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Comparison of iron analysesand iron chlorosis inKentucky bluegrass cultivarsE.E. Pierson a , R.B. Clark a , D.M. Schaaf a &D.T. Lindgren ba Departments of Agronomy and Horticulture ,University of Nebraska , Lincoln, NE, 68583b U.S. Department of Agriculture AgriculturalResearch Service , University of Nebraska ,North Platte, NE, 69101Published online: 21 Nov 2008.

To cite this article: E.E. Pierson , R.B. Clark , D.M. Schaaf & D.T. Lindgren (1986)Comparison of iron analyses and iron chlorosis in Kentucky bluegrass cultivars,Journal of Plant Nutrition, 9:3-7, 867-872

To link to this article: http://dx.doi.org/10.1080/01904168609363487

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JOURNAL OF PLANT NUTRITION, 9(3-7), 867-872 (1986)

COMPARISON OF IRON ANALYSES AND IRON CHLOROSISIN KENTUCKY BLUEGRASS CULTIVARS1

KEY WORDS: Ferrous iron, Total iron, X-ray,o-Phenanthroline, Ferrozine, Mineral nutrition,Poa pratensis

E.E. Pierson, R.B. Clark, D.M. Schaaf, and D.T. Lindgren

Departments of Agronomy and Horticultureand

U.S. Department of AgricultureAgricultural Research Service

University of NebraskaLincoln, NE 68583 and North Platte, NE 69101

ABSTRACT

Iron chlorosis is a common problem for Kentucky bluegrass(Poa pratensis L.) grown on calcareous soils. Ironconcentrations are commonly used to assess cultivar differences tochlorosis. Iron concentrations in leaf clippings determined byx-ray, O-phenanthroline, and ferrozine were used to evaluate visualFe chlorosis ratings of 12 Kentucky bluegrass cultivars over twoyears. Cultivar differences for Fe concentration were greater fortotal Fe (x-ray and o-phenanthroline) than for ferrous Fe(ferrozine). Total Fe concentrations determined by o-phenanthrolinewere higher than by x-ray; ferrous Fe concentrations were relativelylow. Leaf Fe concentrations determined by each method did notcorrelate with cultivar leaf Fe chlorosis ratings. From visualchlorosis ratings, the cultivars Sydsport, Victa, and Glade werefairly tolerant and Park, Wabash, and Delta were fairly susceptibleto Fe chlorosis when grown on a calcareous soil in the field.

1Published as Paper No. 7955, Journal Series, NebraskaAgricultural Division.

867

Copyright © 1986 by Marcel Dekker, Inc. 0190-4167/86/0903-0867$3.50/0

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868 PIERSON ET AL.

INTRODUCTION

Iron chlorosis is a common problem for Kentucky bluegrass grownon calcareous soils (Harivandi and Butler, 1982). Since leafappearance in Kentucky bluegrass is important, reducing chlorosisor finding more resistant or tolerant cultivars has been the objectof various studies (Harivandi and Butler, 1980, 1982; Minner andButler, 1984). Since chlorosis of Kentucky bluegrasses has usuallybeen identified as an Fe deficiency problem, Fe concentrations inleaf material have often been used to evaluate cultivar differencesfor the disorder (Harivandi and Butler, 1982).

Even though many methods can be and have been used for Feanalysis, the objective of this study was to (1) compare total andferrous Fe in Kentucky bluegrass cultivars, and (2) relate Feconcentrations with the visual rating of Fe deficiency chlorosis inKentucky bluegrass cultivars.

MATERIALS AND METHODS

Collection and Handling Samples

Leaf clippings from 12 Kentucky bluegrass (Poa pratensisL.) cultivars were obtained from field plots at the University ofNebraska, West Central Research and Extension Center, North Platteduring 1982 and 1983. The cultivars used were 'Adelphi1, 'Baron1,'Bensun', 'Columbia', 'Delta', 'Fylking', 'Glade', 'Newport','Park', 'Sydsport', 'Victa1, and 'Wabash'. The grass cultivars weregrown on a Cozad silt loam (typic Haplustoll) soil with a pH of 7.9,P and Fe levels of 62 mg (Bray extractable) and 4.4 mg (DTPAextractable) kg" soil, respectively, and a medium level of excesslime in the 0-20 cm soil fraction. The cultivars were establishedin 1979. Samples were taken in August of 1982 and July of 1983 whenthe cultivars were showing relatively severe symptoms of chlorosis.Plots were replicated four times in a randomized complete blockdesign.

The leaf clippings were cut 3.0 cm from the soil surface withstainless steel scissors and immediately frozen. Visual chlorosisratings were made the same day samples were obtained [1 = green to 9= very severe chlorosis]. Samples were stored and transported at-5 C and freeze dried (-40 C) for 48 h before being masceratedin a fast-action reciprocal shaker (teflon pellets inside 25 mmdiameter x 50 mm long plastic containers).

Iron Analyses

Total Fe was determined in samples using energy-dispersivex-ray fluorescence (EDXRF) and colorimetric 1,10-o-phenanthroline(OPH) methods and ferrous Fe by a ferrozine (FZ) method. For eachmethod of analysis, three subsamples of each grass sample were

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KENTUCKY BLUEGRASS CULTIVARS 869

assayed. For EDXRF analysis, 100 mg was pressed into a 13 mmdiameter pellet and analyzed for Fe as described by Knudsen et al.(1981). Colorimetric total Fe was performed using a OPH methoddescribed by Pierson and Clark (1984a, 1984b) and Pierson et al.(1984b). The 100 mg subsamples of each grass sample were digestedwith 2.0 mL each of concentrated HpSO. and 30% H„02 beforesamples were assayed for Fe. Ferrous Fe in leaf tissue wasextracted in each subsample (100 mg) with 2.0 mL of 10.4 uMferrozine [3-(2-pyridyl)-5,6-bis-(4-phenylsulfuricacid)-l,2,4-triazine] in concentrated HC1 plus 15.0 mL of distilledwater and Fe determined in the samples as described by Pierson andClark (1984a, 1984b).

RESULTS AND DISCUSSION

Kentucky bluegrass cultivars showed differences for visualchlorosis ratings (Table 1). Sydsport, Victa, and Glade had thelowest visual Fe chlorosis ratings and Park, Wabash, and Delta hadthe highest chlorosis ratings both years when grown on thecalcareous soil at North Platte, NE. Some, but not all of thecultivars used in this study, showed similar responses to ironchlorosis when grown on a calcareous soil in Colorado (Harivandi andButler, 1982).

Total Fe determined by the OPH and EDXRF methods both showedfew cultivar differences for leaf Fe concentrations in either yearof the test (Table 1). OPH-Fe values were considerably higher thanthe EDXRF-Fe values. The OPH-Fe concentration values were lower in1982 than 1983. On the other hand, EDXRF-Fe concentrations werehigher in 1982 than 1983. Ferrous Fe (FZ-Fe) concentrations weremuch lower than total Fe concentrations (Table 1). Cultivardifferences for ferrous Fe were minimal. Variability for OPH-Fe andEDXRF-Fe means were greater than for FZ-Fe means.

No significant correlation between total or ferrous Feconcentrations and visual Fe chlorosis was noted for the cultivars(highest r = -0.49 for FZ-Fe in 1983). Other correlation valuesbetween Fe concentration and visual chlorosis ratings were 0.35 orlower.

These results showed that leaf Fe concentrations were notclosely related to the degree of visual leaf Fe chlorosis symptomsin the Kentucky bluegrass cultivars grown in the field. The methodof Fe determination and the source of Fe analyzed for were notparticularly important. Other studies with Kentucky bluegrasscultivars and blends showed Fe to be related to visual chlorosisratings (Harivandi and Butler, 1982). The method of Fedetermination in the latter study was by atomic absorptionspectroscopy which is another method for total Fe. The Feconcentration means also had relatively high variability and only afew genotype differences to Fe were noted (Harivandi and Butler,

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Table 1. Visual Fe deficiency chlorosis ra t ings (VCR) and leaf £-phenanthroline-Fe (OPH-Fe-), x-ray-Fe(EDXRF-Fe), and ferrozine-Fe (FZ-Fe) concentrations of 12 Kentucky c u l t i v a r s .

Cultivar

AdelphiBaronBens unColumbia

DeltaFylkingGladeNewport

ParkSydsportVictaWab ash

Mean

LSD (0.05)

VCRf

5.05.85.25.0

6.05.54.84.8

6.23.84.06.2

5.2

0.6

0PH-Fe

134168141270

140132226144

154218177188

174

54

1982

EDXRF-Fe

76.885.772.199.8

74.479.366.585.7

63.287.277.299.2

80.6

31.9

FZ-Fe

11.311.411.510.4

10.29.8

12.311.4

10.910.610.711.6

11.0

2.0

VCR+

5.06.24.85.5

7.25.23.55.2

7.22.53.87.2

5.3

0 .8

OPH-Fe

476360471430

466458432472

531517694545

488

175

1983

EDXRF-Fe

57.355.522.349.4

57.664.948.541.2

43.145.665.654.6

50.4

22.4

FZ-Fe

10.010.49.69.9

9.99.49.19.9

9.510.010.011.2

9.9

1.2

1 » green, 9 » very severe chlorosis.

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KENTUCKY BLUEGRASS CULTIVARS 871

1982). Field grown plants generally have higher and more variableFe concentrations than laboratory grown plants.

More recently, ferrous Fe has been used to relate plantcultivar tolerance or susceptibility to Fe deficiency chlorosis(Katyal and Sharma, 1980; Pierson et al., 1984a, 1984b; Takkar andKaur, 1984) since ferrous Fe has been considered by someinvestigators to be the "active" form of Fe in plants (Brown et al.,1979; DeKock, 1981; Katyal and Sharma, 1980). The Kentuckybluegrass cultivars used in this study showed few differences toferrous Fe. Concentrations of ferrous Fe were small compared to thetotal Fe concentrations noted. Ferrous Fe values were not onlysmall, but showed little difference among cultivars or with visualchlorosis ratings. Ferrous Fe was not shown to be closely related tovisual Fe deficiency chlorosis ratings in laboratory grown drybeans, soybeans, sorghum, or maize (Pierson et al., 1984a, 1984b).In these latter studies, total Fe was more closely related to visualchlorosis ratings than ferrous Fe. Even though total Fe has beenrelated to visual chlorosis ratings in some plants (Pierson et al.,1984a, 1984b), total Fe was not related to visual Fe deficiencychlorosis ratings in the Kentucky bluegrass cultivars of this studywhen grown in the field.

Studies have shown that chlorotic leaves may contain higher Feconcentrations than green leaves (Bennett, 1945, DeKock, 1981;DeKock et al., 1975; Oserkowsky, 1933). The Fe concentrations notedin the leaves of the Kentucky bluegrass cultivars with relativelysevere visual Fe chlorosis tended to be as high or higher thanthosefound in leaves with less severe Fe chlorosis symptoms. The Feconcentrations noted in the leaves of these Kentucky bluegrasscultivars were as high or higher than Fe concentrations found ingreen leaves of many normal plants (Clark et al., 1981; Harivandiand Butler, 1982). Iron £er_ ̂ e_ did not appear to be limiting,therefore, factors other than Fe appeared to be involved inenhancing the chlorosis symptoms noted in the Kentucky bluegrasscultivars.

REFERENCES

Bennett, J.P. 1945. Iron in leaves. Soil Sci. 60:90-105.

Brown, J.C., H.M. Cathey, J.H. Bennett, and R.W. Thimijan. 1979.Effect of light quality and temperature on Fe3+ reduction, andchlorophyll concentration in plants. Agron. J. 71:1015-1021.

Clark, R.B., J.C. Brown, R.A. Olsen, and J.H. Bennett. 1981.Biological effects on plants, p. 272-338. In Multimediacriteria for iron and compounds. Environmental ProtectionAgency, Cincinnati, OH.

DeKock, P.C. 1981. Iron nutrition under conditions of stress. J.Plant Nutr. 3:513-521.

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DeKock, P.C., A. Hall, and R.H.E. Inkson. 1979. Active iron inplant leaves. Ann. Bot. 43:737-740.

Harivandi, M.A., and J.D. Butler. 1980. Iron chlorosis of Kentuckybluegrass cultivars. HortSci. 15:496-497.

Harivandi, M.A., and J.D. Butler. 1982. Factors associated withiron chlorosis of Kentucky bluegrass cultivars. J. Plant Nutr.5:569-573.

Katyal, J.C., and B.D. Sharma. 1980. A new technique of plantanalysis to resolve iron chlorosis. Plant Soil 55:105-118.

Knudsen, D., R.B. Clark, J.L. Denning, and P.A. Pier. 1981. Plantanalysis of trace elements by x-ray. J. Plant Nutr. 3:61-75.

Minner, D.D., and J.D. Butler. 1984. Correcting iron deficiency ofKentucky bluegrass. HortSci. 19:109-110.

Oserkowsky, J. 1933. Quantitative relation between chlorophyll andiron in green and chlorotic pear leaves. Plant Physiol.8:449-468.

Pierson, E.E., and R.B. Clark. 1984a. Chelating agent differencesin ferrous iron determinations. J. Plant Nutr. 7:91-106.

Pierson, E.E., and R.B. Clark. 1984b. Ferrous iron determinationin plant tissue. J. Plant Nutr. 7:107-116.

Pierson, E.E., R.B. Clark, D.P. Coyne, and J.W. Maranville. 1984a.Plant genotype differences to ferrous and total iron in emergingleaves. II. Dry beans and soybeans. J. Plant Nutr.7:355-369.

Pierson, E.E., R.B. Clark, J.W. Maranville, and D.P. Coyne. 1984b.Plant genotype differences to ferrous and total iron in emergingleaves. I. Sorghum and maize. J. Plant Nutr. 7:371-387.

Takkar, P. N., and N. P. Kaur. 1984. HC1 method for Fe 2 +

estimation to resolve iron chlorosis in plants. J. Plant Nutr.7:81-90.

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