The Evaluation of Iron Deficiency Using a Bioassay-type Test1

PHILLIP BARAK AND YONA CnEN2

ABSTRACTExperiments to establish a bioassay-type pot test for the evaluation

of iron deficiency in soils were performed. Peanut plants cv. Shulamitwere grown in small pots (500 g of soil) for 5 weeks in six soils varyingin CaCO3 content from 3.6 to 63.0%. Control and FeEDDHA (ethyl-enediamine di-o-hydroxyphenylacetic acid) treatments were em-ployed. Chlorophyll content of the leaves was measured at the endof the growth period. A significant linear correlation was found be-tween the relative chlorophyll content of leaves (FeEDDHA = 100)and the total CaCO, or active lime content (r = 0.892 and 0.940,respectively). Chlorosis was completely remediable with the additionof FeEDDHA. A mountain rendzina soil containing 63% CaCO3caused the most severe chlorosis. An experiment was conducted inwhich the chlorophyll content of FeEDDHA-treated and untreatedpeanut plants grown on this soil was compared. This experiment wasrepeated at three different times of the year. Some variability wasfound in chlorophyll content of either treated or untreated plantswhen absolute numbers were compared. However, when chlorophyllcontent of the untreated plants was expressed as percent of that ofFeEDDHA-treated ones, the resulting relative values for the threegrowth periods were as close as 46.8, 41.7, and 45.0%. The samesoil was used for further experimentation with the screening test.Three genotypes of peanuts were tested for Fe-sensitivity: Congo Red,Virginia Bunch Improved, and Shulamit. Congo Red was found tobe the most sensitive cultivar as known from former field studies.Iron-enriched peat was prepared in our laboratories and tested asa possible remedy for iron deficiency of Shulamit peanuts grown onthe rendzina soil. A positive response curve of plant chlorophyll toiron-enriched peat dosage was obtained reaching levels comparableto FeEDDHA.

The growth chamber pot test described provides a simple andefficient tool for the following purposes: (i) screening soils for potentialchlorosis problems in peanuts and possibly other plants; (ii) screeningpeanut cultivars for iron efficiency; and (iii) screening iron-containingfertilizers, and roughly estimating their efficiency and applicationrates. The iron-enriched peat which was first tested in this workexhibited positive and promising effects.

Additional Index Words: iron nutrition, iron-enriched peat, Fe-EDDHA, peanuts.

Barak, P., and Y. Chen. 1982. The evaluation of iron deficiencyusing a bioassay-type test. Soil Sci. Soc. Am. J. 46:1019-1022.

T IME-INDUCED chlorosis due to defective iron nutritionI j has long been known to cause yield reductionand even crop failure in calcareous soils. In the 1950s,an effective remedy for lime-induced chlorosis wasdeveloped but the high cost of FeEDDHA (ethyl-enediamine di-o-hydroxyphenylacetic acid), marketedby Ciba-Geigy Agricultural Chemicals as Sequestrene138, has both hindered its extensive use for field cropsand spawned intensive efforts by the chemical andfertilizer industries to develop an inexpensive substi-tute. The FeEDDHA has become the standard towhich all alternative fertilizers are compared.

Thorough testing of proposed iron chelates requireslarge-pot and field experiments. Such experiments re-quire an entire growing season and are clearly notsuited for rapid screening of chelates and applicationrates. Many field experiments designed to screen ironchelates and/or cultivars are reported, including recentpublications (Hartzook et al., 1974; Lachover andEbercon, 1972; Rodriguez de Cianzio et al., 1979).

For this reason we felt that the concept of a short-term, low cost, and simple bioassay would be desired.

1 Contribution from the Seagram Centre for Soil and Water Sci-ences, Faculty of Agriculture, The Hebrew University of Jerusalem,Rehovot, FOB 12, 76100, Israel. Received 2 Feb. 1982. Approved28 May 1982.

2 Graduate Student and Senior Lecturer of Soil Chemistry, Dep.of Soil and Water Science, Faculty of Agric., FOB 12, Rehovot,76100, Israel.

1020 SOIL SCI. SOC. AM. J. , VOL. 46, 1982

Table 1—Selected properties of experimental soils.

Samplinglocation

GevulotHabessorGilatBeit ZeraKfar RuppinMitzpeh Massua

Soil type

Loess-like sandLoess-like sandLoessValley RendzinaValley RendzinaMountain Rendzina

Texture

SandSandSandy loamHeavy clayLight dayLight clay

CEC

meq/100 g4.45.56.6

51.216.718.0

CaCO,

—————— f3.63.7

13.133.258.263.0

Activelime

fo

2.72.26.4

15.725.634.2

Specificsurface area

m!/g374470

302147130

DTPA-extractable Fe

ppm0.90.81.6

11.32.71.1

Therefore, experiments were performed to establisha bioassay-type pot test for the evaluation of irondeficiency and its remedy.

Criteria in evaluating the test were: (i) rapid de-velopment of chlorosis; (ii) severe chlorosis remedi-able with FeEDDHA; (iii) small pot size for conven-ience and high degree of utilization of fertilizer; and(iv) reproducibility without constraints of the growingseason. To this end, a suitable combination of highlime soil where chlorosis is a problem and Fe-sensitiveplant has been sought.

Peanuts (Arachis hypogaea L.) have been shownto be prone to lime-induced chlorosis in Israel andabroad (Lachover and Ebercon, 1972; Young, 1967).The sufficiency range for iron content in peanut plantshas been variously given as ranging from 50 to 300ppm Fe in dry matter for various plant parts, variousages, and varying mineral composition (Small andOhlrogge, 1973). Numerous reports find little or nocorrelation between iron contents of plants and chlo-rotic appearance as measured by chlorophyll concen-tration of leaves (Chen and Barak, 1982). Gopal (1973)has shown specifically for peanuts that the iron-to-chlorophyll ratio in leaves varies with age and positionduring the lifespan of the plant. Jacobson and Oertli(1956) have found that a correlation between iron andchlorophyll contents exists only when the iron contentof nutrient solutions is held strictly constant. Sinceambient supplies of iron are almost certainly the rulerather than the exception in soils, total iron contentof plants is not likely to represent well the iron nu-trition status of plants. On the other hand, chlorophyllcontent of leaves has been shown to be well correlatedwith iron concentration of nutrient solutions (Agar-wala and Sharma, 1961; Bar-Akiva and Lavon, 1968;Rio et al., 1978) and with chlorotic appearance of thewhole plant (Rodriguez de Cianzio et al., 1979).

Six calcareous soils of Israel and three genotypesof peanut were tested to develop the pot test. An iron-enriched peat which has been produced in our labo-ratory as a proposed replacement for FeEDDHA wasthen tested on the optimal soil-cultivar combination.Iron nutrition status was determined by comparingchlorophyll contents of treatments to those ofFeEDDHA.

MATERIALS AND METHODS

Experiment I—SoilsSix calcareous soils of varying texture, CaCO3, and active

lime contents were evaluated for the degree of chlorosisdeveloped by Arachis hypogaea cv. Shulamit. Selected soilproperties are presented in Table 1.

The bottoms of plastic pots (10-cm diameter, 9-cm height)were lined with limestone gravel. Five hundred grams ofsoil (<2 mm) were poured into the pots. Four seeds wereplanted in each pot and sufficient deionized water was addedto bring the soil to field capacity. The pots were placed ina growth chamber maintaining a temperature of 27 ± 2°C.Ten days after planting, the plants were thinned to leavethe two most vigorous seedlings. Pots were irrigated dailyby weight. Illumination was provided for 12 h per day bya bank of four Sylvania "Growlux" bulbs 40 cm above thesoil surface. The FeEDDHA at a rate of 4 mg Fe/kg of soilwas administered to one-half of the pots of each soil byaddition to irrigation water on the 14th day after seeding.

The plants were cut off 1 cm above the soil surface 35d after planting (or about 26 d after emergence). Samplesfor chlorophyll analysis were taken by removing one leafletfrom each leaf, alternating upper and lower leaflets, fromthe fifth leaf on the central stem and upward to the apex.Leaflets from both plants in a pot were combined to givea sample weighing 0.5 to 1.0 g and composed of 5 to 10leaflets.

Chlorophyll content of leaf samples was analyzed andcalculated by the method of MacKinney (1941). The leaveswere homogenized in an 80:20 acetone-to-water mixture.The homogenate was transferred to 100-ml volumetric flaskand made to volume with solvent. The leaves and extractantwere kept in the dark overnight. The optical density of theclear green extract was determined at 645 and 663 nm witha Bausch and Lomb Spectronic 210UV spectrometer. WhenOD^ exceeded 0.7, the sample was diluted with solvent

Table 2—Chlorophyll contents of Shulamit peanuts grown onsix calcareous soils with and without FeEDDHA. f

Soil

Habessor

Gevulot

Gilat

Beit Zera

Kfar Ruppin

Mitzpeh MassuaA

B

C

Treatmentt

ControlFeEDDHAControlFeEDDHAControlFeEDDHAControlFeEDDHAControlFeEDDHA

ControlFeEDDHAControlFeEDDHAControlFeEDDHAControl-

Chlorophyllcontent,

mg/g fresh weight

2.26 ± 0.072.44 ± 0.162.17 ± 0.062.61 ± 0.192.92 ± 0.173.46 ± 0.132.17 ±0.172.63 ± 0.041.75 ± 0.252.69 ± 0.07

1.41 ± 0.153.01 ± 0.221.21 ±0.122.90 ± 0.200.86 ± 0.091.91 ± 0.05

-

Relative chloro-phyll content,

FeEDDHA = 100

92.6 ± 6.7 a*(100)

83.1 ± 6.7 ab*(100)

84.4 ± 5.8 ab*(100)

82.5 ± 6.6 ab*(100)

65.1 ± 9.4 be(100)

46.8 ± 6.0(100)

41.7 ± 5.0(100)

45.0 ± 4.9(100)

44.5 ± 3.1 cpooled(A,B,C)

t Asterisk indicates control treatment not different than FeEDDHAtreatment (a = 0.05). Results are reported as mean ± SE.

t Application rate of Fe in the form of FeEDDHA was 4 mg/kg of soil.

BARAK & CHEN: EVALUATION OF IRON DEFICIENCY USING A BIOASSAY-TYPE TEST 1021

before measurement. Chlorophyll concentration of the ex-tracts was calculated by the following equation:

chlorophyll (mg/L) = 8.04 OD^ + 20.30 OD^.Results were expressed on a leaf sample fresh weight orarea basis.

The pot test was performed with four replicates. Theexperiment was repeated on Mitzpeh Massua soil in thegrowth chamber for three different times of the year whichwere labelled A, B, and C.

Experiment II—CultivarsThree genotypes of peanuts were tested for Fe-sensitivity:

Shulamit, Virginia Bunch Improved, and Congo Red. Potswere filled with 500 g of Mitzpeh Massua soil. To facilitatecomparison between cultivars, chlorophyll contents weredetermined on a leaf surface-area basis as well as on a freshweight basis. Other details are as above in Experiment I.

Experiment III—Iron-enriched PeatIron-enriched peat preparations (FeP) were dry-mixed

into Mitzpeh Massua soil at rates of 0.6, 1.2, and 2.4 g/pot.Four seeds of peanuts cv. Shulamit were planted in eachpot. As control one set of pots received no iron. Otherdetails are as above in Experiment I.

RESULTS AND DISCUSSION

Experiment I—SoilsIn treatments and soils where chlorosis developed,

chlorosis typically developed in the second or thirdleaf and became more severe in the fourth and fifthleaf. This phenomenon is thought to be due to ex-haustion of iron contained in the seed and the sub-sequent switch to dependence upon iron uptake fromthe deficient soil. Severest chlorosis was visible in theuntreated plants grown on the Mitzpeh Massua soil;the leaves ranged from yellowish green with greencoloring immediately adjacent to leaf veins to com-pletely yellowish green. Untreated plants grown inKfar Ruppin and Beit Zera soils had progressivelymore green areas adjacent to the veins; Gilat, Gevulot,and Habessor soils produced no discernable chloroticareas between veins. The FeEDDHA completely pre-vented chlorosis in Mitzpeh Massua, Kfar Ruppin,and Beit Zera soils.

The chlorophyll contents of peanuts grown with andwithout FeEDDHA in the six soils are given in Table2. Chlorophyll contents were found to express quan-titatively the differences among treatments and soilswhich have been noted qualitatively above. As maybe seen in Table 2, the three separate growth periodsfor Mitzpeh Massua, A, B, and C, sown November1979, March 1980, and August 1980, respectively,produced somewhat different values for chlorophyllcontents on a fresh weight basis. However, when chlo-rophyll contents of the untreated plants are expressedas percent of chlorophyll in FeEDDHA-treated plants,the relative chlorophyll contents of control treatmentsfor growth periods A, B, and C are 46.8, 41.7, and45.0%, respectively. The relative chlorophyll contentis therefore found to be reproducible regardless ofseason.

The relative chlorophyll content of the six untreatedsoils (as a percent of chlorophyll content of Fe-

£100

60

Z 40

Y = 9151-1.254 Xr = 0.940r2 = 0.884

J_ I _L

Y = 92.49-0.5879 Xr = 0.892r2 = 0.796

_L J_ _L(0 20 30 20 40 60

(00

80

60

ACTIVE LIME (%) CoC03 (%)

Fig. 1—Correlation of relative chlorophyll contents of peanuts grownon six calcareous soils, with active lime and CaCO, contents (av-erage ±SE).

EDDHA treatment) ranged from 44.5% (pooled) forMitzpeh Massua to 92.6% for Habessor. The MitzpehMassua soil was not under cultivation but nearby pas-ture and avocado groves were chlorotic. Agriculturalpractice at Kfar Ruppin and Beit Zera includes regularuse of FeEDDHA. At the Habessor ExperimentalStation, on the other hand, Hartzook (1972) found nosignificant yield improvement for Shulamit peanutsfertilized with FeEDDHA. The relative chlorophyllcontent of the control treatments in this pot experi-ment therefore seems to be in general agreement withthe need for and/or the response to FeEDDHA fer-tilization for sensitive crops in open fields.

Of the six soil properties presented in Table 1 > onlyCaCO3 content and active lime content (followingDrouineau, 1942) correlate well with the relative chlo-rophyll contents of the control treatments of the sixsoils (Fig. 1). In general, control treatments are morechlorotic as CaCO3 or active lime contents increase;since CaCO3 and active lime in these six soils arehighly correlated (r = 0.988), the data does not permita clear-cut decision as to which parameter is in factthe cause of lime-induced chlorosis.

Experiment II—CultivarsVisual ratings of chlorosis showed Congo Red to

be more subject to lime-induced chlorosis than Vir-ginia Bunch Improved and Shulamit. The chlorophyll

Table 3—Chlorophyll contents of peanut cultivars grownin Mitzpeh Massua soil.f

Chlorophyll content

Peanutcultivar

CongoRed

Treat-ment^

ControlFeEDDHA

FWmg/g

0.89 ±0.073.06 ±0.07

SA0ig/cm!

13.8±0.940.9 ±0.6

Relative chlorophyllcontent

(FeEDDHA = 100|

FW

29.1 ±2.4(100)

SA

28.2 ±2.3(100)

VirginiaBunch Control

Improved FeEDDHAShulamit Control

FeEDDHA

1.41 ±0.033.33 ±0.17

23.0 ±0.856.6 ±3.7

0.86±0.09 15.4±1.61.91 ±0.05 34.9±0.9

42.3 ±2.3 40.6 ±3.0(100) (100)

45.3 ±4.9 44.1 ±4.7(100) (100)

t Data are presented based on leaf fresh weight (FW] or surface area (SA).Chlorophyll contents of all control treatments are significantly lowerthan FeEDDHA (a = 0.05).

} Application rate of Fe in the form of FeEDDHA was 4 mg/kg of soil.

1022 SOIL SCI. SOC. AM. J., VOL. 46, 1982

1ZO

100

gI8 soJrJJw

38

H

a

60

40

*cd

*bc

ii 2.4 48

FeP ADDED (g/kg soil)Fig. 2—Response of relative chlorophyll content to iron-enriched peat

(FeP) added to Mitzpeh Massua soil (average ±SE). Lower-caseletters indicate results of a Newman-Keuls multiple range test.Asterisk indicates treatment not significantly different than Fe-EDDHA (a = 0.05).

contents of three peanut cultivars are presented inTable 3 on fresh weight and surface-area bases. Rel-ative chlorophyll contents of control treatments ofeach cultivar (as percent of FeEDDHA-treatmentchlorophyll content) are virtually identical whether ona fresh weight or surface-area basis.

All three cultivars demonstrated a statistically sig-nificant response to FeEDDHA. Congo Red is themost Fe-inefficient of the three cultivars in this potexperiment. This is in accord with field experimentsreported by Hartzook et al. (1974) who found CongoRed to show greater yield response to FeEDDHA thaneither Shulamit or Virginia Bunch Improved. BecauseShulamit is the major variety of peanut grown in Is-rael, experimentation with iron-peat preparations con-tinued with Shulamit rather than Congo Red.

Experiment III—Iron-enriched PeatThe response of plant chlorophyll to iron-enriched

peat (FeP) dosage is plotted in Fig. 2. Chlorophyllcontents were almost doubled at the lowest additionof FeP (1.2 g/kg). At 2.4 g FeP/kg, chlorophyll con-tents equalled that of FeEDDHA. Considering thelarge standard error, it is doubtful that further doublingof FeP levels to 4.8 g/kg improved chlorophyll content.It is noteworthy to mention that the dosages of FePtested in this experiment were used as a guideline infield experiments which were conducted. In these ex-periments, application rates of 1 to 2 g FeP/kg of soilwere successfully used for the remedy of iron defi-ciency in peanuts and gladioli grown on calcareoussoils. Results are reported elsewhere (Chen, Navrot,and Barak, 1982; Chen, Steinitz, Cohen, and Elber,1982).

SUMMARY AND CONCLUSIONSA bioassay-type pot test for the evaluation of iron

deficiency problems in soils and their remedy has beendeveloped. Peanuts grown for 5 weeks in small pots(500 g of soil) on a highly calcareous soil (63.0%CaCO3) in a growth chamber provided a simple andefficient system for the study of plant response to iron-containing fertilizers. In the absence of fertilizer,severe chlorosis is developed remediable with Fe-EDDHA or other efficient iron source. Degree of chlo-rosis was correlated with the chlorophyll content ofthe leaves. The experiments were conducted in growthchambers under constant temperature (about 27°C)and artificial light to ensure reproducibility betweenexperiments without constraint of growing season.The test was found to be useful for the estimation ofthe degree of severeness of iron deficiency in varioussoils or for screening plant cultivars for iron efficiency.In addition, the test provides a tool for the evaluationof iron-containing fertilizers. Iron-enriched peat seemsto be a promising fertilizer when adequate rates areapplied.