Summary Investigations into iron deficiency have been hindered by the lack of a satisfactory diagnostic tissue test, which in turn results from the total iron content ...
Plant and Soil 97, 353-359 (1987).
Ms. 6652
9 1987 Martinus NijhoffPublishers, Dordrecht. Printed in the Netherlands.
D i a g n o s i s o f iron deficiency in groundnut, Arachis hypogaea L. J.K. RAO, K.L. SAHRAWAT** and J.R. BURFORD
International Crops Research Institute for the Semi-Arid Tropics (ICRISA 7"), ICRISA T Patancheru P.O., A.P. 502324, India Received 25 November 1985. Revised June 1986
Key words iron
Extractable iron
Fresh tissue analysis
Leaf age
Ortho-phenanthroline
Total
Summary Investigations into iron deficiency have been hindered by the lack of a satisfactory diagnostic tissue test, which in turn results from the total iron content of plant tissue commonly being an unreliable index of the iron status. Our measurements of chlorotic and normal leaves of field grown groundnut (Arachis hypogaea L.) showed that total iron was unsatisfactory as the measure of iron status of plant tissue. It was found that iron status was better assessed from an estimate of the ferrous iron content of fresh leaf materials obtained by extraction with o-phenanthroline. Extractable iron content increased with leaf age. Chlorotic buds or the first fully opened leaf always contained less than 6/tg extractable-Fe/g fresh tissue.
Introduction Chlorosis is widespread in groundnut (ArachishypogaeaL.) grown on calcareous and alkaline soils in India. It is suspected that alkalinityinduced iron deficiency is the primary cause because the visual symptoms are very consistent with those caused by iron deficiency, yet the response to iron applications has been very variable. It is possible that other nutrient deficiencies may have been present7; diagnostic tests are therefore needed to aid in interpreting the reasons for success or otherwise of the amelioration techniques. There is not a widely-accepted satisfactory diagnostic tissue test for iron, because total iron concentrations of plant tissue do not provide a suitable index of the iron status of p l a n t s 4 , 8,9,13"
Several techniques based on plant tissue analysis have been proposed for diagnosis of iron deficiency in plants (for review seeS'l~). Various extractants have been proposed to extract the fraction of total iron, which is metabolically active and is related to occurrence of iron chlorosis. These extractants include water, dilute acids (hydrochloric acid, acetic acid, oxalic acid and citric acid), dilute NaOH, chelating agents such as EDTA, DTPA, tartaric acid and some organic solvents including 2,2' dipyridyl and its derivatives, o-phenanthroline and several other compounds. * Approved for publication as ICRISAT Journal Article No. 307. ** Corresponding author.
353
354
RAO, SAHRAWAT AND BURFORD
Bar-Akiva e t al. 3 suggested that peroxidase activity was a useful tissue test for diagnosing iron deficiency in vegetable crops. Later on the technique proposed for measuring peroxidase activity was modified 2, wherein some derivatives of benzidine rather than benzidine itself were suggested for developing blue colour which is indicative of the enzymatic activity and the active iron in the leaf tissue. Mehrotra et al. H tested 14 extractants to evaluate the iron status of maize plant grown in sand culture and supplied with graded concentration of iron in solution ranging from deficiency to sufficiency. They found that the concentration of Fe in 1 N HC1, 1 N oxalic acid, 1 N citric acid, 0.1 N EDTA and 0.1 N DTPA was positively correlated with Fe supply in the medium. However, examination of their data (Fig. 111) clearly indicates that 1 N acetic acid was perhaps the best extractant to assess the iron status of maize tissue and to distinguish between deficiency and sufficiency. However, recent studies have shown that iron deficiency in rice could be satisfactorily diagnosed by measurement of the iron extracted from the fresh tissue by 0-phenanthroline; this extraction method estimates the ferrous iron content of the tissue 5. As a preliminary evaluation of the applicability of the method for groundnut, we have investigated the relationship between the occurrence of chloresis in groundnut and ophenanthroline-extractable iron content ('extractable iron') of the leaves. Materials and methods
Three separate series of measurements and observations were made on groundnut crops established by other scientists at the Institute. On each of two occasions in 1980, leaf samples were collected randomly from plants ('cv TMV 2') on an Alfisol to provide duplicate composite samples representing chlorotic and healthy growth. The surface soil was sampled at the same time; six cores were taken from each of the chlorotic and non-chlorotic areas and bulked to provide composite samples. During the rainy season in 1981, the iron content of young leaves of cv. TMV 2 on an Alfisol was monitored by collecting samples at intervals of 2 14 days; on each sampling occasion, two rows each consisting of 5 plants were harvested, and the leaves of the same age bulked for each plot. There were four replications. All leaves from the first three samplings were analysed to provide an indication of variability in extractable iron content with leaf age. Also in the 1981 rainy season, a single sampling was made on 1 September when 8 separate breeding lines of groundnut were collected and analysed; these 8 entries represented duplicate samples of materials with extremes of both overall growth and chlorosis. Ten plants were removed from each plot, and young leaves of the same age bulked for each plot. There were 3 replicates. This last experiment was located on a Entisol (pH 8.5 in surface 0-15 cm). The leaf samples after collection were washed thoroughly in tap water, followed by washing in acidified (HCI) water and distilled water. They were blotted dry and used for o-phenanthroline extractable iron. For total iron and other elemental analysis, the samples after washing were dried in an oven at 60~ and ground in a mini Wiley mill with stainless steel blades to pass through 40-mesh sieve. Ferrous iron in leaf tissue was estimated by extracting the fresh tissue with 1.5% o-phenanthroline solution (pH 3.0) as described by Katyal and SharmaS; this is subsequently termed 'extract-
355
D I A G N O S I S OF I R O N D E F I C I E N C Y IN G R O U N D N U T
able iron'. Total iron and other nutrients in oven-dried (60~ ground samples of plant tissue were analysed by the tri-acid digestion method 6. Available iron, zinc, and manganese in dry soil were estimated by extraction with DTPA reagent ~~ All other analyses were done as described by Jackson 6 .
Results and discussion The chlorosis that occurs at ICRISAT Center is very variable. It usually develops very suddenly, over a period of 1-3 days, and from a distance it is noticeable as irregular patches of yellow-green foliage amongst larger areas of normal green foliage. The chlorosis is identical to that described by Agarwala and Sharma' for iron deficiency; it consists of a characteristic interveinal yellowing in the youngest leaves, with complete bleaching of these leaves in extreme cases. Initial comparisons of the iron contents of leaves from non-chlorotic and chlorotic areas, in an Alfisol experimental field in the 1980 rainy season, gave results (Table 1) which were in agreement with those obtained for rice by other workers; total iron content was not lower in leaves from chlorotic areas, but the extractable-iron content was much lower. Analysis of the composite soil samples showed that there was no marked difference in the properties of the soils taken from the vicinity of the affected and healthy plants (Table 2).
Table 1. Total and o-phenanthroline extractable iron contents of cv T M V 2 groundnut leaves collected from chlorotic and normal areas in 1980 rainy season* Source of leaf material
Extractable-iron (~g/g fresh tissue)
Total iron dry wt, (#g/g)
Chlorotic areas Normal areas SE +
5.9 11,1 0.53
95 69 3.6
* Sampled on August 1 and 6.
Table 2, Analysis of surface soil samples (0-15cm) taken from areas with normal and chlorotic growth of groundnut* Source of soil sample
Normal areas Chlorotic areas
pH**
8.3 8.3
* Sampled on I and 6, August 1980. ** 1:2 Soil:water.
Olsen-P (#g/g)
12 12
DTPA-extractable nutrients (,ug/g) Fe
Mn
Zn
2.5 2.7
3.5 3.6
1,2 1,3
356
RAO, S A H R A W A T A N D B U R F O R D
Table 3. M e a n extractable and total iron contents of groundnut leaves of different age (cv T M V 2)*
Concentration of iron (gg/g)
Leaf sample
Extractable
Main bud Lateral bud Leaf 1 Leaf 2 Leaf 3 Leaf 4 Leaf 5 SE •
Total
Fresh weight basis
Dry weight basis
Dry weight basis
4.6 5.1 5.9 6.6 7.2 7.6 7.9 0.38
34.5 38.2 37.3 36.2 37.2 39.0 40.5 2.08
267 408 217 206 229 315 313 34.6
* Sampled on 29 and 31 July, and 3 August, 1981. Main and lateral buds were healthy on 29 July and chlorotic on 31 July a n d 3 August.
The development of chlorosis in the youngest leaves indicated that the extractable-iron content of these should provide a better index of the iron status of a plant than total iron content. The first three samplings of the monitoring program in 1981 were therefore used to determine the iron content of all leaves on plants of cv TMV 2. The extractable-iron content of the fresh tissue consistently increased as a leaf aged (Table 3), but there was no consistent relationship between leaf age and total iron, or the extractable iron content expressed on a dry weight basis. ExtractTable 4. Extractable and total iron in lateral buds (Lb), main bud (Mb) and first fully opened leaf (L-I) of groundnut (cv T M V 2) in the rainy season, 1981 Date 1981
Jul 29 Jul 31 Aug 3 Aug 5 Aug 7 A u g 10 A u g 12 A u g 14 A u g 17 A u g 19 A u g 26 Sep 7 Sep 11 Sep 25 SE •
O-phenanthroline-extractable iron (pg/g)
Total iron (~g/g)
Fresh wt. basis
Dry wt. basis
Dry wt. basis
Lb.
Mb
L-1
Lb
Mb
L-I
Lb
Mb
L-I
6,0 5,0" 4.2" 6.7 4.7 5.4 6.2 5.6 5.2 8.3 7.0 0.28
5.4 4.3* 4.1" 6.3 5.1 6.0 6.2 6.0 5.0 8.2 7.9 4.7* 5.6 0.43
7.2 5.5 4.9 8.3 5.5 6.0 7.7 8.2 6.3 14.0 8.3 10.1 5.3* 6.7 0.42
35.6 45.2* 33.2* 47.8 26.2 27.8 34.7 35.7 32.7 49.0 37.1 2.49
34.4 35.6* 33.2* 42.8 26.8 29.1 33.3 35.3 28.9 44.3 36.2 29.1 32.5* 2.36
40.5 39.3 32.0 42.5 26.5 21.4 31.8 30.9 28,2 49.3 31.3 39.5 20.8* 26.5 1.93
236 456* 531" 279 253 181 232 260 154 147 159 -
233 230* 340* 317 264 242 215 175 157 177 177 77* 55* 27.8
246 188 217 212 239 132 139 206 160 106 128 124 52* 81 14.7
* M a r k e d chlorosis.
24.9
357
DIAGNOSIS OF IRON DEFICIENCY IN GROUNDNUT
able iron content decreased in each of the three samplings, reflecting the onset of chlorosis, but similar decreases were observed in leaves of all ages. The monitoring program in 1981 was restricted to the youngest leaf material (the main bud, lateral buds, and the first opened leaf). Chlorosis developed less frequently and less severely in 1981 than in previous seasons. However, the extractable-iron content of buds and the first opened leaf was usually lowest during or shortly after the few occasions that chlorosis did develop (Table 4). Again, there was no consistent relationship between total iron content and the incidence of chlorosis. Severe chlorosis developed during the 1981 rainy season in one experiment that contained a large number of breeding lines. The growth of individual plants, and the severity of chlorosis, was consistent within a breeding line, but there was very wide variation across the lines. The most diverse lines for growth and chlorosis were selected for sampling and analysis. The results again showed that the youngest leaves of chlorotic plants contained less extractable iron than those from healthy plants (Table 5). Analysis of plant samples for other elements such as Mn showed that their contents were above the deficiency levels both in green and chlorotic tissue (unpublished data). The consistently lower extractable iron and higher total iron in chlorotic than in normal leaves (Tables 1, 4 and 5) showed that the iron deficiency was due to poorer utilization of iron within a leaf rather than to decreased absorption of iron by the root or translocation from the Table 5. Content of extractable and total iron (/tg/g) in main bud (Mb) and first fully opened leaf (L-I) of different groundnut breeding lines* Extent of chlorosis~
Plant growthw
Breeding entry
Extractable Fe** Mb L- 1
Total Fe*** Mb L- 1
Severe
Poor
Severe
Good
Nil
Poor
Nil
Good
FESR 12-P5 FESR 12-P6 NCAC 664 U-I-2-1 TMV-2 Krapovikas 16 C. No. 501 E. Runner
4.0 4.1 4.8 4.1 5.4 6.5 5.8 6.0 0.36
413 438 416 429 286 267 231 252 15.1
SE +
4.4 5.0 4.5 4.4 9.0 11.4 9.9 10.3 0.58
302 225 325 371 196 174 202 263 7.2
* Leaves sampled on 1 September 1981, 72 days after sowing. ** Fresh weight basis. *** Dry weight basis. wExtent of chlorosis and plant growth scores made on a scale of 0-10; the highest value was given for maximum growth or maximum chlorosis.
RAO, SAHRAWAT AND BURFORD
358
root to the leaf. This pattern is consistent with the common association of chlorosis in our fields with high soil moisture content, resulting from either rainfall or irrigation. High soil moisture content, by reducing aeration would cause an increase in the ferrous iron in the soil solution, thus promoting iron uptake and an increase in the total iron content of the leaf tissue. Reduced aeration would also restrict carbon dioxide escape from the soil thus causing, in these alkaline soils, increased bicarbonate concentration in the soil solution, which is known to reduce the availability of iron in the plant 1~. Although the extractable iron content of fresh leaf tissue increased with leaf age, the first fully-opened leaf appears to exhibit a wider range in extractable iron content (Tables 4 and 5), than the younger unopened leaves (main bud or lateral buds). More detailed studies are needed to determine whether the first fully opened leaf or a bud is the most reliable test organ for diagnostic work. From the data in both Tables 4 and 5, chlorosis was observed in the present work only when the extractableiron content of the buds of first opened leaf was less than 6 #g/g fresh tissue. Conclusions
The results with groundnut are consistent with those from recent studies on rice, which indicate that the o-phenanthroline extractable iron content of fresh leaf tissue appears to be a much better index of the iron status of the plant than total iron content. Extractable-iron in chlorotic buds or the first fully opened leaf was always less than 6 #g/g of fresh tissue.
References
1
2 3 4 5 6 7
Agarwala S C and Sharma C P 1979 Recognising micronutrient disorders of crop plants on the basis of visible symptoms and plant analysis. Botany Department, Lucknow University, Lucknow, India. Bar-Akiva A 1984 Substitutes for benzidine in the peroxidase assay, for rapid diagnosis of iron deficiency in plants. Commun. Soil Sci. Plant Anal. 15, 929-934. Bar-Akiva A, Maynard D N and English J E 1978 A rapid tissue test for diagnosing iron deficiency in vegetable crops. HortScience 13, 284-285. Chen Y and Barak P 1982 Iron nutrition of plants on calcareous soils. Adv. Agron. 35, 217-240. Gupta U C 1968 Studies on the o-phenanthroline method for determining iron in plant materials. Plant and Soil 28, 298-305. Jackson M L 1967 Soil Chemical Analysis. Prentice Hall (India), New Delhi. Jones J B Jr., Pallas J E Jr., and Stansell J R 1980 Tracking the elemental content of leaves and other plant parts of the peanut under irrigated culture in the sandy soils of South Georgia. Commun. Soil Sci. Plant Anal. 11, 81-92.
DIAGNOSIS OF IRON DEFICIENCY IN GROUNDNUT 8 9 10 11 12 13
359
Katyal J C and Sharma B D 1980 A new technique of plant analysis to resolve iron chlorosis. Plant and Soil 55, 105-119. Leeper G W 1952 Factors affecting availability of inorganic nutrients in soils with special reference to micronutrient metals. Ann. Rev. Plant Physiol. 3, 1-16. Lindsay W L and Norvell W A 1978 Development of a DTPA soil test for zinc, iron, manganese and copper. Soil Sci. Soc. Am. J. 42, 421-428. Mehrotra S C, Sharma C P and Agarwala S C 1985 A search for extractants to evaluate the iron status of plants. Soil Sci. Plant Nutr. 31, 155-162. Porter L K and Thorne D W 1955 Interrelation of carbon dioxide and bicarbonate ions in causing chlorosis. Soil Sci. 79, 373-382. Wallace A, Wood R A and Soufi S M 1976 Cation-anion balance in lime-induced chlorosis. Commun. Soil Sci. Plant Anal. 7, 15-26.
