Ms. 4605

FOLIAR MACRONUTRIENTS (N, P, K, Ca AND Mg) IN LIME (TILIA SPP)

I. S A M P L I N G T E C H N I Q U E S

by H. INSLEY, R. C. B O S W E L L and J. B. H. G A R D I N E R

Forestry Commission, Forest Research Station, Wrecclesham, Farnham, Surrey, U.K.

KEY WORDS

Leaf size Nutrient concentration Nutrient variation Sampling precision Tilia x x europaea, L.

SUMMARY

The effect of sampling technique upon the measurement of variation in nutrient (N, P, K, Ca and M g) concentration was examined. Sampling and analytical precision over two years were indicated by coefficients of variation which ranged from 5.7 to 14.0 per cent for sampling error and 4.3 to 16.1 per cent for analytical error. Concentration of N, P and K in the leaves was found to be independent of sampling height. Ca and Mg concentrations, however, decreased with increased height on the crown. The differences in concentration between the bottom and top of the crown were 15 and 19 per cent for Ca and Mg respectively. The nutrient concentrations of samples taken from basal coppice shoots were significantly different from those in the true crown. Nutrient concentration was independent of aspect. Leaf area varied throughout the season, but the largest leaves were always at the base of the crown and the smallest at the top.

INTRODUCTION

Genera l foliar sampl ing of lime trees (Tilia spp.) was carried out over a wide range

of site types and soils in Southern Eng land and Wales dur ing 1975 s. Analysis of

these samples indicated the range of concent ra t ions of foliar macronu t r i en t s (N,

P, K, Ca and Mg) which might be expected dur ing the usually accepted midsum-

mer sampl ing period. The main factors likely to cause var ia t ion in the concen-

t ra t ion of foliar macronu t r i en t s were believed to be seasonal variat ion, age of

tree, soil (nutr ient status, pH, structure and physical condition), and factors

relating to the sampl ing such as height on the crown, aspect and the size of leaves

collected. Shade leaves were always avoided, as were those damaged by leaf

eat ing insects and those soiled by birds. Dust from the air has been shown to have little effect on mac ronu t r i en t concent ra t ions 3.

Limes were intensively sampled from 1975 to 1978 so that foliar macronu t r i en t

377

Plant and Soil 61, 377-389 (1981). 0032-079X/81/0613-0377501.95. �9 1981 Martinus Nijhoff/Dr W.. Junk Publishers, The Haoue. Printed in The Netherlands.

378 H. INSLEY, R. C. BOSWELL AND J. B. H. GARDINER

Plate 1. A hydraulic platform with a 10 m reach to collect the leaves.

FOLIAR MACRONUTRIENTS IN LIME. I 379

variation and its causes could be investigated. This was done with a view to developing foliar analysis as a diagnostic technique for determining the nutrient status of broadleaved amenity trees. Variation which results from the sampling process is examined in this paper - seasonal variation will be discussed in a

further paper.

FIELD M E T H O D S

Sampling followed the usually accepted procedure for deciduous broadleaved trees with some modifications to take account of the findings of G u h a and Mitchell 3'4. Except where seasonal variation was being examined leaves were collected during the mid to late season period (July- August). Leaf samples were taken from unshaded branches with a southerly aspect, at or slightly above the widest part of the crown (except where variation with aspect and height were being investigated). The sample branches were removed using a jointed set of a luminium poles fitted with a rope-operated pruning head 2. Some trees were too large for the desired height to be reached using poles so lower branches were sampled. Where variation with height was being examined a hydraulic platform with a 10 m reach was used to supplement the poles (Plate 1).

Approximately 600 cm 2 of healthy, fully expanded, unshaded leaves were taken from the branch for each sample. Care was taken to exclude soiled, dead, diseased or damaged leaves from the samples and to avoid leaves from the ends of the shoots.

The length and breadth (Fig. 1) of the four largest leaves in each sample were measured. These leaves were then returned to the sample which was placed in a clean polythene bag and taken to the

B B

A

I

Fig. 1. Axes used to measure leaf length (A) and leaf breadth (B).

3 8 0 H. INSLEY, R. C. BOSWELL AND J. B. H. GARDINER

laboratory for analysis. A subsample of leaves of widely varying size was subsequently used to determine the relationship between leaf area and the leaf lengths and breadth.

The trees used to study variation in foliar macronutr ients resulting from the sampling methods were an avenue of 30 C o m m o n limes (Tilia x europaea L.) at Avington, Hampshire and four C o m m o n limes situated at Shortfield Green, Surrey. The trees were known to have been planted in 1930 at the first site and were therefore approximately 50 years old at the time of sampling. The Shortfield Green trees were thought to have been approximately 30 years old. At Avington the trees were growing on a silty brown earth of alluvial origin, while at Shortfield Green the trees were growing on soil over Lower Greensand.

Variation due to samplin9 and analysis

As described in part II samples were collected from the Avington trees at intervals throughout the summers of 1977 and 1978 so that within season variation of foliar macronutrients could be studied. A randomised series of duplicate samples was collected throughout the season in both years in order to estimate variation caused by the sampling technique. The samples in each pair were analysed separately so that a coefficient of variation due to sampling error could be calculated.

As a cheek on the variation caused by the laboratory analysis further samples from the Avington trees were selected and divided into duplicate subsamples. These were separately analysed and the results used to calculate a coefficient of variation due to analytical error.

Effect of height

Variation in macronutr ient level with height was studied using both the Avington and Shortfield Green trees. At Avington samples were collected from three positions on the crown approximating to the edge of the lower crown, halfway up the outside of the crown and as near to the highest point of the crown as could be reached (Plate 1). At Shortfield Green samples were collected once yearly during early August from the same three positions on the crown as at Avington but with additional samples collected from the basal coppice sprouts.

Effect of aspect

The influence of aspect on macronutr ient level was investigated over a three year period from 1976 to 1978 using the Shortfield Green trees. Leaf samples were collected during early August each year from the four positions on the tree described above and from the north, south, east and west aspects.

L AB OR AT OR Y M E T H O D S

The leaf samples were oven dried at 100~ overnight before the petioles and large veins were removed. The leaves were then ground up and stored in glass tubes until ready for analysis. Before analysis the samples were re-dried at 100~ for one hour. A subsample of 100rag of the ground leaf was then weighed into a test tube and dissolved in 1 ml of concentrated sulphuric acid. Two shots of 0.4 ml of hydrogen peroxide were added to this solution before heating at 330~ for 30 rain. After cooling for 10 mins a further 0.2 ml of hydrogen peroxide was added. The solution was then reheated at 330~ for 10 min before being made up to 15 ml with deionised water. After thorough shaking, to mix the solution, subsamples were taken for analysis. N concentration was measured by a colorimetric method (alkaline phenol) using an autoanalyser based on the Autoanalyser 2 system. P, K, Ca and Mg were assessed by plasma emission spectroscopy using a Spectraspan III.

FOLIAR MACRONUTRIENTS IN LIME. I 381

RESULTS

The range of macronutrient contents found in the duplicate foliar samples collected at Avington in 1977 and 1978 was as follows:

1977 1978

N 1.65-5.49 1.85~l.62 P 0.11~0.58 0.15~0.75 K 0.32-1.73 0.50-1.83 Ca 1.1~4.98 0.90-4.23 Mg 0.18-0.45 0.22-0.40

A total of 25 and 30 pairs ~f samples were collected during 1977 and 1978 respectively and following macronutrient analysis used for calculation of the coefficient of variation due to sampling. The results of this are presented in Table 1.

In addition to the use of duplicate samples to estimate sampling error further samples were selected at random from among those collected at Avington during the seasonal variation work and used to estimate analytical error. This was done by analysing duplicate milled subsamples (26 samples in 1977, 29 in 1978). The results were then used to calculate the coefficient of variation due to analytical error (Table 1). Guha and Mitchell 3 used 5 pairs of samples and duplicate subsamples to measure the coefficients of variation due to sampling and analysis. For P, K, Ca and Mg they obtained coefficients of variation (sampling plus analysis) for the leaf blade ranging from 1.1 to 10.4 per cent. For variation due to analytical error they obtained coefficients ranging from 0.5 per cent for Mg to 2.9 per cent for P. They concluded that the coefficients of variation due to analytical

Table 1. Percentage coefficients of variation due to sampling and analytical errors, for foliar N, P, K, Ca and Mg concentration in the Avington trees during 1977 and 1978

1977 1978 Element per cent Sampling Analysis Sampling Analysis

N 5.7 10.1 7.5 4.3 P 6.0 8,4 9.1 7.6 K 12.4 12.3 14.0 8.5 Ca 12.3 16,1 7.5 13.4 Mg 10.9 15.0 10.9 8.0

3 8 2 H. INSLEY, R. C. BOSWELL A N D J. B. H. GARDINER

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Fig. 2. Variation in foliar Ca concentration with height on the crown as measured in 15 Til ia x e u r o p a e a at Avington, Hamshire during 1977 and 1978.

F O L I A R M A C R O N U T R I E N T S I N LIME. I 3 8 3

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Fig. 3. Variation in foliar Mg concentration with height on the crown as measured in 15 Tilia x e u r o p a e a at Avington, Hampshire during 1977 and 1978. See Fig. 2 for legend.

384 H. INSLEY, R. C. B O S W E L L A N D J. B. H . G A R D I N E R

determinants seldom exceeded half those due to sampling. This was not found to be the case in the present work. The coefficients of variation ranged from 5.7 to 14.0 per cent for sampling and from 4.3 to 16.1 per cent for analytical error (See Table 1). In 1977 the coefficient of variation due to analytical error was-greater than that due to sampling for N, P, Ca and Mg. This was almost certainly due to less precise laboratory work during a period of heavy workload in 1977. In 1978 the analytical precision was greater than the sampling precision for all macronut- rients except Ca.

Effect of height

Some data were missing from the second half of the avenue of Common limes at Avington so only the 15 for which a full data set was available were included in the statistical analysis. This allowed the use of a balanced analysis of variance for comparison of nutrient concentrations at the three sampling heights on the crown. The analyses of variance were done for each sampling date separately because of the trends in nutrient concentrations throughout the season and the possibility of changing error variances.

Comparison of the concentrations of N, P and K at the top, middle and lower edge of the crown showed that there were no consistent trends at any time during the 1977 and 1978 seasons. When the nutrient concentrations were significantly different at the three heights on the crown the differences were usually rendered meaningless by significant year x position interactions.

However, both Ca and Mg concentrations showed a significant trend with height on the crown throughout the season in both 1977 and 1978 (Figs. 2 and 3). The combined results for both seasons showed that there were very highly significant differences in Ca concentration with height in the crown on 10 out of the 15 sampling dates. On one other date the differences were significant at the 1 per cent level and on the four other sampling dates there were significant year • position interactions because of differences between the two seasons.

The differences in concentration of Mg were very highly significant on 12 out of the 15 sampling dates. On two of the other sampling dates, although the

differences in Mg concentration with sample height on the crown were very highly significant, they were significant (at the 5 per cent level) year x position interactions. Both Ca and Mg exhibited similar and consistent trends with the highest concentrations at the bot tom of the canopy and the lowest concen- trations at the top. Ca concentrations at the top of the crown were on average 15 per cent lower than those at the outside edge of the lower canopy. The concen-

F O L I A R M A C R O N U T R I E N T S IN LIME. I 385

trations of Mg at the top were an average of 19 per cent lower than those at the base.

The results for the Shortfield Green trees agree with those from the Avington trees in all but a few aspects, although they were less precise because they involved only four trees sampled once during mid-summer. There were no consistent trends in nutrient concentration with height in the true crown, although in 1978 only the concentrations of all five nutrients decreased with height in the crown. There were no significant differences between the concen- trations of the nutrients at different heights on the true crown except for P in 1977 when the difference was only significant at the 5 per cent level. However, when the concentrations of P, K and Mg in the basal coppice shoots were compared with those in the tree canopy very highly significant differences were always found. The differences in the concentration of N in the basal shoots and canopy were very highly significant in 1976 and 1978 but not in 1977. For Ca the differences were only significant (all at 5 per cent level) in 1976.

Effect o f aspect

Nutrient variation with aspect was examined using the four Common limes at Shortfield Green. Samples were taken at varying heights on the trees, as described in the preceding sections, and at the four cardinal points of the compass. Analysis of these showed that within the crown of the trees there was no evidence of any variation in nutrient concentration with aspect during the three years 1976 to 1978.

The results of the analysis of the samples collected from the basal coppice shoots showed some rather spurious relationships between aspect and nutrient concentration in 1976 and 1977 only. In 1978 there were no significant differences in nutrient concentration of the basal shoot samples with aspect. In 1976 there were higher concentrations of P in the basal shoot samples from the north and east aspects than from the south and west samples. The concentration of K was

Table 2. Summary of the differences in nutrient concentration between samples collected from the basal coppice shoots and true crown of 4 Tilia x europaea at Shortfield Green, Surrey 1976-1978

N P K Ca Mg

1976 *** *** *** * *** 1977 - *** *** - *** 1978 *** *** *** - ***

386 H. INSLEY, R. C. B O S W E L L A N D J. B. H . G A R D I N E R

significantly higher in the sample from the N aspect. Samples from the other three aspects were not significantly different.

In 1977 the concentration of N in the basal shoot samples was greater on the east and west than on the north and south aspects. Basal samples from the southern aspect contained significantly lower P and K concentrations than samples from the other aspects. These differences in concentration in the basal shoot samples were significant only at the 5 per cent level.

The relationship between leaf size and nutrient concentration

The length and breadth of the four largest leaves in each sample taken from the Avington trees were measured and subsequently these parameters were used to calculate mean leaf area. The calculation of mean leaf area was done by averaging the estimated areas of the four leaves, not by calculating a single area using the mean length and breadth measurements.

The relationship for estimated area obtained from a subsample of 40 leaves was:

Area -- A x B • 0.64368 + 0.1927

See Fig. 1 for an explanation of A and B. Leaf area was found to vary with sampling height and could be expected to

vary between trees. An unbalanced analysis of variance incorporating all samples collected was used to compare the mean leaf area of each sample (Fig. 4). This showed that there was some evidence of a position x date interaction, but very highly significant date and sampling position effects. There were also significant differences in leaf area between trees.

The variation in leaf area due to sampling position and date was removed from the data in order to examine the relationship between leaf area and nutrient concentration and the residuals were then related to the foliar N, P, K and Mg concentrations in the leaves. There was no evidence from tfiis of any relationship between leaf size and nutrient concentration in 1977 or 1978. A further at tempt was made to find relationships between leaf area and nutrient concentration by examining every date and sampling combination, with a maximum of 30 trees in each combination. Although this was only tried for the first two dates, the percentage variance accounted for was very low and negative. This indicated that trying to fit a regression coefficient was less satisfactory than using the mean values as an estimate. The results thus confirmed the previous conclusion that there were no relationships between leaf size and nutrient concentration. This

Fig. 4.

FOLIAR MACRONUTRIENTS IN LIME. I 387

80- Estimated leaf area (cm 2)

70-

60-

50-

40-

30-

t

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The relationships between sampling date, position on crown and estimated leaf area for 30 Tilia x europaea at Avington, during 1978. See Fig. 2 for legend.

result was som6what surprising especially in the case of N where relationships with leaf size are well known.

DISCUSSION

The sampling and analytical methods used took account of the results of previous work. In particular the petioles and larger veins were removed before analysis so that the material analysed was almost wholly composed of laminae. It has been shown for both sycamore (Acer pseudoplatanus L.) and Horse chestnut (Aesculus hippocastanun L.) that the concentration of some elements in the petiole and lamina are appreciably different 3.

Although apparently less precise than the estimates of previous workers using smaller samples 3, the sampling and analytical methods used in this study were thought to be accurate enough to detect seasonal and within tree variation as well as for diagnostic purposes. Compared with the several-fold differences in nut- rient concentration between trees, and the large within season changes in concen- tration the precision indicated by the percentage coefficients of variation for sampling and analytical error is good.

Changes in concentration of Ca and Mg with height on the crown were

388 H. INSLEY, R. C. BOSWELL AND J. B. H. GARDINER

indicated by the Avington results but not those for the Shortfield Green trees. Guha and Mitchell 4 found that Ca concentrations were fairly constant at all heights in sycamore and Horse chestnut but that in beech (Fayus sylvatica L.) the concentration decreased with height, as shown for Common lime in the present work. The fall in Ca concentration between the bot tom and top of the crown in beech indicated by their figures was about 39 per cent compared with the 15 per cent in C o m m o n lime. The results for P, K and Mg also agreed with those of Guha and Mitchell 4. For beech, sycamore and Horse chestnut they found little change in P and K concentration with height on the crown and a decrease of up to 20 per cent in Mg concentration between the bot tom and top of the crown. In the present work the Common limes at Avington showed no consistent significant trends in N, P or K concentration with change in sample height. Mg concen- tration decreased on average by 19 per cent between the bot tom and top of the crown.

The absence of any significant change in nutrient concentration with aspect on the crown is also in accord with previous results for temperate deciduous broadleaved trees t.3. The occurrence of some rather spurious differences of nutrient concentration with aspect on the basal coppice shoots of the Shortfield Green limes serves as a reminder that such atypical material should be avoided. Quite apart from their other differences these leaves from basal coppice shoots are more susceptible to contamination than those on the true crown.

The practical implications of this lack of any significant variation in concen- tration with height or aspect are, that where routine sampling to assess N, P and K status is being carried out, foliage may be taken from any part of the lower crown provided that the leaves are clean and in full light.

The consistency through the season of the differences or lack of differences in nutrient concentration with sampling height is not unexpected. There was a consistent difference in the estimated leaf area with sampling height (Fig. 4). It has previously been shown that the rate of dry weight increase of leaves of hickory (Carya spp.) and oak (Quercus spp.) is dependent of height on the crown 6. This means that the relationship between the nutrient concentrations at different heights on the crown will also remain constant in spite of changes in the concentrations themselves, provided that the supply of nutrients does not vary with height on the crown. The present work confirms this and therefore when nutrient concentration is being assessed for diagnostic purposes leaf size and the consequent total nutrient content are of less importance,

Received 3 December 1980

FOLIAR MACRONUTRIENTS IN LIME. I 389

REFERENCES

1 Auchmoody, L. R. and Hammack, K .P . 1975 Foliar nutrient variation in four species of upland oaks. NE For. Exp. Stn. U.S.D.A. For. Serv. Res. Pap. NE 331.

2 Everard, J. 1973 Foliar analysis, sampling methods, interpretation and application of the results. Q. J. For. 67, 51~56.

3 Guha, M. M. and Mitchell, R.L. 1965 The trace and major element composit ion of the leaves of some deciduous trees. I. Sampling techniques. Plant and Soil 23, 323-338.

4 Guha, M. M. and Mitchell, R.L. 1966 The trace and major element composit ion of the leaves of some deciduous trees. II. Seasonal changes. Plant and Soil 24, 90-112.

5 Insley, H. and Gardiner, J. B.H. 1980 Foliar nutrient levels in lime. Arboriculture Res. Note 19. For. Comm. Farnham.

6 Mitchell, H. L. 1936 Trends in the nitrogen, phosphorus, potassium and calcium content of the leaves of some forest trees during the growing season. Black Rock Forest Pap. 1, 3~44 .