K, Mg, and Mn

chemical tissue tests from corn and soybean samples, and (a) determine

its relationship to plant composition, soil fertility, and yield data

for the same nutrient, (b) to establish critical sap nutrient concentra-

tions when possible, and (c) examine some "cross-nutrient" relationships.

MATERIALS AND METHODS

Corn and soybean yield, tissue test, and leaf composition data

were obtained from 102 experimental plots at the Brownstown and Toledo

Experiment Fields in 1968 in south central Illinois. At early tassel,

when silk was just beginning to appear, corn leaf samples (the leaf oppo-

site and below the ear) were gathered from six randomly selected plants

from plots receiving fertilizer rates ranging from 0 to 269, 0 to 58,

and 0 to 141 kg/ha of N, P, and K, respectively. Six soybean plants

per plot were sampled at Toledo when they were in the early pod stage,

utilizing the youngest mature leaf and its petiole below the crown. Soil

test data for P, K, and pH were determined by modified Bray Pj/and P2

methods11, the cobaltinitrite K method12, and the glass electrode pH

meter13, respectively. At maturity corn was harvested from each

plot and adjusted to 15.5% moisture. Individual plots were about 10 m

wide and 20 m long with rows 76 cm apart. Primary soil types at both

locations were in the Hoyleton-Cisne-Huey Association. Plant populations

for corn averaged about 56,000 plants per hectare.

39

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SYLTIE, MELSTED, AND WALKER

All leaf samples were washed and dried a short time in the field.

Three leaves from each plot were bagged, oven dried and ground, prior to

laboratory analysis. Nitrogen was determined by standard Kjeldahl

procedures and P, K, Mg and Mn were analyzed on ashed samples using

emission spectroscopy.

Tissue Testing Methods. Sap concentrations for nitrate-N, P, K, Mg and

Mn were determined using the rapid tissue tests on each of three leaves

from each plot and the values averaged. Standard color charts were

used to obtain readings as precise as possible. Standard solutions used

in preparation of the color charts were adjusted to the range of nutrient

concentrations known to be present within the corn and soybean sap. For

the K and Mg tests the actual reagents were used, while for N, P and Mn

pencil colored paper strips that matched the actual colors of the standard

were used since these reagent colors were not stable13.

For each test sap was expressed onto a filter paper test strip or

nitrate powder by squeezing the corn leaf midrib, or the base of a soybean

petiole, with a pliers to wet the reagent. Additional reagents, if

required, were applied and developed color was compared to the appro-

priate color standard.

Nitrate-N. The reagent of Nelson, Kurtz, and Bray14 was employed. One

hundred grams of dry BaS04, 10 g of M11SO4.H2O, 2 g of finely powdered

Zn (to reduce nitrate to nitrite), 75 g of citric acid, 4 g of sulfanilic

acid, and 2 g of ry-napthy lamine were finely ground as separate portions

with a pestle and mortar, then thoroughly mixed and stored in a blackened

container. Any degree of red color produced on reaction with plant sap

indicated the presence of nitrates.

Phosphorus. A Mo-blue reaction similar to that employed for color-

imetrically measuring P in the Bray P^ and P2 tests was used to measure

sap P5 . Ten g of ammonium molybdate were dissolved in 85 ml of HoO to

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TISSUE TESTS AS INDICATORS FOR CORN AND SOYBEANS

produce solution A. Solution B was composed of a cold solution of 16 ml

of H2O in 170 ml of concentrated HC1. Finally, solutions A and B were

mixed, and 2 g of boric acid (reagent grade) per 50 ml of solution were

added. A portion of this final solution was diluted 10 times for the test

reagent. A freshly-prepared dilute SnCl2»2H20 solution was used to reduce

the sap spot on the filter paper strip.

Potassium. Melsted's spot test6 method was utilized to determine sap K.

Solution A contained 0.6 g of dipicrylamine (2,2', 4,4', 6,6'-hexanitro-

diphenylamine) and 0.6 g of Na2CO3 boiled in 25 ml of H2O. Solution B was

prepared by diluting 8 ml of A to 25 ml with H2O, and solution C by diluting

10 ml of B to 15 ml with H2O. Three 8 mm diameter spots, one from each

solution A, B, and C were placed along a filter paper strip and dried.

The spots, when treated with plant sap and 0.5 N HCl, were sensitive

to K concentrations of approximately 750 ppm to 3000 ppm and were inter-

polated to as low as 600 ppm and as high as 4000 ppm K with standards.

Magnesium. Cheng and Bray's spot method16 was used to determine sap Mg.

A mixture of 0.15 g of F241 (l-hydroxy-2-naphthylazo)-5-2-naphthol-4-sulfonic

acid) and 0.5 g of sodium borate was finely ground with a pestle and mortar

and dissolved in 25 ml of hot methanol. Complexing solution A was prepared

by dissolving 10 g of potassium oxalate, 0.1 g of sodium borate, and 0.05 g

of NaHCOß in 50 ml of H2O. Complexing solution B contained 10 ml of A in

20 ml of H2O. Three 8 mm spots of the F241 solution were placed along a

filter paper strip and dried. Then a small amount of each complexing

solution was placed over separate F241 spots to produce sensitivities of

from approximately 10 to 100 ppm Mg. A saturated sodium borate solution

was used to buffer sap acidity.

Manganese. Feigl's tetrabase determination16 for Mn was utilized to

determine sap Mn. Reagent I was prepared by dissolving 2 g of K0H and 5 g

of KIO4 in 100 ml of H20. Reagent II contained 0.8 g of NaOH, 0.25 g of

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SYLTIE, MELSTED, AND WALKER

p,p'-methylenebis(N,N-dimethylaniline), and 20 ml of glacial acetic acid in

80 ml of H2O. A drop of reagent I was applied to sap expressed on a filter

paper strip, followed 30 seconds later by a drop of reagent II to give a

blue color in the presence of Mn.

STATISTICAL METHODS

Simple correlation techniques were used for analyzing data. Coefficients

of variability for each tissue test were also calculated from the three

individual plant analyses with 20 randomly selected plots. Each tissue

test was correlated with plant composition, soil fertility, and yield data,

using specific variables of agronomic interest. The correlation analysis

was performed over both Toledo and Brownstown corn, and over Toledo soybeans.

A limitation in the data analysis was the necessity for fixing absolute

values to tissue test readings at either extreme of a specific test's

range of values, e.g. negative values and those above the highest standard.

Also, there may be some question whether it was appropriate to correlate

the nitrate tissue test, a positive or negative indicator of nitrate with

other variables of interest. Recognizing limitations, correlations of sap

nitrate are presented in this report.

RESULTS AND DISCUSSION

Coefficients of variability (CVs) for tissue test values from the

three leaves selected per plot indicated variability among individual

plants under uniform growing conditions. "-rage CVs and 20 randomly

selected corn and soybean plots were 40%, .„, 10%, 23%, and 37% for the N,

P, K, Mg, and Mn tissue tests, respectively. Sap concentrations of Mn, .,

and Mg were found to be higher, on the average, for corn than for soybeans.

Concentrations of Mn were 10 to 50 or more times higher in corn than

soybeans. Soybean sap P concentrations were usually higher and more uniform

than for corn, while K concentrations varied little between the two plant

species. Some representative sap concentrations are shown in Table 1.

1*2

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TISSUE TESTS AS INDICATORS FOR CORN AND SOYBEANS

Table 1. Sap concentrations of N, P, K, Mg, and Mn for five corn and fivesoybean plots at Toledo, Illinois.

Treatment

N

13567269135202

00000

kg/ha

P

2915292915

2915292915

K

743774037

743774037

N

15712155

0.32.02.02.00.7

P

2881110012

100100100100100

Sap concentrations.

K

Corn

360011004000600

2000

Soybeans

20001800200014001900

ppm

Mg

20002000130020001200

800100060013001500

Mn

321583318

0.10.10.21.30.1

Table 2 displays selected correlation coefficients between tissue

test, plant composition and soil fertility data for the same nutrient, and

between tissue test and yield. Several correlations were significant at

the 1% probability level, while a lesser number were significant at the 5%

probability level. The nitrate tissue test correlated very well with both

plant composition (Figure 1 C) and fertilizer N for corn, but not for

soybeans. This might be expected since the soybean, a symbiotic N fixer,

supplies most of its own N. Phosphorus tissue tests correlate very well

with plant composition for corn (Figure 1 D), although soil test P levels

show only slight relationships with tissue test values in both corn and

soybeans. Soybean P fertilization levels were highly correlated with

the P tissue test. Potassium tissue tests correlate very well across all

but one parameter for corn and soybeans, indicating this tissue test to

be the most quantitatively precise of the five studied. Only soybean

«*3

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SYLTIE, MELSTED, AND WALKER

Table 2. Correlation coefficients between t issue t e s t values and plantcomposition, s o i l , t e s t , f e r t i l i z e r , and yield values for thesame nutr ient for corn and soybeans+

Factors Brownstown Toledo ToledoCorrelatedT Corn Corn Soybeans

N t t vs N 0.447**N c t vs N 0.478**P t t vs P 0.423**P t t vs P 0.273*P t t vs P 0.082P t t vs P 0.155

K t t vs K 0.857*K t t vs K 0.625**K,.,. vs K 0.829**Mgtt vs Mg 0.037Mntt vs Mn 0.957**

N t t vs Yld 0.020P t t vs Yld -0.571**K t t vs Yld 0.316*Mgtt vs Yld -0.223Mntt vs Yld -0.337*

* * P50.01 * O.OKPS0.05 +A dash indicates no correla t ion coefficientvas obtained. TSublet ters mean the following: t t= t i s sue t e s t ; pc=plantcomposition; st=soil test; f=fertilizer treatment; P^ = soil P test(adsorbedP); P2 = soil P test (adsorbed + acid-soluble P); Yld » yield.

soil test K values were not significantly correlated with K tissue tests.

The Mg tissue test showed no significant correlations with total Mg in

the leaf for either corn or soybeans. Corn leaf Mn tissue tests, however,

showed a very good relationship to total Mn in the leaf, but this correla-

tion was not significant in soybeans. This was probably due to the wider

range in soil pH on the corn plots, and thus a wide range in Mn

solubility levels. T he much lower Mn values and narrow range in the soy-

beans also may have tended to obscure the correlation.

Several highly significant tissue test-yield relationships were

observed (Table 2). For corn, the P and Mn tissue tests showed

0.426*0.490*0.628**0.1450.0260.121

0.937**0.904**0.904**0.3650.668**

0.0020.425*0.723**0.2980.485*

0.223

0.2970.373

-0.472*0.616**

0.625**0.3990.783**0.349

-0.088

-0.579**0.4020.728**

-0.206*-0.293

hk

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TISSUE TESTS AS INDICATORS FOR CORN AND SOYBEANS

ccO.B-I

R - 0.937T - 0.33 • O.O0OM9X

0.0looo eooo 300O qooo

TISSUE TEST K. PPM IN SOP

R - 0.147Y - 2.14 • 0.OIJ6X

TISSUE TEST N. PPM IN SflP

2?.5

ena

ccl.O-

0.5.1000

R - 0.625Y - 0.218 « 0.0O086X

0.5

°-0.3ut

CO

>-

<x

1300 1600 1900 2200

TISSUE TEST K. PPM IN SRP

R - 0.122Y - 0.23 « 0.00065X

20 40 SO 80TISSUE TEST P. PPM IN SfiP

F I G . 1

significant negative correlations with yield. Similarly, soybeans

displayed negative yield relationships with sap N and Mg levels, but not

with P and Mn. Only K tissue test values showed positive significant

correlations with yield for both crops. The value of the N, P, and K

tissue tests as related to plant composition, fertility, and yield data

for the same nutrient is summarized in Table 3.

—itoo

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SYLTIE, MELSTED, AND WALKER

Table 3. Summary of the value of tissue tests for corn and soybeans asdeterminants of plant composition, soil fertility, and yieldlevels for the same nutrient*

Plant compositionSoil fertilityYield

Plant compositionSoil fertilityYield

Nitrate

ExcellentGoodN. S.

N. S.N. S,GoodT

Tissue Test

Phosphorus

Corn

ExcellentPoorExcellent"1"

Soybeans

N. S.GoodPoor

Potassium

ExcellentExcellentExcellent

ExcellentGoodExcellent

*Descriptive terms have the following meanings: Excellent = all correlationcoefficients are highly significant; Good •= most correlation coefficientsare highly significant; Poor = only one correlation coefficient is signifi-cant. +This P-Yield correlation is negative, though considered to be anexceptional case. £such a correlation has little or no value for soybeans.

Tentative critical sap nutrient concentrations were established for N,

P, K, and Mn. For certain nutrients, such as N, P, and Mn, any amount of

detectable nutrient in the sap has been shown to be sufficient for normal

plant growth17. This study supports these N, P, and Mn critical levels,

since yields for corn and soybeans were usually seriously reduced only when

one or more of these tissue tests was negative. Figure 1 C illustrates how

tissue test values below a plant composition value of about 2.3% N, near

Melsted, Motto and Peck's18 critical corn leaf composition level of 3.07. N,

were' all negative, indicating plant stress. For nutrients such as K and Mg,

however, sap nutrient deficiency concentrations can be more precisely

determined within the tissue test's range. Critical K sap concentrations

.were determined by using Melsted's et al18 critical plant composition levels

of 1.90% (corn) and 2.20% (soybeans). Extension of these plant composition

values to curves of Figures 1 A, and 1 B, corresponding critical tissue test

1*6

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TISSUE TESTS AS INDICATORS FOR CORK AND SOYBEANS

values of about 3000 ppm (corn) and 2000 ppm (soybeans) were obtained. The

soybean critical value agrees with Melsted's value6, but the corn critical

value is 1000 ppm above his estimation. Critical Mg sap concentrations

could not be determined since Mg plant composition and tissue test values

were not significantly correlated. This was probably due to a narrow range

of Mg tissue and composition values caused primarily by a lack of Mg-

deficient plants at the two locations.

Some highly significant "cross-nutrient" relationships between tissue

test results and plant composition and fertility levels are shown in Table 4.

Table 4. Highly significant "cross-nutrient" relationships between tissuetest results and plant composition or soil fertility levels forToledo and Brownstown soybeans.

FactorsCorrelated* Corn Soybeans

N t t vs P p c

N t t vs MgpcPtt vs Mgpc

Ntt vs Ktt

Ntt vs Kpc

Ntt vs KfPtt vs Npc 0.441+Ptt vs Nf 0.481+

Ptt vs Ktt 0.614Ptt vs Kpc 0.684Ptt vs Kf 0.577Ktt vs Ppc 0.559Ktt vs Mgpc ' 0.867 0.660Ktt vs Mn p c 0.667Mgtt vs P2 0.612Mntt vs NpC 0.659Mntt vs Kst 0.592+Mntt vs pH s t 0.560+

*Subletters mean the following: tt = tissue test; pc = plant composition;st » soil test; f = fertilizer treatment; P2 "».soll.P test (adsorbed +acid-soluble P). "Trom Brownstown corn.

1*7

Positive

0.408

0.807

Negative

correlations

correlations

0.576

0.5400.7100.514

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SYLTIE, MELSTED, AND WALKER

Most of these correlations vere negative, supporting the contention of

Cook et al. 9 and Russell30 that as the concentrations of certain ionic

species increases, the concentration of other ions will decrease within

both plant tissue and sap (ion antagonism). Various other biological

interactions are also known to be operative that may cause these negative

tissue test-plant composition and fertility correlations.

ACKNOWLEDGMENT

This research was supported by the University of Illinois Agricultural

Experiment Station, University of Illinois at Urbana-Champaign, Urbana,

Illinois.

REFERENCES

1. Hoffer, G. N. Purdue U. Agr. Exp. Sta. Bul. 298. 1930.

2. Morgan, F. M. Connecticut Agr. Exp. Sta. Bul. 372. 1935.

3. Emmert, E. M. Kentucky Agr. Exp. Sta. Cir. 43. 1934.

4. Thornton, S. F., S. D. Connor, and R. R. Fraser. Purdue U. Agr. Exp.Sta. Cir. 204. 1945.

5. Bray R. H. Soil Sci. 60: 219-221. 1945.

6. Melsted, S. W. Better Crops with Plant Food 34 (1): 26, 42-45. 1950.

7. Lynd, J. Q., and L. M. Turk. Jour. Amer. Soc. Agron. 40: 940-941.1948.

8. Lynd, J. Q., L. M. Turk, and R. L. Cook. Agron. Jour. 42: 402-407.1950.

9. Shear, G. M. Virginia Agr. Exp. Sta. Tech. Bul. 84. 1943.

10. Yuen, S. H., and A. G. Pollard. Jour. Sei. Food Agr. 2: 537-542.1951.

11. Laverty, J. C. Soil Sci. Soc. Am. Proc. 27: 360-361. 1963.

12. Jackson, M. L. Soil chemical analysis. Prentice-Hall, Inc. EnglewoodCliffs, N. H. 498 p. 1958.

13. Syltie, P. W. Correlation studies of rapid tissue tests with yield andplant and soil analyses. M. S. Thesis, Department of Agronomy,University of Illinois, Urbana, 61801. 1971.

48

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TISSUE TESTS AS INDICATORS FOR CORN AND SOYBEANS

14. Nelson, J.L., L. T. Kurtz, and R. H. Bray. Anal. Chem. 26: 1081-1082.1954.

15. Cheng, K. L., and R. H. Bray. Soil Sci. 72: 449-458. 1951.

16. Feigl, Fritz. Spot tests in inorganic analysis. Elsevier Pub. Co.New York. 1967.

17. Melsted, S. W. Unpublished data.

18. Melsted, S. W., H. L. Motto, and T. R. Peck. Agron. Jour. 61: 17-20.1969.

19. Cook, R. L., L. S. Robertson, K. Lawton, and P. J. Rood. Soil Sci. Soc.Am. Proc. 12: 279-381. 1948.

20. Russell, R. W. Soil conditions and plant growth, 7th Ed. Interscience,New York. 1966.

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