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Response of olive trees to foliar application of humic substances extracted from leonardite
SCIENTIA HORTICULTURR Scientia Horticulturae 66 (1996) 191-200
Response of olive trees to folk application of humic substances extracted from leonardite Femiindez-Escobar *, M. Benlloch, D. Barranco, A. Duefias, J.A. Gutkrez GaSn Departamento de Agronomia, Universidad de Grdoba, Apartado 3048, 14080 Chdoba. Spain
Accepted 1 April 1996
Abstract The effect of foliar applied humic substances extracted from leonardite on olive growth was studied in greenhouse experiments using cuttings and young olive plants, some of which were irrigated with a nutrient solution, and also in field experiments selecting mature trees growing in the drylands or under irrigation. Foliar application of leonardite extracts to young olive plants stimulated shoot growth when they were growing without the addition of mineral elements to the irrigation water, but did not promote growth when applied to plants watered with a nutrient solution, although growth of fertilized plants was greater than that of unfertilized ones. Under field conditions, folk application of leonardite. extracts stimulated shoot growth and promoted the accumulation of K, B, Mg, Ca and Fe in leaves. However, when leaf N and leaf K values were below the threshold limit for the sufficiency range, foliar application of humic substances was ineffective to promote accumulation of these nutrients in leaves. Keywords: Folk
nutrition; Humic acids; Humic substances; Leonardite; Olive; OIea europaea
1. Introduction The beneficial effects of humic substances on plant growth have been observed by man from ancient times. Today, it is recognized that soil organic matter may affect the physical, chemical and microbiological properties of the soil and, indirectly, affects plant growth. Also, it is well documented that humic substances have a direct effect on plant growth (Chen and Aviad, 1990).
* Corresponding author. Fax: 34-57-218569. 0304-4238/%/$15.00 Copyright 0 1996 Elsevier Science B.V. All rights reserved. PII SO304-4238(96)009 14-4
192
R. Fernhdez-Escobar
ei cd./ Scienria Horricdnwae
66 (1996) 191-200
During the 19th century and in the early 20th century, there was controversy among scientists on the role of the humus on plant nutrition. While some of them supported the hypothesis that humus is the only material which supplies nutrients to plants, others accumulated evidence and provided information on the role of mineral elements in plant nutrition. In the first half of the 20th century, Amon and Stout (1939) proposed the criteria for essentiality of a element for plant growth, establishing one of the principles governing modem plant nutrition. However, there are a number of papers reporting information about the enhancement of plant growth promoted by the application of humic substances (Chen and Aviad, 1990). This growth promoting activity seems to be caused by plant hormone-like materials present in the humic substances (O’Donnell, 1973; Casenave de S~~lippo et al., 1990). Humic substances are usually applied to the soil, and favorably affect the soil structure and soil microbial populations. Foliar sprays of these substances also promote growth in a number of plant species such as tomato, cotton and grape (Brownell et al., 1987). Spraying with fulvic acid also increased the yield of wheat grown under dry conditions (Xudan, 1986), suggesting the capability of the humic substances to reduce water stress. In addition, the lower cost of the application of these products by foliar spray has been indicated (Chen and Aviad, 1990). Leonardite is an oxidized form of lignite coal. Humic and fulvic acids may be obtained by extraction from leonardite and, presently, humic preparations from leonardite are available for use in agriculture. The objective of the present work was to study the effect of foliar applied leonardite extracts on growth and yield of olive trees growing in the dryland or under irrigation.
2. Materials and methods A series of greenhouse and field experiments were conducted to study the effect of foliar applied humic substances on olive yield and growth. In all cases, Naturvital-16 (Daymsa, Spain) containing 9% humic acids and 7% fulvic acids extracted from leonardite, and 0.59% N, 3.3% K, 95.7 ppm P, 0.32% Ca, 300 ppm Mg, 0.53% S, 0.3% Fe, 22 ppm MO, 6.9 p.p.m. Cu, 5.2 p.p.m. Zn, 0.3 p.p.m. B, and 8.2 p.p.m. Mn, was used. 2.1. Greenhouse experiments Two different ex~~rnen~ were carried out in the greenhouse with the aim of studing the effect of leonardite extracts concentration on growth of young olive trees. For this purpose, in a preliminary experiment l-year-old olive plants cv. Picual growing in 1.5 1 plastic pots were placed in a greenhouse at 30/15’C (day/night) with a 14-h photoperiod. The plants received one foliar application of Naturvital 16 at concen~ations of 0%, 0.5%, I%, 2%, 4%, 8% or 16%. There were two replications (plants) per treatment. In a second experiment, mist-rooted ‘Picual’ olive cuttings were transferred to 2-l plastic pots containing a mixture of river sand and peat (2:l by volume), and placed in a
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greenhouse under the same growth conditions described above. Before the initiation of treatments and after one month of acclimatization, the cuttings were pruned to a single shoot per plant. The plants were arranged in a split-plot experiment. The whole-plot factor consisted of the application of mineral nutrients or no application of mineral nutrients. Plants that received mineral fertilization were watered with a solution of 1 g 1-r of Hakaphos fertilizer containing 15% N, 4.8% P, 12% K, 0.8% Mg, 9% S, 0.03% B, 0.1% Mn, 0.06% Zn, and 0.004% MO. The leonardite extract concentration was the subplot factor. Each subplot consisted of four plants and subtreatments were one foliar application of Naturvital 16 at concentrations of 0%, 0.25%, 0.5% or 1%. The experiment was developed from April-July 1991, and during this time plants were watered twice a week with 250 ml of water. Shoot length was measured on six dates during the experiment on each experimental plant. 2.2. Field experiments Four trials were conducted in three olive orchards located in three different areas of Andalusia, Southern Spain. Two experiments were carried out in a commercial orchard located near Cordoba. Drip-irrigated, 5-year-old ‘Manzanillo’ trees and 5-year-old ‘Hojiblanca’ olive trees were selected for the experiments. Trees were spaced 8 X 6 m apart. The third experiment was conducted in the Experimental Farm of La Mina at Cabra, Southern C6rdoba province. Non-irrigated, lo-year-old ‘Hojiblanca’ trees spaced 7 X 7 m apart were selected for the experiment. Annual rainfall in this area was 450 mm. The fourth experiment was conducted in a commercial orchard located in Ubeda, Jaen province. Non-irrigated, 15-year-old ‘Picual’ trees spaced 10 X 10 m apart were selected. Annual rainfall in the area was 300 mm. For all field experiments, a randomized block design was used with four blocks and three treatments. Each plot consisted of one tree in the irrigated ‘Manzanillo’ and ‘Hojiblanca’ trials located in Cordoba, and of five trees in the other trials. The treatments were a nontreated control and foliar applications of Naturvital-16 at 0.5% or 1%. The treatments were applied in April, approximately 1 month before bloom. The Cordoba experiments were developed during 1991 and repeated in 1992; the other experiments were conducted during 1992. Vegetative growth was determined at the end of the growing season by measuring shoot length on ten tagged shoots per experimental tree. Also, the above-ground tree volume, a variable used as covariate to adjust yield, was calculated from height and spread measurements as indicated by Westwood (1978). The effect of treatments on cropping was evaluated by measuring yield per tree at harvest and the number of fruits per shoot on those tagged to determine vegetative growth. A sample of approximately 1 kg of fruits per plot was taken at harvest to determine fruit size and oil content. Fruit oil content was determined by nuclear magnetic resonance (NMR) after milling the fruit sample. The effect of leonardite extracts on the nutritional status of the olive trees was determined by leaf analysis of a sample of 100 leaves per plot collected in July, according to Beutel et al. (1983). Fully-expanded, mature leaves from the middle portion of nonbearing, current season shoots were removed for analysis of mineral nutrients.
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Leaves were collected in paper bags and stored in a portable ice chests. Once in the laboratory, leaves were washed with 0.03% Triton X-100, rinsed in deionized water, dried at 80°C for 48 h, ground, and stored in an oven at 60°C until analysis. Nitrogen was determined by the Kjeldahl procedure. For other elemental determinations, the stored samples were ashed in a muffle furnace at 600°C for 12 h, and dissolved in 0.1 N HCl. Total P was determined by calorimetry using the method described by Murphy and Riley (1962). Boron was determined in the extract by calorimetry (Greweling, 1976). The remaining elements (K, Mg, Ca, Zn, Mn, Fe and Cu) were measured using an atomic absorption spectrophotometer. Analyses of variance and regression analyses were performed on the data from each experiment, with the exception of yield that was analyzed by analysis of covariance using the total above-ground tree volume as covariate.
3. Results 3.1. EfSect of leonardite extracts concentration
on growth of young olive plants
Foliar application of leonardite extracts to l-year-old olive plants at concentrations higher than 1% of the commercial product Naturvital-16, inhibited plant growth (data not shown). Concentrations of 0.5% and 1% improved olive cutting growth when plants were growing without the addition of mineral elements to the irrigation water (Fig. l(b)), but there was no promoting effect when leonardite extracts was applied to plants receiving mineral nutrients (Fig. l(a)) or when Naturvital-16 was applied at 0.25% (Fig. l(b)). However, growth of fertilized plants was greater than that of plants that did not receive additional fertilizers. 3.2. Effect of leonardite extracts on vegetative growth ana! yield of olive trees
Vegetative growth was affected by foliar application of leonardite extracts to mature olive trees. Shoot growth of ‘Hojiblanca’ trees growing under irrigation (Table 1) and also growing in the drylands (Table 21, significantly increased when leonardite extracts were applied to leaves at concentrations of 0.5% or l%, compared to untreated trees. In the experiment of 1991, shoot growth of ‘Hojiblanca’ treated trees was more than twice that of control trees (Table 1). However, no significant differences were found either on ‘Manzanillo’ and ‘Picual’ trees or on ‘Hojiblanca’ trees in the 1992 experiments. Lack of significance might be due to the high variability obtained in these experiments. Fruit set increased significantly after foliar application of leonardite extracts only in the ‘Hojiblanca’ experiment conducted under no irrigation (Table 2), and was unaffected by treatments in the other cultivars and experiments. The quadratic response indicates an effect of leonardite extracts on fruit set only at the lower concentration. For fruit size, there was a similar quadratic response in the ‘Manzanillo’ experiment conducted in 1991 (Table l), but no significant effect was observed in the other cultivars and experiments. Yield and fruit oil content were unaffected by the treatments (Tables 1 and 2).
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A
100
60
.
60
r
4o
-
20
/Y
’
/:
5 ? 2
O loo
0 0
20
40
60
60
Days after treatments Fig. 1. Effect of the concentration of foliar applied Naturvital-16 on shoot growth of olive plants irrigated with a nutrient solution (A) or without a nutrient solution (B). Vertical bars indicate the SE.
3.3. Effect of leonardite extracts on leaf nutrient concentration Leaf analysis of samples collected in July showed a nutritional olive tree status characterized by K values below the threshold limit of 0.8% for the sufficiency range in the experiments conducted with ‘Picual’ and ‘Hojiblanca’ under no irrigation (Table 3), and also in the irrigated ‘Hojiblanca’ experiment of 1992 (Table 4). Also, leaf N values were below the sufficiency threshold of 1.5% in the irrigated experiment of ‘Manzanillo’ and ‘Hojiblanca’ conducted in 1991 (Table 4). No other nutritional disorder was observed in the experiments. Folk application of leonardite extracts affected leaf nutrient concentration. Leaf K values significantly increased in experiments conducted with ‘Manzanillo’ in 1992 and with ‘Hojiblanca’ in 1991 (Table 4). Also, leaf Ca, Mg, B and Fe concentrations were significantly affected by the treatments (Tables 3 and 4). However, leaf nutrient values
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Table 1 The effect of foliar application cultivars
of leonardite Shoot growth (cm)
Treatment
1991
‘Manzanillo’ Control Leonatdite extracts 0.5% Leonardite extracts 1% Significance Coefficient of variation (o/o)
8.27 10.72 15.25 NS 30.9
‘Hojiblanca’ Control Leonardite extracts 0.5% Leonardite extracts 1% Significance Coefficient of variation (%I
6.25 14.02 13.47 L’ 29.9
1992
extracts
on vegetative
growth
Fruit set (fruits/cm) 1991
Fruit size
1992
0.29 0.24 0.19 NS 29.4
1991
1992
1991
1992
3.96 4.55 4.14 4.0
3.62 3.33 3.36 NS 11.7
31.0 30.3 31.0 NS 23.3
32.1 35.3 24.2 NS 28.6
3.61 3.58 3.91 NS 7.3
3.43 3.29 3.82 NS 7.2
42.6 38.4 38.3 NS 22.1
53.2 60.3 50.9 NS 16.3
Q*
0.39 0.43 0.39 NS 10.6
olive
Yield (Kg tree-‘)
(g)
0.27 0.23 0.28 NS 16.6
7.64 8.55 7.71 NS 7.1
and yield of two irrigated
NS, nonsignificant; * P = 0.05. L, linear; Q, quadratic.
in all these experiments were above the sufficiency threshold, including control trees. In contrast, trees with leaf N and leaf K values below the threshold limit were unaffected by folk applications of leonardite extracts.
Table 2 The effect of folk cultivars
application
of leonardite extracts on vegetative Shoot growth
growth and yield of two nonirrigated
olive
(cm)
Fruit set (fruits cm- ’ )
Fruit size (g)
Yield (leg tree- ‘1
Fruit oil content (%I
‘Picual’ Control Leonardite extracts 0.5% Leonardite extracts 1% Significance Coefficient of variation (%)
5.82 6.41 6.51 NS 8.4
0.17 0.13 0.15 NS 15.6
3.45 3.56 3.57 NS 5.4
21.42 28.40 24.65 NS 17.0
16.68 18.15 17.45 NS 5.8
‘Hojiblanca’ Control Leonardite extracts 0.5% Leonardite extracts 1% Significance Coefficient of variation (%b)
6.64 8.08 8.03 L’ 9.8
0.42 0.55 0.44
3.92 3.78 3.85 NS 7.5
24.61 29.08 25.49 NS 14.2
20.63 21.35 21.20 NS 1.3
Treatment
NS, nonsignificant; P = 0.05. L, linear; Q, quadratic. l
Q’ 12.2
NS 5.0
0.10 0.09 0.09
NS 1.8
0.10 0.10 0.09
P (o/o)
* P = 0.05; * * P = 0.01.
NS 1.4
Significance Coefficient of variation (o/o)
NS, nonsignificant; L, linear.
1.7 1.7 1.7
‘Hojiblanca’ Control Leonardite extracts 0.5% Leonardite extracts 1%
1.7
I .I
1.7
N (%)
NS 4.6
0.4 0.4 0.4
NS 7.6
0.6 0.6 0.6
K (o/o)
L’ 3.5
1.8 1.6 1.5
l
1.2 1.3 1.5 L ’ 3.6
Ca (o/o)
0.1 0.1 0.1 NS 5.2
NS 6.8
0.1 0.1 0.1
Mg (%)
of leonardite extracts on leaf nutrient concentration
NS 1.4
extracts 0.5% extracts 1%
application
Significance Coefficient of variation (%)
‘Picual’ Control Leonardite Leonardite
Treatment
Table 3 The effect of folk
36.1 36.5 35.0 NS 6.5
25.5 28.0 31.6 L’ 4.2
B (p.p.m.)
of two nonirrigated
olive cultivars.
39.5 39.3 38.8 NS 4.0
48.9 45.8 51.1 NS 5.1
Mn (p.p.m.)
13.6 15.8 13.9 NS 4.6
8.8 11.1 10.0 NS 8.8
Zn (p.p.m.1
31.6 33.3 34.6 NS 4.2
30.3 38.4 37.3 NS 8.2
Cu (p.p.m.1
46.2 48.8 47.1 NS 5.2
37. I 43.2 58.1 L’ 5.8
Fe (p.p.m.)
l
NS, nonsignificant; P= L, linear, Q, quadratic.
1.8 I .8 1.8 NS 1.9
0.05; * * P=
1.3 1.3 1.2 NS 5.1
‘Hojiblanca’ Control Leonardite extracts 0.5% Leonardite extracts 1% Significance Coefficient of variation(%)
1.6 1.6 1.6 NS 2.4
0.01.
0.1 0.1 0.1 NS 8.9
0.1 0.1 0.1 NS 21.8
1991
1991
0.1 0.1 0.1 NS 1.4
0.1 0.1 0.1 NS 3.5
1992
0.81 0.84 1.00 L * 8.8
0.84 0.84 0.88 NS 10.8
1991
K (%)
0.58 0.57 0.63 NS 4.2
0.84 0.96 1.03 L * 3.8
1.6 1.7 1.7 NS 4.0
1.8 1.6 1.8 NS 8.0
1991
Ca (%)
concentration
1992
extracts on leaf nutrient
PC%)
1992
of leonardite
N (%I
1.2 1.2 1.2 NS 3.3
application
‘Manzanillo’ Control Leonardite extracts 0.5% Leonardite extracts 1% Significance Coefficient of variation (%)
Treatment
Table 4 The effect of folk
Q’* 6.5
6.8
3.9
1.5
I .7 Q . . Q’*
0.1 0.1 0.1 NS 4.9
1992
0.08 0.14 0.12
0.1 0.1 0.1 NS 14.7
1991
0.1 0.2 0.1
1.2
1.5 1.5 1.4 NS 3.6
1992
Mg (%)
of two irrigated
8.9
L”
24.4 27.9 31.0
35.6 36.0 42.4 NS 10.6
1991
23.7 27.0 29.3 L’ 4.6
40.6 44.5 45.0 NS 3.2
1992
B (p.p.m.1
olive cultivars
46.1 44.9 43.7 NS 11.4
51.2 44.9 51.8 NS 16.4
1991
30.1 38.6 40.0 NS 7.4
56.9 52.7 54.6 NS 4.9
1992
Mn (p.p.m.)
11.6 12.4 12.8 NS 5.8
10.1 11.3 10.2 NS 8.6
1991
9.3 10.0 10.3 NS 6.2
11.7 13.0 12.6 NS 6.2
1992
Zn (p.p.m.)
33.3 15.9 16.2 NS 50.7
21.1 13.2 17.2 NS 46.3
1991
45.4 35.7 28.5 NS 14.7
38.5 43.4 45.7 NS 7.8
1992
Cu (p.p.m.)
1991
48.0 63.3 51.9 NS 5.1
62.8 58.5 55.3 NS 7.8
1992
Fe (p.p.m.)
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4. Discussion Foliar application of humic substances clearly stimulate vegetative growth of olive trees. The results are in agreement with those reported for a wide number of plant species (Chen and Aviad, 1990). The promotion of growth was independent of the culture system, because the effect was observed in both irrigated and non-irrigated olive trees. However, no effect was observed on yield, even in experiments where fruit size or fruit set were improved, and in experiments conducted in 2 successive years when vegetative growth was promoted. The stimulating effect of humic substances on plant growth has been related, at least in part, to enhanced uptake of mineral nutrients. Increase of uptake of macro and microelements influenced by humic substances have been reported in a large number of publications and in different plant species (Rauthan and Schnitzer, 1981; Chen and Aviad, 1990; Fagbenro and Agboole, 1993). This effect has been observed when humic substances were applied to the soil and also when they were mixture in a nutrient solution (Lee and Bartlett, 1976; Vaughan and Malcom, 1985; David et al., 1994), suggesting the existence of a synergistic effect of combined applications of mineral nutrients and humic substances. In this study, the synergistic effect was not observed when a nutrient solution applied to the irrigation water was combined with foliar applications of leonardite extracts to young olive plants. This disagreement with the reported results might be due to the fact that the nutrient solution and the humic acids were not mixed, making impossible the formation of chelates and complexes that directly or indirectly could affect nutrient uptake. On the other hand, foliar sprays of leonardite extracts significantly influenced leaf K, Ca, Mg, B and Fe concentrations under field conditions, indicating that humic substances may affect nutrient leaf values by mechanisms other than the direct formation of complexes and chelates in the soil. The plant-hormone like activity attributable to humic substances (O’Donnell, 1973; Casenave de Sanfilippo et al., 1990) might explain the phenomenon. It should be noted, moreover, that nutrient accumulation in leaves caused by the treatment was evident only when leaf nutrient concentration in control trees was above the threshold limits established for the sufficiency range in olives (Beutel et al., 1983), and that leaf K values were unaffected by treatments when they were below that limit. These results seems to indicate that foliar application of humic substances do not influence the nutritional status of the olive trees and, therefore, do not compensate for the lack of mineral nutrition.
References Amon, D.I. and Stout, P.R., 1939. The essentiality of certain elements in minute quantity for plants with special reference to copper. Plant Physiol., 14: 371-375. Bet&l, J., Uriu, K. and Lilleland, 0.. 1983. Leaf analysis for California deciduous fruits. In: Soil and plant tissue testing in California. University of California, Bull., 1879. Brownell, J.R., Nordstrom, G., Marihart, J. and Jorgensen, G., 1987. Crop responses from two new leonardite extracts. Sci. Total Environ., 62: 491-499. Casenave de Sanfllippo, E., Argiiello, J.A., Abdala, G. and Orioli, G.A., 1990. Content of auxin-, inhibitorand gibberellin-like substances in humic acids. Biol. Plant., 32: 346351.
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et al. / Scientia Horticulturae 66 (1996) 191-200
Chen, Y. and Aviad, T., 1990. Effects of humic substances on plant growth. In: Humic substances in soil and crop science; Selected Readings, American Society of Agronomy and Soil Science Society of America, Madison, pp. 161- 186. David, P.P., Nelson, P.V. and Sanders, D.C., 1994. A humic acid improves growth of tomato seedling in solution culture. J. Plant Nutr., 17: 173-184. Fagbenro, J.A. and Agboole, A.A., 1993. Effect of different levels of humic acid on the growth and nutrient uptake of teak seedlings. J. Plant Nutr., 16: 1465-1483. Grewehng, T., 1976. Chemical analysis of plant tissue. Search and Agriculture, Agronomy 6. Cornell University, Ithaca, NY, pp. l-35. Lee, Y.S. and Bartlett, R.J., 1976. Stimulation of plant growth by humic substances. Soil Sci. Sot. Am. J., 40: 876-879. Murphy, J. and Riley, J.P., 1962. A modified single solutions method for the determination of phosphate in natural waters. Anal. Chem. Acta, 27: 31-36. O’Donnell, R.W., 1973. The auxin-like effects of humic preparations from leonardite. Soil Sci., 116: 106-l 12. Rauthan, B.S. and Schnitzer, M., 1981. Effects of a soil fulvic acid on the growth and nutrient content of cucumer (Cucumis satiuus) plants. Plant Soil, 63: 491-495. Vaughan, D. and Malcom, R.E., 1985. Influence of humic substances on growth and physiological processes. In: D. Vaughan and R.E. Malcom (Editors), Soil Organic Matter and Biological Activity. Martinus Nijhoff/Dr. W. Junk Publ., Dordrecht, pp. 37-75. Westwood, M.N., 1978. Temperate zone pomology. Freeman, San Francisco, CA, 428 pp. Xudan, X., 1986. The effect of fohar application of fulvic acid on water use, nutrient uptake and wheat yield. Austr. J. Agric. Res., 37: 343-350.
Response of olive trees to folk application of humic substances extracted from leonardite Femiindez-Escobar *, M. Benlloch, D. Barranco, A. Duefias, J.A. Gutkrez GaSn Departamento de Agronomia, Universidad de Grdoba, Apartado 3048, 14080 Chdoba. Spain
Accepted 1 April 1996
Abstract The effect of foliar applied humic substances extracted from leonardite on olive growth was studied in greenhouse experiments using cuttings and young olive plants, some of which were irrigated with a nutrient solution, and also in field experiments selecting mature trees growing in the drylands or under irrigation. Foliar application of leonardite extracts to young olive plants stimulated shoot growth when they were growing without the addition of mineral elements to the irrigation water, but did not promote growth when applied to plants watered with a nutrient solution, although growth of fertilized plants was greater than that of unfertilized ones. Under field conditions, folk application of leonardite. extracts stimulated shoot growth and promoted the accumulation of K, B, Mg, Ca and Fe in leaves. However, when leaf N and leaf K values were below the threshold limit for the sufficiency range, foliar application of humic substances was ineffective to promote accumulation of these nutrients in leaves. Keywords: Folk
nutrition; Humic acids; Humic substances; Leonardite; Olive; OIea europaea
1. Introduction The beneficial effects of humic substances on plant growth have been observed by man from ancient times. Today, it is recognized that soil organic matter may affect the physical, chemical and microbiological properties of the soil and, indirectly, affects plant growth. Also, it is well documented that humic substances have a direct effect on plant growth (Chen and Aviad, 1990).
* Corresponding author. Fax: 34-57-218569. 0304-4238/%/$15.00 Copyright 0 1996 Elsevier Science B.V. All rights reserved. PII SO304-4238(96)009 14-4
192
R. Fernhdez-Escobar
ei cd./ Scienria Horricdnwae
66 (1996) 191-200
During the 19th century and in the early 20th century, there was controversy among scientists on the role of the humus on plant nutrition. While some of them supported the hypothesis that humus is the only material which supplies nutrients to plants, others accumulated evidence and provided information on the role of mineral elements in plant nutrition. In the first half of the 20th century, Amon and Stout (1939) proposed the criteria for essentiality of a element for plant growth, establishing one of the principles governing modem plant nutrition. However, there are a number of papers reporting information about the enhancement of plant growth promoted by the application of humic substances (Chen and Aviad, 1990). This growth promoting activity seems to be caused by plant hormone-like materials present in the humic substances (O’Donnell, 1973; Casenave de S~~lippo et al., 1990). Humic substances are usually applied to the soil, and favorably affect the soil structure and soil microbial populations. Foliar sprays of these substances also promote growth in a number of plant species such as tomato, cotton and grape (Brownell et al., 1987). Spraying with fulvic acid also increased the yield of wheat grown under dry conditions (Xudan, 1986), suggesting the capability of the humic substances to reduce water stress. In addition, the lower cost of the application of these products by foliar spray has been indicated (Chen and Aviad, 1990). Leonardite is an oxidized form of lignite coal. Humic and fulvic acids may be obtained by extraction from leonardite and, presently, humic preparations from leonardite are available for use in agriculture. The objective of the present work was to study the effect of foliar applied leonardite extracts on growth and yield of olive trees growing in the dryland or under irrigation.
2. Materials and methods A series of greenhouse and field experiments were conducted to study the effect of foliar applied humic substances on olive yield and growth. In all cases, Naturvital-16 (Daymsa, Spain) containing 9% humic acids and 7% fulvic acids extracted from leonardite, and 0.59% N, 3.3% K, 95.7 ppm P, 0.32% Ca, 300 ppm Mg, 0.53% S, 0.3% Fe, 22 ppm MO, 6.9 p.p.m. Cu, 5.2 p.p.m. Zn, 0.3 p.p.m. B, and 8.2 p.p.m. Mn, was used. 2.1. Greenhouse experiments Two different ex~~rnen~ were carried out in the greenhouse with the aim of studing the effect of leonardite extracts concentration on growth of young olive trees. For this purpose, in a preliminary experiment l-year-old olive plants cv. Picual growing in 1.5 1 plastic pots were placed in a greenhouse at 30/15’C (day/night) with a 14-h photoperiod. The plants received one foliar application of Naturvital 16 at concen~ations of 0%, 0.5%, I%, 2%, 4%, 8% or 16%. There were two replications (plants) per treatment. In a second experiment, mist-rooted ‘Picual’ olive cuttings were transferred to 2-l plastic pots containing a mixture of river sand and peat (2:l by volume), and placed in a
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greenhouse under the same growth conditions described above. Before the initiation of treatments and after one month of acclimatization, the cuttings were pruned to a single shoot per plant. The plants were arranged in a split-plot experiment. The whole-plot factor consisted of the application of mineral nutrients or no application of mineral nutrients. Plants that received mineral fertilization were watered with a solution of 1 g 1-r of Hakaphos fertilizer containing 15% N, 4.8% P, 12% K, 0.8% Mg, 9% S, 0.03% B, 0.1% Mn, 0.06% Zn, and 0.004% MO. The leonardite extract concentration was the subplot factor. Each subplot consisted of four plants and subtreatments were one foliar application of Naturvital 16 at concentrations of 0%, 0.25%, 0.5% or 1%. The experiment was developed from April-July 1991, and during this time plants were watered twice a week with 250 ml of water. Shoot length was measured on six dates during the experiment on each experimental plant. 2.2. Field experiments Four trials were conducted in three olive orchards located in three different areas of Andalusia, Southern Spain. Two experiments were carried out in a commercial orchard located near Cordoba. Drip-irrigated, 5-year-old ‘Manzanillo’ trees and 5-year-old ‘Hojiblanca’ olive trees were selected for the experiments. Trees were spaced 8 X 6 m apart. The third experiment was conducted in the Experimental Farm of La Mina at Cabra, Southern C6rdoba province. Non-irrigated, lo-year-old ‘Hojiblanca’ trees spaced 7 X 7 m apart were selected for the experiment. Annual rainfall in this area was 450 mm. The fourth experiment was conducted in a commercial orchard located in Ubeda, Jaen province. Non-irrigated, 15-year-old ‘Picual’ trees spaced 10 X 10 m apart were selected. Annual rainfall in the area was 300 mm. For all field experiments, a randomized block design was used with four blocks and three treatments. Each plot consisted of one tree in the irrigated ‘Manzanillo’ and ‘Hojiblanca’ trials located in Cordoba, and of five trees in the other trials. The treatments were a nontreated control and foliar applications of Naturvital-16 at 0.5% or 1%. The treatments were applied in April, approximately 1 month before bloom. The Cordoba experiments were developed during 1991 and repeated in 1992; the other experiments were conducted during 1992. Vegetative growth was determined at the end of the growing season by measuring shoot length on ten tagged shoots per experimental tree. Also, the above-ground tree volume, a variable used as covariate to adjust yield, was calculated from height and spread measurements as indicated by Westwood (1978). The effect of treatments on cropping was evaluated by measuring yield per tree at harvest and the number of fruits per shoot on those tagged to determine vegetative growth. A sample of approximately 1 kg of fruits per plot was taken at harvest to determine fruit size and oil content. Fruit oil content was determined by nuclear magnetic resonance (NMR) after milling the fruit sample. The effect of leonardite extracts on the nutritional status of the olive trees was determined by leaf analysis of a sample of 100 leaves per plot collected in July, according to Beutel et al. (1983). Fully-expanded, mature leaves from the middle portion of nonbearing, current season shoots were removed for analysis of mineral nutrients.
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Leaves were collected in paper bags and stored in a portable ice chests. Once in the laboratory, leaves were washed with 0.03% Triton X-100, rinsed in deionized water, dried at 80°C for 48 h, ground, and stored in an oven at 60°C until analysis. Nitrogen was determined by the Kjeldahl procedure. For other elemental determinations, the stored samples were ashed in a muffle furnace at 600°C for 12 h, and dissolved in 0.1 N HCl. Total P was determined by calorimetry using the method described by Murphy and Riley (1962). Boron was determined in the extract by calorimetry (Greweling, 1976). The remaining elements (K, Mg, Ca, Zn, Mn, Fe and Cu) were measured using an atomic absorption spectrophotometer. Analyses of variance and regression analyses were performed on the data from each experiment, with the exception of yield that was analyzed by analysis of covariance using the total above-ground tree volume as covariate.
3. Results 3.1. EfSect of leonardite extracts concentration
on growth of young olive plants
Foliar application of leonardite extracts to l-year-old olive plants at concentrations higher than 1% of the commercial product Naturvital-16, inhibited plant growth (data not shown). Concentrations of 0.5% and 1% improved olive cutting growth when plants were growing without the addition of mineral elements to the irrigation water (Fig. l(b)), but there was no promoting effect when leonardite extracts was applied to plants receiving mineral nutrients (Fig. l(a)) or when Naturvital-16 was applied at 0.25% (Fig. l(b)). However, growth of fertilized plants was greater than that of plants that did not receive additional fertilizers. 3.2. Effect of leonardite extracts on vegetative growth ana! yield of olive trees
Vegetative growth was affected by foliar application of leonardite extracts to mature olive trees. Shoot growth of ‘Hojiblanca’ trees growing under irrigation (Table 1) and also growing in the drylands (Table 21, significantly increased when leonardite extracts were applied to leaves at concentrations of 0.5% or l%, compared to untreated trees. In the experiment of 1991, shoot growth of ‘Hojiblanca’ treated trees was more than twice that of control trees (Table 1). However, no significant differences were found either on ‘Manzanillo’ and ‘Picual’ trees or on ‘Hojiblanca’ trees in the 1992 experiments. Lack of significance might be due to the high variability obtained in these experiments. Fruit set increased significantly after foliar application of leonardite extracts only in the ‘Hojiblanca’ experiment conducted under no irrigation (Table 2), and was unaffected by treatments in the other cultivars and experiments. The quadratic response indicates an effect of leonardite extracts on fruit set only at the lower concentration. For fruit size, there was a similar quadratic response in the ‘Manzanillo’ experiment conducted in 1991 (Table l), but no significant effect was observed in the other cultivars and experiments. Yield and fruit oil content were unaffected by the treatments (Tables 1 and 2).
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A
100
60
.
60
r
4o
-
20
/Y
’
/:
5 ? 2
O loo
0 0
20
40
60
60
Days after treatments Fig. 1. Effect of the concentration of foliar applied Naturvital-16 on shoot growth of olive plants irrigated with a nutrient solution (A) or without a nutrient solution (B). Vertical bars indicate the SE.
3.3. Effect of leonardite extracts on leaf nutrient concentration Leaf analysis of samples collected in July showed a nutritional olive tree status characterized by K values below the threshold limit of 0.8% for the sufficiency range in the experiments conducted with ‘Picual’ and ‘Hojiblanca’ under no irrigation (Table 3), and also in the irrigated ‘Hojiblanca’ experiment of 1992 (Table 4). Also, leaf N values were below the sufficiency threshold of 1.5% in the irrigated experiment of ‘Manzanillo’ and ‘Hojiblanca’ conducted in 1991 (Table 4). No other nutritional disorder was observed in the experiments. Folk application of leonardite extracts affected leaf nutrient concentration. Leaf K values significantly increased in experiments conducted with ‘Manzanillo’ in 1992 and with ‘Hojiblanca’ in 1991 (Table 4). Also, leaf Ca, Mg, B and Fe concentrations were significantly affected by the treatments (Tables 3 and 4). However, leaf nutrient values
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Table 1 The effect of foliar application cultivars
of leonardite Shoot growth (cm)
Treatment
1991
‘Manzanillo’ Control Leonatdite extracts 0.5% Leonardite extracts 1% Significance Coefficient of variation (o/o)
8.27 10.72 15.25 NS 30.9
‘Hojiblanca’ Control Leonardite extracts 0.5% Leonardite extracts 1% Significance Coefficient of variation (%I
6.25 14.02 13.47 L’ 29.9
1992
extracts
on vegetative
growth
Fruit set (fruits/cm) 1991
Fruit size
1992
0.29 0.24 0.19 NS 29.4
1991
1992
1991
1992
3.96 4.55 4.14 4.0
3.62 3.33 3.36 NS 11.7
31.0 30.3 31.0 NS 23.3
32.1 35.3 24.2 NS 28.6
3.61 3.58 3.91 NS 7.3
3.43 3.29 3.82 NS 7.2
42.6 38.4 38.3 NS 22.1
53.2 60.3 50.9 NS 16.3
Q*
0.39 0.43 0.39 NS 10.6
olive
Yield (Kg tree-‘)
(g)
0.27 0.23 0.28 NS 16.6
7.64 8.55 7.71 NS 7.1
and yield of two irrigated
NS, nonsignificant; * P = 0.05. L, linear; Q, quadratic.
in all these experiments were above the sufficiency threshold, including control trees. In contrast, trees with leaf N and leaf K values below the threshold limit were unaffected by folk applications of leonardite extracts.
Table 2 The effect of folk cultivars
application
of leonardite extracts on vegetative Shoot growth
growth and yield of two nonirrigated
olive
(cm)
Fruit set (fruits cm- ’ )
Fruit size (g)
Yield (leg tree- ‘1
Fruit oil content (%I
‘Picual’ Control Leonardite extracts 0.5% Leonardite extracts 1% Significance Coefficient of variation (%)
5.82 6.41 6.51 NS 8.4
0.17 0.13 0.15 NS 15.6
3.45 3.56 3.57 NS 5.4
21.42 28.40 24.65 NS 17.0
16.68 18.15 17.45 NS 5.8
‘Hojiblanca’ Control Leonardite extracts 0.5% Leonardite extracts 1% Significance Coefficient of variation (%b)
6.64 8.08 8.03 L’ 9.8
0.42 0.55 0.44
3.92 3.78 3.85 NS 7.5
24.61 29.08 25.49 NS 14.2
20.63 21.35 21.20 NS 1.3
Treatment
NS, nonsignificant; P = 0.05. L, linear; Q, quadratic. l
Q’ 12.2
NS 5.0
0.10 0.09 0.09
NS 1.8
0.10 0.10 0.09
P (o/o)
* P = 0.05; * * P = 0.01.
NS 1.4
Significance Coefficient of variation (o/o)
NS, nonsignificant; L, linear.
1.7 1.7 1.7
‘Hojiblanca’ Control Leonardite extracts 0.5% Leonardite extracts 1%
1.7
I .I
1.7
N (%)
NS 4.6
0.4 0.4 0.4
NS 7.6
0.6 0.6 0.6
K (o/o)
L’ 3.5
1.8 1.6 1.5
l
1.2 1.3 1.5 L ’ 3.6
Ca (o/o)
0.1 0.1 0.1 NS 5.2
NS 6.8
0.1 0.1 0.1
Mg (%)
of leonardite extracts on leaf nutrient concentration
NS 1.4
extracts 0.5% extracts 1%
application
Significance Coefficient of variation (%)
‘Picual’ Control Leonardite Leonardite
Treatment
Table 3 The effect of folk
36.1 36.5 35.0 NS 6.5
25.5 28.0 31.6 L’ 4.2
B (p.p.m.)
of two nonirrigated
olive cultivars.
39.5 39.3 38.8 NS 4.0
48.9 45.8 51.1 NS 5.1
Mn (p.p.m.)
13.6 15.8 13.9 NS 4.6
8.8 11.1 10.0 NS 8.8
Zn (p.p.m.1
31.6 33.3 34.6 NS 4.2
30.3 38.4 37.3 NS 8.2
Cu (p.p.m.1
46.2 48.8 47.1 NS 5.2
37. I 43.2 58.1 L’ 5.8
Fe (p.p.m.)
l
NS, nonsignificant; P= L, linear, Q, quadratic.
1.8 I .8 1.8 NS 1.9
0.05; * * P=
1.3 1.3 1.2 NS 5.1
‘Hojiblanca’ Control Leonardite extracts 0.5% Leonardite extracts 1% Significance Coefficient of variation(%)
1.6 1.6 1.6 NS 2.4
0.01.
0.1 0.1 0.1 NS 8.9
0.1 0.1 0.1 NS 21.8
1991
1991
0.1 0.1 0.1 NS 1.4
0.1 0.1 0.1 NS 3.5
1992
0.81 0.84 1.00 L * 8.8
0.84 0.84 0.88 NS 10.8
1991
K (%)
0.58 0.57 0.63 NS 4.2
0.84 0.96 1.03 L * 3.8
1.6 1.7 1.7 NS 4.0
1.8 1.6 1.8 NS 8.0
1991
Ca (%)
concentration
1992
extracts on leaf nutrient
PC%)
1992
of leonardite
N (%I
1.2 1.2 1.2 NS 3.3
application
‘Manzanillo’ Control Leonardite extracts 0.5% Leonardite extracts 1% Significance Coefficient of variation (%)
Treatment
Table 4 The effect of folk
Q’* 6.5
6.8
3.9
1.5
I .7 Q . . Q’*
0.1 0.1 0.1 NS 4.9
1992
0.08 0.14 0.12
0.1 0.1 0.1 NS 14.7
1991
0.1 0.2 0.1
1.2
1.5 1.5 1.4 NS 3.6
1992
Mg (%)
of two irrigated
8.9
L”
24.4 27.9 31.0
35.6 36.0 42.4 NS 10.6
1991
23.7 27.0 29.3 L’ 4.6
40.6 44.5 45.0 NS 3.2
1992
B (p.p.m.1
olive cultivars
46.1 44.9 43.7 NS 11.4
51.2 44.9 51.8 NS 16.4
1991
30.1 38.6 40.0 NS 7.4
56.9 52.7 54.6 NS 4.9
1992
Mn (p.p.m.)
11.6 12.4 12.8 NS 5.8
10.1 11.3 10.2 NS 8.6
1991
9.3 10.0 10.3 NS 6.2
11.7 13.0 12.6 NS 6.2
1992
Zn (p.p.m.)
33.3 15.9 16.2 NS 50.7
21.1 13.2 17.2 NS 46.3
1991
45.4 35.7 28.5 NS 14.7
38.5 43.4 45.7 NS 7.8
1992
Cu (p.p.m.)
1991
48.0 63.3 51.9 NS 5.1
62.8 58.5 55.3 NS 7.8
1992
Fe (p.p.m.)
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4. Discussion Foliar application of humic substances clearly stimulate vegetative growth of olive trees. The results are in agreement with those reported for a wide number of plant species (Chen and Aviad, 1990). The promotion of growth was independent of the culture system, because the effect was observed in both irrigated and non-irrigated olive trees. However, no effect was observed on yield, even in experiments where fruit size or fruit set were improved, and in experiments conducted in 2 successive years when vegetative growth was promoted. The stimulating effect of humic substances on plant growth has been related, at least in part, to enhanced uptake of mineral nutrients. Increase of uptake of macro and microelements influenced by humic substances have been reported in a large number of publications and in different plant species (Rauthan and Schnitzer, 1981; Chen and Aviad, 1990; Fagbenro and Agboole, 1993). This effect has been observed when humic substances were applied to the soil and also when they were mixture in a nutrient solution (Lee and Bartlett, 1976; Vaughan and Malcom, 1985; David et al., 1994), suggesting the existence of a synergistic effect of combined applications of mineral nutrients and humic substances. In this study, the synergistic effect was not observed when a nutrient solution applied to the irrigation water was combined with foliar applications of leonardite extracts to young olive plants. This disagreement with the reported results might be due to the fact that the nutrient solution and the humic acids were not mixed, making impossible the formation of chelates and complexes that directly or indirectly could affect nutrient uptake. On the other hand, foliar sprays of leonardite extracts significantly influenced leaf K, Ca, Mg, B and Fe concentrations under field conditions, indicating that humic substances may affect nutrient leaf values by mechanisms other than the direct formation of complexes and chelates in the soil. The plant-hormone like activity attributable to humic substances (O’Donnell, 1973; Casenave de Sanfilippo et al., 1990) might explain the phenomenon. It should be noted, moreover, that nutrient accumulation in leaves caused by the treatment was evident only when leaf nutrient concentration in control trees was above the threshold limits established for the sufficiency range in olives (Beutel et al., 1983), and that leaf K values were unaffected by treatments when they were below that limit. These results seems to indicate that foliar application of humic substances do not influence the nutritional status of the olive trees and, therefore, do not compensate for the lack of mineral nutrition.
References Amon, D.I. and Stout, P.R., 1939. The essentiality of certain elements in minute quantity for plants with special reference to copper. Plant Physiol., 14: 371-375. Bet&l, J., Uriu, K. and Lilleland, 0.. 1983. Leaf analysis for California deciduous fruits. In: Soil and plant tissue testing in California. University of California, Bull., 1879. Brownell, J.R., Nordstrom, G., Marihart, J. and Jorgensen, G., 1987. Crop responses from two new leonardite extracts. Sci. Total Environ., 62: 491-499. Casenave de Sanfllippo, E., Argiiello, J.A., Abdala, G. and Orioli, G.A., 1990. Content of auxin-, inhibitorand gibberellin-like substances in humic acids. Biol. Plant., 32: 346351.
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et al. / Scientia Horticulturae 66 (1996) 191-200
Chen, Y. and Aviad, T., 1990. Effects of humic substances on plant growth. In: Humic substances in soil and crop science; Selected Readings, American Society of Agronomy and Soil Science Society of America, Madison, pp. 161- 186. David, P.P., Nelson, P.V. and Sanders, D.C., 1994. A humic acid improves growth of tomato seedling in solution culture. J. Plant Nutr., 17: 173-184. Fagbenro, J.A. and Agboole, A.A., 1993. Effect of different levels of humic acid on the growth and nutrient uptake of teak seedlings. J. Plant Nutr., 16: 1465-1483. Grewehng, T., 1976. Chemical analysis of plant tissue. Search and Agriculture, Agronomy 6. Cornell University, Ithaca, NY, pp. l-35. Lee, Y.S. and Bartlett, R.J., 1976. Stimulation of plant growth by humic substances. Soil Sci. Sot. Am. J., 40: 876-879. Murphy, J. and Riley, J.P., 1962. A modified single solutions method for the determination of phosphate in natural waters. Anal. Chem. Acta, 27: 31-36. O’Donnell, R.W., 1973. The auxin-like effects of humic preparations from leonardite. Soil Sci., 116: 106-l 12. Rauthan, B.S. and Schnitzer, M., 1981. Effects of a soil fulvic acid on the growth and nutrient content of cucumer (Cucumis satiuus) plants. Plant Soil, 63: 491-495. Vaughan, D. and Malcom, R.E., 1985. Influence of humic substances on growth and physiological processes. In: D. Vaughan and R.E. Malcom (Editors), Soil Organic Matter and Biological Activity. Martinus Nijhoff/Dr. W. Junk Publ., Dordrecht, pp. 37-75. Westwood, M.N., 1978. Temperate zone pomology. Freeman, San Francisco, CA, 428 pp. Xudan, X., 1986. The effect of fohar application of fulvic acid on water use, nutrient uptake and wheat yield. Austr. J. Agric. Res., 37: 343-350.