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					Effect of humic substances on nutrient uptake
by herbage and on production and nutritive value
of herbage from sown grass pastures
G. Verlinden*, T. Coussens*, A. De Vliegher†, G. Baert* and G. Haesaert*
*Faculty of Biosciences and Landscape Architecture, University College Ghent, Ghent University, Ghent, Belgium
and †ILVO-Plant, Merelbeke, Belgium




Abstract
                                                                Keywords: humic substances, leonardite, grass, yield,
The effect of humic substances on the nutrient uptake,          nutrients, nutritive value
herbage production and nutritive value of herbage from
sown grass pastures was studied in six field experi-
ments. Commercial humic substances were applied in
                                                                Introduction
combination with mineral fertilizer or slurry, either as a      Many European agricultural regions with high inputs of
solution (HF liquid; 8Æ3 kg humic substances ha)1) or           inorganic fertilizers or organic fertilizer as slurry are
incorporated into the mineral fertilizer (HF incorpo-           faced with decreasing organic matter (OM) content of
rated; 3Æ6 to 6Æ4 kg humic substances ha)1). A series of        their soils (Vleeshouwers and Verhagen, 2002). In
cuts, ranging from two to five cuts, was taken during            Belgium, the OM content of more than 0Æ50 of arable
the growing season. The general response in herbage             land and permanent grasslands is lower than the
production to application of humic substances was an            optimal value (Sleutel et al., 2003; Boon et al., 2009).
increase in herbage mass of dry matter (DM) at the first         As soil OM is essential for sorption and gradually release
cut although this was only significant in two experi-            of nutrients, large amounts of external organic sources
ments for the HF incorporated treatment. Total herbage          are needed to increase the OM content of the soil.
production of DM over the growing season, however,              However, application of high quantities of organic
was similar for treatments with or without application          materials, which are usually rich in nitrogen (N) and
of humic substances. The overall effect of HF incorpo-          phosphorus (P), is in conflict with legislation on
rated and HF liquid on the herbage mass of DM at the            nutrient inputs in many countries in north-west
first cut across the experiments was calculated using a          Europe.
meta-analysis technique and it was shown that there                The use of concentrated products of humic substances
was a significant proportional increase of 0Æ14                  (humic and fulvic acids) in agriculture is one solution as
(P < 0Æ05) with the HF incorporated treatment and a             many of the beneficial characteristics of soil humus are
non-significant increase of 0Æ08 with the HF liquid              associated with the presence of humic and fulvic acids.
treatment compared to the control treatment. The                These humic substances are recognized as the most
nutritive value of the herbage at the first cut was              chemically active compounds in soils, with cation and
similar across all treatments. In general nitrogen,             anion exchange capacities far exceeding those of clays
phosphorus and potassium uptake at the first grass cut           (Stevenson, 1994; Koopal et al., 2005).
was higher after application of humic substances but               Many beneficial effects of humic substances on the
only in one experiment was this increase statistically          growth of plants are recognized as they can produce
significant.                                                     various morphological, physiological and biochemical
                                                                effects on plants (see reviews by Visser, 1986; Chen and
                                                                Aviad, 1990 and Nardi et al., 2002). Numerous studies
                                                                have shown that the addition of a specific amount of
Correspondence to: G. Verlinden, Faculty of Biosciences and     humic substances can enhance the growth of roots,
Landscape Architecture, University College Ghent, Ghent         shoots and leaves, and encourage nutrient absorption
University, Voskenslaan 270, B-9000 Ghent, Belgium.             by plants. Extended reviews were published by Vara-
E-mail: greet.verlinden@hogent.be                               nini and Pinton (2001) and Nardi et al. (2002). The
Received 19 June 2009; revised 16 November2009                  effects of humic substances on growth and metabolism




doi: 10.1111/j.1365-2494.2009.00726.x                     Ó 2010 Blackwell Publishing Ltd. Grass and Forage Science, 65, 133–144   133
134 G. Verlinden et al.




of maize have been studied by many authors (Canellas             weight solution). The HF liquid was sprayed on the
et al., 2002; Sharif et al., 2002; Quagiotti et al., 2004;      grass herbage at the start of the growing season. Besides
Nardi et al., 2007; Eyheraguibel et al., 2008; Carletti         the liquid form, the solid form, Humifirst WG, contain-
et al., 2008; Canellas et al., 2008; Verlinden et al., 2009).   ing 0Æ68 of humic substances, was also used in the
Other crops studied have been barley (Ayuso et al.,             experiments. Humifirst WG was incorporated into
1996), wheat (Delfine et al., 2005; Jones et al., 2007),         mineral fertilizers at varying concentrations (Table 1)
potato (Verlinden et al., 2009), olive plants (Fernandez-       and will be subsequently referred to as HF incorporated.
Escobar et al., 1996), tomato (Atiyeh et al., 2002;             The humic substances in both products were extracted
Yildirim, 2007), lettuce (Tufencki et al., 2006) and            from Canadian leonardite which is highly oxidized
strawberry (Pilanah and Kaplan, 2003; Arancon et al.,           lignite with more than 0Æ85 of humic acids.
2006). Comparison of the different studies showed that
the effects of humic substances may be affected by
                                                                Site and experimental designs
the origin, concentration and molecular weight of the
humic substances, and the species and variety of the            Field experiments with humic substances were carried
plant treated (Visser, 1986; Chen and Aviad, 1990;              out in 2006, 2007 and 2008 at four locations in
Varanini and Pinton, 2001; Nardi et al., 2002).                 Flanders, the northern part of Belgium. In 2006
   The effect of humic substances in grass pastures has         Experiments BOT1 and BOT2 were set up at Bottelare
rarely been reported. However, if OM content of grass           (L. multiflorum pasture), Experiment HOOG was set up
pastures continues to decrease, grass pastures could also       at Hoogstraten (L. perenne pasture) and Experiment
benefit from the use of humic substances. Cooper et al.          MER1 was set up at Merelbeke (L. perenne pasture). In
(1998) showed that humic substances could increase              2007 Experiment BOT2 was continued with the same
root mass of creeping bentgrass (Agrostis stolonifera L.)       treatments and Experiment HOOG was continued with
proportionately by 0Æ45 in the 0–10 cm soil layer and           some additional treatments (Table 1). In 2008, two new
increase P concentration of the herbage proportionately         experiments were set up at Merelbeke (MER2, L. perenne
by 0Æ03–0Æ05. Hafidi et al. (1997) measured an increased         pasture) and at Moortsele (MOOR, L. perenne pasture).
P absorption by 0Æ19–0Æ35 in Italian ryegrass herbage           Soil characteristics on each location are shown in
(Lolium multiflorum L.) following application of different       Table 2.
extracts of humic substances. The experiments of                   The humic acid treatments in each experiment are
Cooper et al. (1998) and Hafidi et al. (1997) were set           shown in Table 1. In Experiments BOT1, BOT2, MER2
up in greenhouse conditions and did not assess the              and MOOR, three treatments were imposed: (i) a
effect of humic substances on herbage production and            control treatment with mineral fertilizer according to
its mineral composition.                                        fertilizer recommendations based on soil chemical
   The aim of this study was a simultaneous assess-             analyses, (ii) mineral fertilizer + 50 L ha)1 HF liquid
ment of the effect of humic substances on nutrient              and (iii) mineral fertilizer with HF incorporated. In
uptake, herbage production and the nutritive value of           Experiment MER1 only two treatments were imposed:
the herbage. Field experiments were preferred to                (i) a control treatment with mineral fertilizer according
greenhouse experiments in order to interpret the                to fertilizer recommendations based on soil chemical
effects of humic substances in the light of environ-            analyses and (ii) mineral fertilizer with HF incorpo-
mental conditions. General conclusions about the                rated. In Experiment HOOG the same treatments as in
effect of humic substances on herbage production,               MER1 were applied as well as three treatments with
nutrient uptake and nutritive value of the herbage              slurry application: (i) slurry application plus mineral
between the different experiments were obtained by              fertilizer, (ii) slurry application mixed with 50 L ha)1
applying the meta-analysis technique of Hedges et al.           HF liquid + mineral fertilizer and (iii) slurry application
(1999) and Gurevitch and Hedges (2001). This                    plus mineral fertilizer with HF incorporated. For all
method allows the integration of results of indepen-            experiments, a randomized complete block design was
dent field experiments.                                          used with four replicates. Plot size varied between
                                                                19 m2 for Experiment MER1 and 102 m2 for Experi-
                                                                ment HOOG, with an average plot size of 45 m2. On all
Materials and methods
                                                                locations, except in Experiment BOT2, the experiments
                                                                were set up on grasslands of more than 1 year old
Humic substances
                                                                which were not included in a crop rotation. The
A liquid mixture of humic and fulvic acids (HumifirstÒ;          exception was Experiment BOT2 where grass seed
Tradecorp, Madrid, Spain) was used as an organic                was sown in April 2006. Consequently, results of a full
amendment. The mixture, termed HF liquid, contained             growing season could only be assessed in 2007 at this
0Æ12 of humic acids and 0Æ03 of fulvic acids (weight ⁄          location.




                                                                 Ó 2010 Blackwell Publishing Ltd. Grass and Forage Science, 65, 133–144
                                                                                        Effects of humic substances on grass pastures 135




Table 1 Experiments and treatments, including amounts of humic substances (HS) in the form of Humifirst (HF) as a liquid or
incorporated into mineral fertilizer. Amount of mineral fertilizer based on chemical soil analysis and recommendations by the Soil
Service of Belgium. Amounts of mineral fertilizer in each experiment are given as the amounts at the beginning of the season or after
each cut in the sequence below and the values are expressed in kg ha)1: Experiment BOT1, N = 100 ⁄ 80, P2O5 = 80 ⁄ 0,
K2O = 100 ⁄ 100; Experiment BOT2 in 2006, N = 90 ⁄ 80 ⁄ 80, P2O5 = 80 ⁄ 0 ⁄ 0, K2O = 130 ⁄ 80 ⁄ 0; Experiment BOT2 in 2007:
N = 90 ⁄ 80 ⁄ 80, P2O5 = 60 ⁄ 0 ⁄ 0, K2O = 130 ⁄ 80 ⁄ 80; Experiment HOOG in 2006 and 2007: N = 100 ⁄ 60 ⁄ 50 ⁄ 40, P2O5 =
 0 ⁄ 0 ⁄ 30 ⁄ 30, K2O = 170 ⁄ 100 ⁄ 100; Experiment MER1, N = 120 ⁄ 100 ⁄ 100 ⁄ 80, P2O5 = 56 ⁄ 60 ⁄ 30 ⁄ 24, K2O = 120 ⁄ 100 ⁄
 100 ⁄ 80; Experiment MER2, N = 100 ⁄ 80 ⁄ 70, P2O5 = 70 ⁄ 20 ⁄ 0, K2O = 120 ⁄ 100 ⁄ 80; Experiment MOOR, N = 90 ⁄ 70 ⁄ 50,
P2O5 = 70 ⁄ 0 ⁄ 20, K2O = 90 ⁄ 80 ⁄ 70.

                                                                                 Treatments

                                                                 Amount of HS                                                 Amount of HS
Experiment                          2006                         (kg ha)1)                            2007                    (kg ha)1)

BOT1             1. Mineral fertilizer                                     0
                 2. Mineral fertilizer                                     8Æ3
                     + 50 L ha)1 HF liquid
                 3. Mineral fertilizer                                     6Æ4
                     + 0Æ015 HF incorporated
BOT2             1. Mineral fertilizer                                     0           1. Mineral fertilizer                      0
                 2. Mineral fertilizer                                     8Æ3         2. Mineral fertilizer                      8Æ3
                     + 50 L ha)1 HF liquid                                                  + 50 L ha)1 HF liquid
                 3. Mineral fertilizer                                     3Æ6         3. Mineral fertilizer                      3Æ6
                     + 0Æ015 HF incorporated                                                + 0Æ015 HF incorporated
HOOG             1. Mineral fertilizer                                     0           1. Mineral fertilizer                      0
                 2. Mineral fertilizer                                     5Æ7         2a. Mineral fertilizer                     0
                     + 0Æ035 HF incorporated                                                + 0Æ035 HF incorporated in 2006
                                                                                       2b. Mineral fertilizer                     5Æ7
                                                                                            + 0Æ035 HF incorporated
                                                                                        in 2006 and 2007
                 3. Slurry application                                     0           3. Slurry application                      0
                      (20 tons ha)1) + mineral fertilizer                                   + mineral fertilizer
                 4. Slurry application                                     8Æ3         4. Slurry application                      8Æ3
                      + mineral fertilizer                                                  + mineral fertilizer
                      + 50 L ha)1 HF liquid                                                 + 50 L ha)1 HF liquid
                 5. Slurry application                                     5Æ7         5a. Slurry application                     0
                      + mineral fertilizer                                                  + mineral fertilizer
                      + 0Æ035 HF incorporated                                               + 0Æ035 HF incorporated in 2006
                                                                                       5b. Slurry application                     5Æ7
                                                                                            + mineral fertilizer
                                                                                            + 0Æ035 HF incorporated
                                                                                            in 2006 and 2007
MER1             1. Mineral fertilizer                                     0
                 2. Mineral fertilizer                                     8Æ3
                     + 0Æ035 HF incorporated

                                                                       2008                                                   Amount of HS
                                                                                                                                (kg ha)1)

MER2                                       1.   Mineral   fertilizer                                                              0
                                           2.   Mineral   fertilizer   + 50 L ha)1 HF liquid                                      8Æ3
                                           3.   Mineral   fertilizer   + 0Æ015 HF incorporated                                    6Æ4
MOOR                                       1.   Mineral   fertilizer                                                              0
                                           2.   Mineral   fertilizer   + 50 L ha)1 HF liquid                                      8Æ3
                                           3.   Mineral   fertilizer   + 0Æ035 HF incorporated                                    6Æ5




Ó 2010 Blackwell Publishing Ltd. Grass and Forage Science, 65, 133–144
136 G. Verlinden et al.




Table 2 Characterization of the six experimental sites. The soil chemical parameters were determined from samples taken in the
0–6 cm layer of the soil except in Experiment BOT2 where samples were taken in the 0–30 cm soil layer.

                                                                       Experiment

Soil variable            BOT1                 BOT2               HOOG                MER1                 MER2                 MOOR

Soil type            Loamy sand*          Loamy sand            Sand               Sand               Sandy loam            Sandy loam
Location               Bottelare           Bottelare         Hoogstraten         Merelbeke             Merelbeke             Moortsele
OM content         1Æ1   Very low†      0Æ8 Very low       1Æ8   Very low      1Æ6 Very low         1Æ3   Very low       4Æ8   Fairly high
 (% C)
pH-KCl‡            5Æ4    Optimal       5Æ2    Optimal     4Æ9   Fairly low    6Æ4     High         5Æ1   Fairly low     6       Fairly high
P content          23    Fairly high    13      Low        44       High       16     Optimal       15    Fairly low     18      Fairly low
 (mg 100 g)1)§
K content          8      Fairly low    8        Low       14     Optimal      22       High        9     Fairly low     35          High
 (mg 100 g)1)§
Mg content         14      Optimal      6        Low       9        Low        11     Optimal       6         Low        55      Fairly high
 (mg 100 g)1)§
Ca content         59        Low        67       Low       88     Optimal      71     Optimal       62        Low        265       Optimal
 (mg 100 g)1)§
Na content         1Æ2       Low        1Æ3      Low       2Æ4      Low        2Æ5       Low        1Æ6       Low        3Æ8      Fairly low
 (mg 100 g)1)§

*According to the USDA-triangular diagram of soil textural classes.
†Classification of the respective soil parameter as a function of soil use, soil type and soil organic matter content used by the National
Soil Service of Belgium (Boon et al., 2009). Seven classes are distinguished: very low, low, fairly low, optimal, high, fairly high, very
high.
‡pH-KCl (1M) determined in a soil-solution ratio of 1:1 (1 M potassium chloride).
§Measured in ammonia-lactate extract.

The number of grass cuts at each location depended on                  (cutting height of approximately 5 cm). For each plot, a
the weather and grass conditions on each field and                      subsample of the freshly harvested herbage mass was
varied between two and five cuts per growing season.                    weighed, dried at 60°C for 72 h and re-weighed to
The aim was to have a herbage production between 2Æ5                   determine the dry matter (DM) content and to calculate
and 4Æ5 tons DM ha)1 for each cut. Only two cuts were                  the herbage mass of DM of each plot at each date.
taken in experiment BOT1 in 2006 (9 May and 22                         Herbage mass of DM of the different cuts were counted
June) due to the very dry period in July 2006 (Figure 1)               up to calculate total herbage production during the
and the subsequent degradation of L. multiflorum. In                    growing season.
BOT2 four cuts were taken in 2006 and 2007, respec-                      The mineral composition and nutritive value of the
tively on 22 June, 25 July, 5 September and 17 October                 herbage harvested was measured using subsamples
in 2006 and 28 March, 26 April, 5 June and 9 July in                   collected to determine herbage mass on all cutting
2007. The first cut in 2007 was performed just after                    dates. Mineral composition was measured on all cutting
winter before fertilizer and HF application as grass                   dates and nutritive value was only measured at the first
growth on this field was substantial during the mild                    cut. The subsamples were ground using a sample mill
winter period of 2006–2007. This situation provided a                  (Retsch) to pass through a 0Æ5-mm sieve. Macro-
good opportunity to measure residual effects of humic                  nutrients (P, K, Mg, Ca and Na) concentrations in
substances. In experiment HOOG four cuts were taken                    herbage were determined by ICP-AES after dry-ashing
in 2006 (8 May, 6 June, 13 July and 5 September) and                   of the samples. Nitrogen was determined using a Vario
in 2007 (20 April, 22 May, 30 July and 12 September).                  Max analyzer after combustion of the samples in an
Both in experiment MER1 and MER2 five cuts were                         oxygen-rich atmosphere at high temperatures (Dumas
performed (MER1, 2006: 11 May, 21 June, 8 August, 8                    method). Crude protein and crude fibre concentrations
September and 26 October; MER2, 2008: 13 May, 16                       and digestibility of OM were determined using near-
June, 22 July, 3 September and 28 October) while only                  infrared reflectance spectroscopy (Infrared Spectrome-
three cuts were carried out in experiment MOOR in                      ter XDS; FOSS-Benelux, Amersfoort, The Netherlands).
2008 (14 May, 24 June and 17 November).                                The NIR spectrum in this method was calibrated with
   Fresh herbage mass at each cut was determined by                    results obtained with reference methods for each
collecting and weighing a subplot of at least 10 m2                    parameter (Kjeldahl-method for crude protein content




                                                                        Ó 2010 Blackwell Publishing Ltd. Grass and Forage Science, 65, 133–144
                                                                                                         Effects of humic substances on grass pastures 137




                                                                     140           2006                                                                     25
                                                                     120
                                                                                                                                                            20




                                                                                                                                                                 Temperature (°C)
                                                                     100




                                                    Rainfall (mm)
                                                                      80                                                                                    15

                                                                      60                                                                                    10
                                                                      40
                                                                                                                                                            5
                                                                      20
                                                                       0                                                                                    0
                                                                           J         F        M      A     M       J   J    A       S       O   N   D

                                                                     140           2007                                                                     25
                                                                     120
                                                                                                                                                            20




                                                                                                                                                                    Temperature (°C)
                                                                     100
                                                     Rainfall (mm)


                                                                      80                                                                                    15

                                                                      60                                                                                    10
                                                                      40
                                                                                                                                                            5
                                                                      20
                                                                       0                                                                                    0
                                                                           J         F        M      A     M       J   J    A       S       O   N   D

                                                                     140           2008                                                                     25

                                                                     120
                                                                                                                                                            20
                                                                     100




                                                                                                                                                                   Temperature (°C)
                                                     Rainfall (mm)




                                                                      80                                                                                    15

Figure 1 Mean daily temperature (solid                                60                                                                                    10
line) and rainfall (bars) for 2006, 2007 and
2008 at location Bottelare. Each month is                             40
divided in period 1 ‘day 1 to 15’ and                                                                                                                       5
                                                                      20
period 2 ‘day 16 to end of the month’.
The data for location Bottelare are                                    0                                                                                    0
representative of the other locations.                                         J          F          M         A       M        J       J       A       S



with a nitrogen to protein conversion factor of 6Æ25,                                             iment (Version 4Æ1, SAS package; SAS Institute Inc.,
Scharrer and Kurschner method (1931) for crude fibre                                               Cary, NC, USA). A two-sample t-test was used to test if
content and De Boever et al. method (1986) for digest-                                            the average of the differences between the observations
ibility).                                                                                         (with and without HF application) was significantly
                                                                                                  different.
                                                                                                     To compare the results between experiments, a meta-
Data analysis
                                                                                                  analysis was performed in accordance with Hedges et al.
In all data analyses, a distinction was made between the                                          (1999) and Gurevitch and Hedges (2001) using the log
effects of HF liquid and HF incorporated. First, the                                              response ratio or the logarithm of the ratio of some
change in herbage production, nutrient uptake and                                                 measured quantity in an experimental and control
nutritive value due to the application of HF was                                                  group, as an index of effect. In this case, it is desirable to
calculated for each experiment as a proportion of the                                             perform the statistical analysis in the metric of the
value of the control treatment (Tables 3, 5 and 6).                                               natural logarithm of the response ratio as the distribu-
Analysis of variance was used to study the effect of                                              tion of the logged response ratio is much more normal
treatment (HF application) on DM production, nutrient                                             in small samples than that of the unlogged ratio. In this
uptake and nutritive value for each individual exper-                                             meta-analysis the treatments with mineral fertilizer




Ó 2010 Blackwell Publishing Ltd. Grass and Forage Science, 65, 133–144
                                                                         Table 3 Mean herbage mass of dry matter (DM) with standard deviation of mean in parentheses for the Control treatment of the cuts on the experimental sites. Humic substances
                                                                         in the form of Humifirst as a liquid (HF liq) or incorporated into mineral fertilizer (HF inc) were applied as treatments (see Table 1). The herbage mass of DM for these treatments are
                                                                         expressed as a ratio of the herbage mass of DM of the Control treatment.

                                                                                                                                                                               Year

                                                                                                                                      2006                                                            2007                                     2008
                                                                                                                                                                                                                                                                   138 G. Verlinden et al.




                                                                                                                                             HOOG                                                             HOOG
                                                                         Experiment
                                                                         Treatment                  BOT1            BOT2           No slurry     Slurry        MER1             BOT2              No slurry        Slurry           MER2              MOOR

                                                                         Cut 1
                                                                           Control (kg ha)1 DM)   3416 (741)        3668 (264)     2529 (208)     2166 (77)     4140 (304)       1781 (458)        2448 (704)       2436 (178)       3139 (61)        2367 (328)
                                                                           HF liq                    1Æ12              0Æ96             –           1Æ18             –              1Æ16            –      –         –    1Æ03‡        1Æ06              1Æ11
                                                                           HF inc                    1Æ03              0Æ85          1Æ42**        1Æ66**          1Æ09             1Æ03          1Æ08† 1Æ06‡      0Æ96† 0Æ95‡         1Æ11              1Æ05
                                                                         Cut 2
                                                                           Control (kg ha)1 DM)   5711 (537)        2089 (225)     2175 (369)    1977 (243)     3814 (231)        3584 (90)        1886 (93)        1825 (142)       2943 (143)       4353 (341)
                                                                           HF liq                    0Æ94              1Æ04             –           0Æ92             –              1Æ01            –      –         –    1Æ23*        1Æ13**            0Æ92
                                                                           HF inc                    0Æ89              0Æ91           0Æ87          0Æ90           1Æ03             0Æ98          1Æ07   1Æ10*     1Æ05    1Æ05         0Æ99             1Æ02
                                                                         Cut 3
                                                                           Control (kg ha)1 DM)                      2785 (58)     1701 (491)     1520 (72)       819 (36)       3944 (426)       2751 (1172)      3195 (1365)       4331 (306)       2813 (453)
                                                                           HF liq                                      1Æ07             –           0Æ85             –              0Æ91            –      –         –    1Æ04          1Æ17             0Æ88
                                                                           HF inc                                      1Æ07           0Æ86          1Æ03           1Æ18*            0Æ97          1Æ14   1Æ08      1Æ14   0Æ96          1Æ02             1Æ05
                                                                         Cut 4
                                                                           Control (kg ha)1 DM)                     1347 (228)     1707 (146)    1940 (217)       2082 (26)      1549 (430)        1578 (142)       1731 (125)       2432 (187)
                                                                           HF liq                                      0Æ95             –           1Æ00              –             0Æ69            –      –         –     0Æ96         0Æ87
                                                                           HF inc                                      0Æ87           1Æ14          1Æ00            1Æ05            0Æ75          0Æ91   0Æ77*     0Æ97    0Æ85         0Æ93
                                                                         Cut 5
                                                                           Control (kg ha)1 DM)                                                                   1401 (22)                                                          1210 (190)
                                                                           HF liq                                                                                     –                                                                 0Æ95
                                                                           HF inc                                                                                  0Æ79**                                                               0Æ80
                                                                         Total herbage production
                                                                           Control (kg ha)1 DM) 9127 (1163)         9889 (475)     8113 (796)    7603 (448)    12 256 (167)     10 857 (1220)      8663 (1524)      9187 (1390)     14 056 (447)      9534 (490)
                                                                           HF liq                    1Æ01              1Æ01             –           1Æ00             –               0Æ95           –       –        –     1Æ06‡        1Æ07             0Æ96
                                                                           HF inc                    0Æ94              0Æ93           1Æ10         1Æ17**          1Æ04              0Æ95         1Æ06† 1Æ02‡      1Æ04† 0Æ96‡          1Æ00             1Æ04

                                                                         *, P < 0Æ05; **, P < 0Æ01 are significant differences between HF and control treatment.
                                                                         †HF applied in 2006.
                                                                         ‡HF applied in 2006 and 2007.




Ó 2010 Blackwell Publishing Ltd. Grass and Forage Science, 65, 133–144
                                                                                   Effects of humic substances on grass pastures 139




without humic substances were considered as the                          experiments and the use of a random effect model
control group (Xc) and the treatments with application                   (Gurevitch and Hedges, 2001). The cumulated mean
                                                                                                                 Ã
of humic substances as the experimental group (Xe).                      effect size or mean log response ratio L is a weighted
The meta-analysis was performed separately for HF                        mean of the log response ratios of the different
liquid (class 1) and HF incorporated (class 2) and was                   experiments or
carried out using the experimental data of the total                                                                   P
                                                                                                                       k
growing season and the data of the first cut as this cut is                                                            wià à Li
the most important in grassland exploitation through its                                                     Ã ¼ i¼1
                                                                                                             L                 ;
                                                                                                                    P Ã
                                                                                                                      k
high nutritive value. Table 4 shows the selection of the                                                                wi
experiments which were included in the meta-analysis.                                                                      i¼1

As data within each class have to be independent, only                   where k is number of experiments and wià ¼ ðvi þr2 Þ, the
                                                                                                                            1
                                                                                                                              k
one experimental year per experiment could be                            reciprocal of the total variance of Li. The total variance
included in the meta-analysis. The first full growing                     of Li consists of two components of variation, the
season was chosen per experiment.                                        sampling variation for each experiment, vi, and the
   Both herbage production and nutrient uptake were                      between-experiment variation, rk. The combination of
considered as an index of effect of humic substances. In                 experimental data is rejected if the between-experiment
the following paragraphs the calculations for the index                  variation is too large compared to the average within-
‘herbage production’ will be described in detail. The                    experiment sampling-error variation. This was the case
first step in the meta-analysis was the calculation of the                for the nutrient uptake data in this meta-analysis and
log response ratio Li as the natural logarithm of the                    thus calculations were only undertaken for the herbage
mean herbage production of the experimental group                        production data. The 100(1 ) a) percentage confidence
(X e ), or herbage production with application of humic                  interval for the mean log response ratio, lk, is given by
                                                                          Ã            Ã           Ã           Ã
substances, to the mean herbage production of the                        L À za=2 SEðL Þ lk L þ za=2 SEðL Þ where
control group (X c ) or the herbage production without                              vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
application of humic substances. The 100 (1 ) a)                                    u
                                                                                    u                                                              P Ã
                                                                                                                                                    k
                                                                                    u                                                        Ã                          Ã
percentage confidence interval for the individual log
                                                    pffiffiffi                            u 1                         X 1 w à 2 wi ðði¼1 wi Þ À wi Þ
                                                                                                                   k
                                                                           SEðL Þ ¼ uð k
                                                                               Ã
response ratio parameter k is given by L À za=2 v                                   u P           Þð1 þ 4                    ð iÞ                                           Þ
                pffiffiffi                                                                t                                   dfi wi                       P k
k L þ za=2 v with za ⁄ 2 the 100(1 ) a ⁄ 2) percentage                                       wià                 i¼1                               ð wià Þ
                                                                                                 i¼1                                                    i¼1
point of the standard normal distribution and variance
vi ¼ ðSDe2Þ þ ðSDc2Þ (where n is sample size and SD is
              2         2

           
        ne Xe        
                  nc Xc                                                  and dfi is the number of degrees of freedom in the ith
standard deviation of mean). The corresponding un-                       experiment (ne + nc ) 2).
logged response ratio and its confidence interval is
obtained by taking the antilogs of the log response ratio
and of the confidence limits for the log response ratio.
                                                                         Results
The following step in the meta-analysis is the calcula-
                                                                         Effects of humic substances on herbage
tion of the cumulated mean effect size of humic
                                                                         production
substances on herbage production across the different
                                                                         For the first cut in Experiments BOT1, MER1, MER2,
Table 4 The experiments contained in the two classes of the              HOOG and MOOR, application of humic substances in
meta-analysis of the experiments, with class 1containing the             combination with mineral fertilizer resulted in a
comparisons between the Control treatment and the HF liquid              positive response in herbage mass of DM on all pastures
treatment and class 2 containing the comparisons between the
                                                                         but few of the responses were statistically significant. In
Control treatment and the HF incorporated treatment. See
                                                                         Experiment HOOG (2006) the proportional increase in
Table 1 for description of treatments. Data within each class
                                                                         herbage mass of DM at the first cut of 0Æ42 for HF
have to be independent and, therefore, only one experimental
                                                                         incorporated compared to control was highly statistical
year per location can be included in the meta-analysis. The first
full growing season was chosen per location.                             significant and in Experiment MER2, HF incorporated
                                                                         increased the herbage mass of DM significantly by 0Æ11.
Class 1 – HF liquid                   Class 2 – HF incorporated          The application of HF liquid in combination with
                                                                         mineral fertilizer did not increase herbage mass of DM
Experiment    BOT1 in 2006            Experiment    BOT1 in 2006
                                                                         significantly (Table 3).
Experiment    BOT2 in 2007            Experiment    BOT2 in 2007
                                                                            In the meta-analysis of the data on the first cut, a
Experiment    MER2 in 2008            Experiment    HOOG in 2006
                                                                         summary of the log response ratios calculated for the
Experiment    MOOR in 2008            Experiment    MER1 in 2006
                                                                         individual experiments and the mean log response
                                      Experiment    MER2 in 2008
                                                                         ratio calculated across the experiments is shown in
                                      Experiment    MOOR in 2008
                                                                         Figure 2a,c. Application of HF incorporated resulted in




Ó 2010 Blackwell Publishing Ltd. Grass and Forage Science, 65, 133–144
                                                                         Table 5 Nutrient uptake (N, P2O5 and K2O) at the first cut and total uptake during growing season with standard deviation of mean in parentheses for the Control treatment of
                                                                         the cuts on the experimental sites. Humic substances in the form of Humifirst as a liquid (HF liq) or incorporated with mineral fertilizer (HF inc) were applied as treatments
                                                                         (see Table 1). The nutrient uptakes for these treatments are expressed as a ratio of the nutrient uptakes of the Control treatment.

                                                                                                                                  Humic acids
                                                                                                                                                                                                                                                                  140 G. Verlinden et al.




                                                                                                                                  application                      N uptake                         P2O5 uptake                          K2O uptake

                                                                                                            Slurry                                  Control                              Control                              Control
                                                                         Year    Experiment      Cut        application     2006     2007   2008    (kg ha)1)         HF liq   HF inc    (kg ha)1)       HF liq   HF inc      (kg ha)1)        HF liq   HF inc

                                                                         2006        BOT1         Cut 1          No           x                      80Æ4   (13Æ0)     1Æ08    1Æ08      26Æ0   (12Æ6)    1Æ01      1Æ11       81Æ0   (4Æ9)     1Æ10    1Æ11
                                                                                                  Total                                             187Æ1   (15Æ5)     1Æ05    1Æ04      59Æ7   (28Æ7)    0Æ95      1Æ00      213Æ0   (8Æ3)     0Æ96    0Æ98
                                                                                     BOT2         Cut 1          No           x                     100Æ0   (10Æ5)     1Æ01    0Æ93      24Æ8   (3Æ7)     0Æ96      0Æ88      153Æ0   (7Æ6)     1Æ00    0Æ93
                                                                                                  Total*                                            267Æ1   (12Æ8)     1Æ01    0Æ92*     55Æ4   (7Æ5)     1Æ03      0Æ96      375Æ3   (11Æ6)    1Æ01    0Æ98
                                                                                    HOOG          Cut 1          No           x                      57Æ1   (4Æ8)        –     1Æ72***   19Æ7   (1Æ7)       –       1Æ84***    66Æ4   (10Æ2)      –     1Æ97**
                                                                                                                 Yes          x                      53Æ2   (6Æ0)      1Æ40    1Æ84**    19Æ9   (1Æ0)     1Æ30      1Æ83***    71Æ9   (5Æ6)     1Æ28    1Æ81***
                                                                                                   Total         No           x                     194Æ6   (22Æ6)       –     1Æ19*     76Æ6   (7Æ2)       –       1Æ16*     299Æ1   (37Æ7)      –     1Æ16
                                                                                                                 Yes          x                     191Æ5   (3Æ3)      1Æ01    1Æ20***   74Æ9   (1Æ6)     1Æ00      1Æ22***   292Æ9   (3Æ1)     0Æ97    1Æ22*
                                                                         2007       BOT2          Cut 1          No           x        x             26Æ1   (5Æ7)      1Æ15    1Æ05       8Æ4   (2Æ0)     1Æ25    111          33Æ8   (5Æ5)     1Æ29    1Æ14
                                                                                    HOOG          Cut 1          No           x        x             74Æ9   (15Æ0)       –     1Æ06
                                                                                                                 No           x        –                                 –     1Æ12
                                                                                                                 Yes          x        x              77Æ9 (4Æ7)       1Æ03    0Æ93
                                                                                                                 Yes          x        –                                 –     0Æ95
                                                                                                   Total         No           x        x            233Æ5 (36Æ8)         –     1Æ02
                                                                                                                 No           x        –                                 –     1Æ00
                                                                                                                 Yes          x        x            251Æ3 (37Æ3)       1Æ04    0Æ92
                                                                                                                 Yes          x        –                                 –     1Æ03
                                                                         2008       MOOR          Cut 1          No           –        –        x     53Æ7 (6Æ0)       1Æ08    1Æ01      19Æ1 (2Æ5)       1Æ10      1Æ05       82Æ3 (13Æ3)      1Æ12    1Æ10
                                                                                                 Total†          No           –        –        x     205Æ7 (–)        0Æ95    1Æ07       75Æ5 (–)        0Æ93      1Æ01       302Æ7 (–)        0Æ97    1Æ06
                                                                                    MER2          Cut 1          No           –        –        x     78Æ4 (3Æ2)       1Æ05    1Æ10      33Æ1 (1Æ4)       1Æ07      1Æ10      125Æ8 (7Æ1)       0Æ97    1Æ11
                                                                                                 Total†‡         No           –        –        x     316Æ0 (–)        1Æ09    1Æ06       120Æ0 (–)       1Æ08      1Æ03       493Æ0 (–)        1Æ03    1Æ08

                                                                         *Total of 3 cuts, no data available for cut 4.
                                                                         †Only at cut 1, analysis was performed for each grass sample replication. At cut 2 to cut 4, analysis was performed for a mixed grass sample made of the 4 replications per treatment.
                                                                         ‡Total of 4 cuts, no data available for cut 5.
                                                                         *, P < 0Æ05; **, P < 0Æ01; ***, P < 0Æ001 are significant differences between HF and control treatment.




Ó 2010 Blackwell Publishing Ltd. Grass and Forage Science, 65, 133–144
                                                                                                                                                                                                                                                                                                                                                 Effects of humic substances on grass pastures 141




                                                                                                                                                                                                                                                                                                                                          a mean log response ratio of 0Æ13 or an unlogged mean




                                                                                                                                                                                                                             HF inc
MOOR and MER2. Values in parentheses are standard deviation of mean. Humic substances in the form of Humifirst as a liquid (HF liq) or incorporated with mineral fertilizer (HF inc)
Table 6 Crude protein (CP) and crude fibre (CF) concentrations and digestibility of organic matter (OM) of herbage from the first cut in Experiments BOT1, BOT2, HOOG,




                                                                                                                                                                                                                                          1Æ00
                                                                                                                                                                                                                                          1Æ03
                                                                                                                                                                                                                                          1Æ03
                                                                                                                                                                                                                                          0Æ99
                                                                                                                                                                                                                                          0Æ99
                                                                                                                                                                                                                                          0Æ99
                                                                                                                                                                                                                                          0Æ99
                                                                                                                                                                                                                                            –
                                                                                                                                                                                                                                                                                                                                          response ratio of 1Æ14 with the unlogged 95 % confi-
                                                                                                                                                                                                                                                                                                                                          dence interval of 1Æ02–1Æ27 for the first cut. In other

                                                                                                                                                                                                 Digestibility of OM
                                                                                                                                                                                                                                                                                                                                          words, the application of HF incorporated significantly



                                                                                                                                                                                                                             HF liq

                                                                                                                                                                                                                                          1Æ00
                                                                                                                                                                                                                                          1Æ00



                                                                                                                                                                                                                                          1Æ00

                                                                                                                                                                                                                                          0Æ99
                                                                                                                                                                                                                                          1Æ00
                                                                                                                                                                                                                                                                                                                                          (a = 0Æ05) increased grass production by 0Æ14 at the first




                                                                                                                                                                                                                                            –
                                                                                                                                                                                                                                            –

                                                                                                                                                                                                                                            –
were applied as treatments (see Table 1). The nutritive variables for these treatments are expressed as a ratio of the nutritive variables of the Control treatment.



                                                                                                                                                                                                                                                                                                                                          cut across the different field experiments. The applica-
                                                                                                                                                                                                                                                                                                                                          tion of HF liquid resulted in a mean log response ratio of



                                                                                                                                                                                                                                          (0Æ0 24)
                                                                                                                                                                                                                                          (0Æ004)

                                                                                                                                                                                                                                          (0Æ018)
                                                                                                                                                                                                                                          (0Æ018)
                                                                                                                                                                                                                                          (0Æ025)
                                                                                                                                                                                                                                          (0Æ025)
                                                                                                                                                                                                                                          (0Æ007)
                                                                                                                                                                                                                                          (0Æ009)
                                                                                                                                                                                                                                                                                                                                          0Æ07 or an unlogged mean response ratio of 1Æ08. The
                                                                                                                                                                                                                                                                                                                                          increase in grass production of 0Æ08 compared to the
                                                                                                                                                                                                                             Control


                                                                                                                                                                                                                                                                                                                                          control was not significant. Higher herbage masses of
                                                                                                                                                                                                                                          0Æ834
                                                                                                                                                                                                                                          0Æ907
                                                                                                                                                                                                                                          0Æ892
                                                                                                                                                                                                                                          0Æ892
                                                                                                                                                                                                                                          0Æ907
                                                                                                                                                                                                                                          0Æ907
                                                                                                                                                                                                                                          0Æ827
                                                                                                                                                                                                                                          0Æ780
                                                                                                                                                                                                                                                                                                                                          DM at the first cut after application of humic substances
                                                                                                                                                                                                                                                                                                                                          in combination with mineral fertilization were often
                                                                                                                                                                                                                             HF inc




                                                                                                                                                                                                                                                                                                                                          followed by lower herbage masses of DM in the

                                                                                                                                                                                                                                          1Æ04*
                                                                                                                                                                                                                                          0Æ99
                                                                                                                                                                                                                                          1Æ02
                                                                                                                                                                                                                                          1Æ03
                                                                                                                                                                                                                                          1Æ00
                                                                                                                                                                                                                                          1Æ03

                                                                                                                                                                                                                                          0Æ99
                                                                                                                                                                                                                                            –




                                                                                                                                                                                                                                                                                                                                          following cuts (Table 3).
                                                                                                                                                                                                                                                                                                                                             The effect of humic substances in combination with
                                                                                                                                                                                                 CF concentration




                                                                                                                                                                                                                                                                                                                                          mineral fertilizer on total herbage production of DM at
                                                                                                                                                                                                                             HF liq

                                                                                                                                                                                                                                          0Æ97
                                                                                                                                                                                                                                          1Æ01



                                                                                                                                                                                                                                          1Æ01

                                                                                                                                                                                                                                          1Æ03
                                                                                                                                                                                                                                          0Æ98
                                                                                                                                                                                                                                                                                                                                          the end of the growing season was variable. Mean total
                                                                                                                                                                                                                                            –
                                                                                                                                                                                                                                            –

                                                                                                                                                                                                                                            –




                                                                                                                                                                                                                                                                                                                                          herbage production of DM on plots with humic
                                                                                                                                                                                                                                                                                                                                          substances and mineral fertilizer varied from )0Æ07 to
                                                                                                                                                                                                                       (g kg)1 DM)




                                                                                                                                                                                                                                                                                                                                          +0Æ10 compared to the means on the control plots
                                                                                                                                                                                                                                          (6Æ0)
                                                                                                                                                                                                                                          (2Æ0)
                                                                                                                                                                                                                                          (8Æ0)
                                                                                                                                                                                                                                          (8Æ0)
                                                                                                                                                                                                                                          (5Æ0)
                                                                                                                                                                                                                                          (5Æ0)
                                                                                                                                                                                                                                          (2Æ0)
                                                                                                                                                                                                                                          (5Æ0)
                                                                                                                                                                                                                       Control




                                                                                                                                                                                                                                                                                                                                          (Table 3). In the meta-analysis the mean log response
                                                                                                                                                                                                                                          236
                                                                                                                                                                                                                                          222
                                                                                                                                                                                                                                          176
                                                                                                                                                                                                                                          176
                                                                                                                                                                                                                                          175
                                                                                                                                                                                                                                          175
                                                                                                                                                                                                                                          230
                                                                                                                                                                                                                                          283




                                                                                                                                                                                                                                                                                                                                          ratio across the experiments was estimated to be zero
                                                                                                                                                                                                                                                                                                                                          for HF liquid and 0Æ03 for HF incorporated. This means
                                                                                                                                                                                                                                                                                                                                          that in general the application of humic substances had
                                                                                                                                                                                                                             HF inc




                                                                                                                                                                                                                                                                                                                                          no effect on total herbage production of DM during the
                                                                                                                                                                                                                                          1Æ01
                                                                                                                                                                                                                                          0Æ99
                                                                                                                                                                                                                                          1Æ02
                                                                                                                                                                                                                                          0Æ97
                                                                                                                                                                                                                                          0Æ99
                                                                                                                                                                                                                                          0Æ97
                                                                                                                                                                                                                                          1Æ00
                                                                                                                                                                                                                                            –




                                                                                                                                                                                                                                                                                                                                          growing season.
                                                                                                                                                                                                 CP concentration




                                                                                                                                                                                                                                                                                                                                             In Experiment HOOG, humic substances were
                                                                                                                                                                                                                             HF liq




                                                                                                                                                                                                                                                                                                                                          applied in combination with mineral fertilizers and
                                                                                                                                                                                                                                          0Æ94
                                                                                                                                                                                                                                          1Æ03



                                                                                                                                                                                                                                                        0Æ99

                                                                                                                                                                                                                                                        0Æ98
                                                                                                                                                                                                                                                        0Æ98
                                                                                                                                                                                                                                            –



                                                                                                                                                                                                                                                          –




                                                                                                                                                                                                                                                                                                                                          slurry as well. The general trend observed by applica-
                                                                                                                                                                                                                                                                                                                                          tion of humic substances, as described above, persisted.
                                                                                                                                                                                                                                                                                                                                          Incorporation of HF into the mineral fertilizer resulted
                                                                                                                                                                                                                       (g kg)1 DM)

                                                                                                                                                                                                                                           165 (9Æ0)
                                                                                                                                                                                                                                          188 (11Æ0)
                                                                                                                                                                                                                                          195 (21Æ0)
                                                                                                                                                                                                                                          195 (21Æ0)
                                                                                                                                                                                                                                          200 (11Æ0)
                                                                                                                                                                                                                                          200 (11Æ0)
                                                                                                                                                                                                                                          156 (10Æ0)
                                                                                                                                                                                                                                           163 (6Æ0)




                                                                                                                                                                                                                                                                                                                                          in a significant increase of 0Æ66 in herbage mass of DM
                                                                                                                                                                                                                       Control




                                                                                                                                                                                                                                                                                                                                          at the first cut. Total herbage production of DM
                                                                                                                                                                                                                                                                 *, P < 0Æ05 is significant difference between HF and control treatment.




                                                                                                                                                                                                                                                                                                                                          increased significantly by 0Æ17 compared to the control
                                                                                                                                                                                                                                                                                                                                          treatment. When HF liquid was mixed into the slurry,
                                                                                                                                                                                                                                                                                                                                          herbage mass of DM at the first cut increased by 0Æ18
                                                                                                                                                                                                                             2008
                                                                                                                                                                                      Humic acids appli-




                                                                                                                                                                                                                                                          x
                                                                                                                                                                                                                                                          x




                                                                                                                                                                                                                                                                                                                                          but total herbage production of DM at the end of
                                                                                                                                                                                                                                                                                                                                          the growing season was comparable to the control
                                                                                                                                                                                           cation



                                                                                                                                                                                                                             2007




                                                                                                                                                                                                                                                                                                                                          treatment.
                                                                                                                                                                                                                                                 x

                                                                                                                                                                                                                                                 x
                                                                                                                                                                                                                                                 –

                                                                                                                                                                                                                                                 –
                                                                                                                                                                                                                                                 –
                                                                                                                                                                                                                                                 –
                                                                                                                                                                                                                             2006




                                                                                                                                                                                                                                                                                                                                          Effect of humic substances on nutrient uptake
                                                                                                                                                                                                                                          x
                                                                                                                                                                                                                                          x
                                                                                                                                                                                                                                          x
                                                                                                                                                                                                                                          x
                                                                                                                                                                                                                                          x
                                                                                                                                                                                                                                          x
                                                                                                                                                                                                                                          –
                                                                                                                                                                                                                                          –




                                                                                                                                                                                                                                                                                                                                          by herbage
                                                                                                                                                                                                                       application




                                                                                                                                                                                                                                                                                                                                          The uptake of N, P2O5 and K2O by the herbage in
                                                                                                                                                                                                                                                                                                                                          control plots (in kg ha)1) and the effect of the applica-
                                                                                                                                                                                                                                          Yes
                                                                                                                                                                                                                                          Yes
                                                                                                                                                                                                                                          No
                                                                                                                                                                                                                                          No
                                                                                                                                                                                                                                          No
                                                                                                                                                                                                                                          No



                                                                                                                                                                                                                                          No
                                                                                                                                                                                                                                          No
                                                                                                                                                                                                                       Slurry




                                                                                                                                                                                                                                                                                                                                          tion of humic substances on the proportional change in
                                                                                                                                                                                                                                                                                                                                          uptake compared to the control is presented in Table 5.
                                                                                                                                                                                                                                                                                                                                          At the first cut, N uptake always increased after
                                                                                                                                                                                                                             Experiment




                                                                                                                                                                                                                                                                                                                                          application of humic substances in combination with
                                                                                                                                                                                                                                                          MOOR
                                                                                                                                                                                                                                          HOOG




                                                                                                                                                                                                                                                          MER2




                                                                                                                                                                                                                                                                                                                                          mineral fertilizer. However, this effect could only be
                                                                                                                                                                                                                                          BOT1
                                                                                                                                                                                                                                          BOT2




                                                                                                                                                                                                                                                                                                                                          statistically demonstrated in Experiment HOOG in 2006
                                                                                                                                                                                                                                                                                                                                          for the HF incorporated treatment. Total N uptake
                                                                                                                                                                                                                                                                                                                                          during the growing season generally increased with
                                                                                                                                                                                                                                                                                                                                          application of humic substances but, again, the
                                                                                                                                                                                                                             Year

                                                                                                                                                                                                                                          2006

                                                                                                                                                                                                                                                 2007




                                                                                                                                                                                                                                                          2008




                                                                                                                                                                                                                                                                                                                                          increased nitrogen uptake was only statistically




Ó 2010 Blackwell Publishing Ltd. Grass and Forage Science, 65, 133–144
142 G. Verlinden et al.




             (a)                         0·6
                                                                                        (b)                     0·6

                                         0·4                                                                    0·4




                                                                                          Log response ratio
              Log response ratio
                                         0·2                                                                    0·2

                                         0·0                                                                    0·0

                                   –0·2                                                                        –0·2

                                   –0·4                                                                        –0·4
                                               HOO BOT1 BOT2 MER1 MER2 MOO   Mean log                                 HOO BOT1 BOT2 MER1 MER2 MOO           Mean log
                                                                             response                                                                       response
                                                                               ratio                                                                          ratio
             (c)                         0·6                                            (d)                     0·6


                                         0·4                                                                   0·4
                    Log response ratio




                                                                                         Log response ratio
                                         0·2                                                                   0·2

                                         0·0                                                                   0·0

                                   –0·2                                                                   –0·2

                                   –0·4                                                                   –0·4
                                               BOT1   BOT2   MER2   MOO      Mean log                                 BOT1   BOT2    MER2    MOO           Mean log
                                                                             response                                                                      response
                                                                               ratio                                                                         ratio

Figure 2 Log response ratios of (a) HF incorporated and (c) HF liquid for the herbage mass of dry matter (DM) at the first cut
and (b) HF incorporated and (d) HF liquid for the total herbage production of DM over the growing season as calculated in the
meta-analysis. In each graph, log response ratios of the individual experiments are shown on the left side and the mean response ratio
across the different experiments is shown on the right side. Error bars indicate 95% confidence intervals.



significant in Experiment HOOG in 2006 for the HF                                                  283 g kg)1 DM at the different cutting dates. The
incorporated treatment.                                                                           digestibility of DM of the herbage ranged from 0Æ78 to
   The uptake of K2O and P2O5 was similar at the                                                  0Æ90. The effect of humic substances on these values at
different locations. The application of humic substances                                          the first cut was very small.
resulted in a positive effect on K2O and P2O5 uptake at
the first cut and total K2O and P2O5 uptake in
Experiment HOOG for the HF incorporated treatment.
                                                                                                  Discussion
The effect of humic substances on MgO, CaO and Na2O                                               The results in the field experiments showed that
uptake was similar to that for the other macro-nutrients                                          application of HF incorporated increased grass produc-
in that uptake MgO, CaO and Na2O at the first cut and                                              tion at the first cut without affecting the nutritive value
the total uptake of MgO, CaO and Na2O was higher in                                               of the herbage although there was no effect on annual
Experiment HOOG for the HF incorporated treatment                                                 herbage production. The shift in total herbage produc-
(data not shown). Since the between-experiment var-                                               tion towards the first cut during the growing season
iation for nutrient uptake data was too large compared                                            after application of humic substances, especially HF
to the average within-experiment variation in sampling                                            incorporated, could be of significance for grassland
error, the combination of experimental data was                                                   management in north-west Europe since the first cut is
rejected by the meta-analysis.                                                                    important as a source of nutrients for the winter as
                                                                                                  silage.
                                                                                                     The higher grass production at the first cut was
The effect of humic substances on nutritive
                                                                                                  generally followed by lower production in the next
value of herbage
                                                                                                  cuts. This decline in herbage production after a high-
Table 6 shows the results of crude protein and crude                                              yielding first cut is a well known phenomenon
fibre concentrations and digestibility of the spring grass                                         (Behaeghe, 1979). It is argued that the herbage uses a
in the different experiments. If analysed, the crude                                              high proportion of its nutrient reserves in the roots for
protein and the crude fibre content of the spring grass                                            the production of spring grass so that regrowth is
varied from 156 to 195 g kg)1 DM and from 176 to                                                  probably hampered by a lack of reserves.




                                                                                                               Ó 2010 Blackwell Publishing Ltd. Grass and Forage Science, 65, 133–144
                                                                                  Effects of humic substances on grass pastures 143




   Significant increases in N uptake by herbage at the                    (IWT) for their financial support, the Soil Service of
first cut associated with the addition of humic sub-                      Belgium for their help with the data collection and the
stances could only be demonstrated in one of the                         team of the experimental farm Bottelare for their
experiments for the HF incorporated treatment and it                     technical assistance.
was not possible to undertake the meta-data analysis
because of the high variability. Increases in yield and
                                                                         References
nutrient uptake following application of humic sub-
stances in combination with mineral fertilizer have                      AR A N C O N N., ED W A R D S C., LE E S. and BY R N E R. (2006)
been reported for barley (Burns et al., 1986; Ayuso                        Effects of humic acids from vermicomposts on plant
et al., 1996), olive trees (Fernandez-Escobar et al., 1996)                growth. European Journal of Soil Biology, 42, S65–S69.
and maize (Pinton et al., 1999). Cooper et al. (1998) and                AT I Y E H R., LE E S., ED W A R D S C., AR A N C O N N. and
                                                                           ME T Z G E R J. (2002) The influence of humic acids derived
Sharif et al. (2002) found that the effect of humic
                                                                           from earthworm-processed organic wastes on plant
substances increased if they were mixed into the                           growth. Bioresource Technology, 84, 7–14.
nutrient solution. This supports the larger effect of HF                 AY U S O M., HE R N A N D E Z T., GA R C I A C. and PA S C U A L J.
incorporated compared to HF liquid since nutrients and                     (1996) Stimulation of barley growth and nutrient
humic substances are in the same granule in the ‘HF                        absorption by humic substances originating from various
incorporated’ product.                                                     organic materials. Biosource Technology, 57, 251–257.
   The reasons for the increase in herbage mass of DM                    BE H A E G H E T.J. (1979) The seasonal variation in the growth of
and N uptake at the first cut with the HF incorporated                      grass (in Dutch). Ghent, Belgium: Ghent University Press.
treatment were not studied. In studies with other crop                   BO O N W., VE R EL S T P., DE C K E R S S., VO G E L S N., BR I E S J.
species, an increase in N uptake may be explained by                       and VA N D E N D R I E S S C H E H. (2009) The chemical soil
                                                                           fertility of the Belgian cropland and pasture areas (2004-2007)
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nitrate uptake via an interaction with plasma mem-
                                                                         BU R N S R., DE L L ’AG N O L A G., MI E L E S., NA R D I S., SA V O I N I
brane H+-ATPase (Maggioni et al., 1987; Pinton et al.,                     G., SC H N I T Z E R M., SE Q U I P., VA U G H A N D. and VI S S E R S.
1999; Canellas et al., 2002; Quagiotti et al., 2004).                      (1986) Humic substances, effect on soil and plants. Milan,
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stimulation of the nitrate uptake capacity and the plasma                  and FA C A N H A A. (2002) Humic acids isolated from
membrane H+-ATPase activity. Quagiotti et al. (2004)                       earthworm compost enhance root elongation, lateral root
showed that humic substances exert direct effects on                       emergence and plasma membrane H+ATPase activity in
gene transcription in roots (genes encoding maize                          maize roots. Plant Physiology, 130, 1951–1957.
                                                                         CA N E L L A S L.P., TE I X E I R A JU N I O R L.R.L., DO B B S L.B., SI L V A
H+-ATPase and thus promoting nitrate influx in roots)
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                                                                           (2008) Humic acids cross-interactions with root and
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leaves). These effects are considered as an important                    CA R L E T T I P., MA S I A., SP O L A O R E B., PO L V E R I N O DE
action of humic substances on plant nutrient acquisition.                  LA U R E T O P., DE ZO R Z I M., TU R E T T A L., FE R R E T T I M.
Another important factor in the uptake of nutrients is the                 and NA R D I S. (2008) Protein expression changes in
root system of plants. Cooper et al. (1998) found that                     maize roots in response to humic substances. Journal of
incorporation of humic substances in the soil stimulated                   Chemical Ecology, 34, 804–818.
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                                                                           selected readings, pp. 161–186. Madison, WI, USA: American
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                                                                           Society of Agronomy and Soil Society of America.
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means that the plants are able to produce more biomass                     creeping bentgrass. Crop Science, 38, 1639–1644.
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We would like to thank the Institute for the Promotion                     Sustainable Development, 25, 183–191.
of Innovation by Science and Technology in Flanders




Ó 2010 Blackwell Publishing Ltd. Grass and Forage Science, 65, 133–144
144 G. Verlinden et al.




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