You are on page 1of 7

Scientia Horticulturae 111 (2007) 319325

Effect of organic and inorganic fertilizers applied during successive

crop seasons on growth and nitrate accumulation in lettuce
Georgios C. Pavlou a,*, Constantinos D. Ehaliotis b, Victor A. Kavvadias a
National Agricultural Research Foundation (N.AG.RE.F.), Olive and Horticultural Crops Institute, Lakonikis 87, Kalamata 24100, Greece
Agricultural University of Athens, Soil Science and Agricultural Chemistry Lab., Iera Odos 75, Athens 11855, Greece
Received 23 December 2005; received in revised form 10 August 2006; accepted 6 November 2006

A romaine-type lettuce (Lactuca sativa L.) cv. Corsica was cultivated during three successive crop seasons (late-spring, late-autumn and late-
winter) in the same soil of an experimental greenhouse in S.W. Peloponnese, Greece. Seven long-term fertilization treatments were tested for their
effect on plant growth and nitrate concentration in the external lettuce leaves. Treatments included: three different doses of organic fertilization
(composted sheep manure) applied at the start of each crop season, three different doses of inorganic N fertilization applied via fertigation during
each crop season, and a control treatment in which no fertilizer was applied. A drip irrigation system was used to water all plants. The highest
nitrate levels were observed in the medium and maximum inorganic fertilization treatments (572664 mg kg1) in all crop seasons. They were
significantly higher compared to the respective organic fertilization treatments (253435 mg kg1) and all other fertilization treatments (148
435 mg kg1). Crop season affected lettuce growth more than nitrate accumulation in the lettuce leaves: lettuce biomass production was the
smallest and most uniform in the late-autumn season and did not respond to the fertilization treatments tested (ranging from 409 to 439 g plant1),
while in the late-spring season biomass production was the highest and most variable (561841 g plant1), it correlated with nitrate concentration
in the leaves and in the medium and maximum inorganic fertilizer doses it significantly exceeded production from all other fertilization treatments
(827841 g plant1). Following the three crop seasons the residual availability of N, P and K was clearly enhanced in the soil receiving the organic
compared to the inorganic fertilization. Nitrate concentration in lettuce leaves was far below the upper limits set by the European Commission in all
fertilization treatments throughout the three crop seasons, a result attributed mainly to the sufficient level of light intensity and duration throughout
the year in Southern Greece.
# 2006 Elsevier B.V. All rights reserved.

Keywords: Lettuce; Lactuca sativa L.; Fertilization; Organic fertilizers; Manure; Nitrates

1. Introduction control of the gut flora (Lundberg et al., 2004; Addiscott and
Benjamin, 2004).
In the temperate zone regions lettuce is cultivated Accumulation of nitrates results from an imbalance between
throughout the year, but production and quality characteristics the uptake and translocation of nitrates by the xylem, and the
benefit from cool weather and high light intensity. The reduction of these nitrates to ammonia which is subsequently
concentration of nitrates in the edible leaves of lettuce are rapidly incorporated into amino acids (Maynard et al., 1976).
regulated by the European Commission Regulation No 563/ However, the internal nitrate concentration in the plant seems to
2002 which has set upper limits in order to protect consumers be controlled by a self-regulatory mechanism exerted either by
from potential toxicological risks following the consumption of negative feedback control on the net nitrate uptake rate
nitrate-rich foods (Maynard et al., 1976; Walker, 1990; (Cardenas-Navarro et al., 1998) or by passive control on nitrate
Bruning-Fann and Kaneene, 1993). Recent studies however, efflux (Scaife, 1989). The viewpoint that non-structural
report on beneficial effects of nitrates mainly related to the carbohydrates and nitrates have a complementary role in
maintaining cell tugor (Blom-Zandstra and Lampe, 1985; Behr
and Wiebe, 1988) offers a credible model for the plant nitrate
* Corresponding author. Tel.: +30 27210 29812; fax: +30 27210 27133. regulation mechanism suggesting the accumulation of nitrates
E-mail address: (G.C. Pavlou). in the vacuole as an alternative osmoticum under low radiation
0304-4238/$ see front matter # 2006 Elsevier B.V. All rights reserved.
320 G.C. Pavlou et al. / Scientia Horticulturae 111 (2007) 319325

conditions (Blom-Zandstra, 1989; Buwalda and Warmenhoven, autumn (second, 26 October5 December 2000), and late-
1999). winter (third, 6 February28 March 2001), at the N.AG.RE.F.-
Accumulation of nitrates in lettuce has been shown to be Olive and Horticultural Crops Institute of Kalamata, S.W.
affected by the soil texture and the source of fertilizer-N (Scaife Peloponnese, Greece (378030 N, 228070 E, altitude 9 m). At the
et al., 1986; Gianquinto et al., 1992; Gunes et al., 1995), the start of each crop season, a romaine lettuce crop cv. Corsica
NH4-N-to-NO3-N fertilizer-N ratio (McCall and Willumsen, (Royal Sluis) was established in a polyethylene covered
1998; Demsar and Osvald, 2003), the timing of fertilizer-N greenhouse (8085% transparency to visible irradiation), with
release (Tesi and Lenzi, 1998), the light intensity and duration 60% side wall cover. The greenhouse soil was a sandy clay
(Behr and Wiebe, 1992; Chadjaa et al., 1999; Drews et al., loam (SCL) with chemical characteristics presented in
1995; Gaudreau et al., 1995), crop season (Gianquinto et al., Table 1.
1992), and lettuce type and cultivar (Blom-Zandstra and Seven long-term fertilization treatments were compared
Eenink, 1986; Siomos, 2000; Escobar-Gutierrez et al., 2002). In including: (i) three different organic fertilization treatments
most types of lettuce, including the romaine type, the highest (O-min, O-mid, O-max) consisting of composted sheep
concentration of nitrates is normally observed in the external manure (Table 1) applied at the start of each cultivation
leaves (Corre and Breimer, 1979; Santamaria et al., 1999; Abu- period, at doses of 13 l plant1 respectively for the first
Rayyan et al., 2004). and second crop seasons, and reduced by 33% for the third
In countries of the Mediterranean basin, sheep manures are crop season (0.67, 1.34, and 2.01 l plant1, respectively), (ii)
traditionally used as an organic fertilization source. Their three different treatments of inorganic-N fertilization (I-
recycling into soils low in organic matter, which are widespread min, I-mid, I-max) applied via drip irrigation during each
in these regions, could benefit soil structure and long term crop season at doses corresponding to 87, 174 and
fertility, and is also an alternative to inorganic fertilizers in the 261 mg N plant1 fertigation1, respectively (Table 2),
growing organic vegetable production business. and (iii) a control treatment in which no fertilizer was
In this work we compare short and long term effects of applied (C). The nutrient elements provided in total during
organic versus inorganic sources of N on the growth and the each crop season by the organic and inorganic fertilization
accumulation of nitrates in the external leaves of a romaine treatments are shown in Table 3. In all three inorganic
lettuce. Composted sheep manure is used for organic fertilization treatments (I-min, I-mid, I-max) the soluble
fertilization whereas standard fertigation applied by farmers commercial fertilizers 153015 and 202020 plus micro-
in the area is used for the comparison to inorganic fertilization. nutrients were applied, each at a rate of 0.25 g plant1
Fertilization from both sources is applied at a minimum, a fertigation1. Extra N fertilization was applied for the I-mid
medium and a maximum dose during three successive crop and I-max treatments with the inorganic fertilizer 34.500
seasons in the same soil. Effects of crop season and long term (NH4NO3) at rates of 0.25 and 0.5 g plant1 fertigation1,
cumulative fertilization on plant and soil are studied. respectively.
Each experimental plot consisted of 54 lettuce plants
2. Materials and methods placed in three rows (30 cm apart) of 18 plants (25 cm
apart). The plants of the two outer rows and the three plants
2.1. Location and design of experiments in each end of the middle row were kept as guard plants.
There were five replicate plots per treatment and the
The experiments were carried out during three successive experiment was set up as a randomized complete block
crop seasons: late spring (first, 6 April17 May 2000), late design.

Table 1
Chemical characteristics of soil and sheep manure used in the experiments
E.C. pH CaCO3 (%) O.M. (%) C.E.C. NKjeldahl POlsen Kexch
(mS cm1) (cmol(+) kg1) (g kg1) (mg kg1) (mg kg1)
Soil 2.6 7.4 13.4 1.4 8.6 0.15 37.0 118.2
Manurea 21.7 8.0 8.4 72.0 36.0 1.64 1.4 (% d.w.) 1.8 (% d.w.)
Manure dry weight = 201 g l1.

Table 2
Nutrient elements provided per fertigation by the inorganic fertilization treatments applied to lettuce
Fertilization Nutrient elements provided in each crop season
N (g plant1 P (g plant1 K (g plant1 Mg (mg plant1 Fe (mg plant1 Mn (mg plant1 Zn (mg plant1 Cu
fertigation1) fertigation1) fertigation1) fertigation1) fertigation1) fertigation1) fertigation1)
I-max 0.261 0.027 0.036 0 0.195 0.081 0.0163 0.0163
I-mid 0.174 0.027 0.036 0 0.195 0.081 0.0163 0.0163
I-min 0.087 0.027 0.036 0 0.195 0.081 0.0163 0.0163
G.C. Pavlou et al. / Scientia Horticulturae 111 (2007) 319325 321

Table 3
Nutrient elements provided per crop season by the fertilization treatments applied to lettuce
Crop season Fertilization treatment Macro-elements (g plant1) Micro-elements (mg plant1)
N P K Mg Fe Mn Zn Cu
First and second O-max 9.840 0.258 4.692 0.102 13.53 13.11 6.69 4.71
O-mid 6.560 0.172 3.128 0.068 9.02 8.74 4.46 3.14
O-min 3.280 0.086 1.564 0.034 4.51 4.37 2.23 1.57
Third O-max 6.593 0.173 3.144 0.068 9.07 8.78 4.48 3.16
O-mid 4.395 0.115 2.096 0.046 6.04 5.86 2.99 2.10
O-min 2.198 0.058 1.048 0.023 3.02 2.93 1.49 1.05
First I-max 3.380 0.356 0.473 0 2.54 1.06 0.21 0.20
I-mid 2.260 0.356 0.473 0 2.54 1.06 0.21 0.20
I-min 1.140 0.356 0.473 0 2.54 1.06 0.21 0.20
Second I-max 2.080 0.218 0.291 0 1.56 0.63 0.13 0.13
I-mid 1.390 0.218 0.291 0 1.56 0.63 0.13 0.13
I-min 0.700 0.218 0.291 0 1.56 0.63 0.13 0.13
Third I-max 2.860 0.301 0.398 0 2.15 0.90 0.18 0.18
I-mid 1.910 0.301 0.398 0 2.15 0.90 0.18 0.18
I-min 0.960 0.301 0.398 0 2.15 0.90 0.18 0.18

2.2. Cultivation and irrigation practices Until around 10 days after transplanting, plants of all
treatments were irrigated with water only (0.2
The outline of practices and climatic conditions are 0.4 l plant1 irrigation1). Afterwards, when plants were well
presented in Table 4. Lettuce seedlings were pre-grown in established in the soil, fertigation was applied with water
190 ml plastic pots filled with the organic substrate Terra quantities of 0.50.6 l plant1 fertigation1. The fertilizer
Nature (Holland). They were transplanted in the greenhouse at concentration in the irrigation water ranged between 0.5%
the five to seven true-leaf stage. Organic fertilizer (sheep and 1%.
manure) had already been incorporated into the soil of the When plants had reached approximately their highest
respective plots down to a depth of about 20 cm, 23 days commercial size they were harvested by cutting the stems at
before transplantation using a milling machine. the soil level. Twelve plants from each plot were harvested soon
Inorganic fertilizers were dissolved in 30 l water tanks and after sunrise on a typical for each season day.
injected by an electric pump at a pressure slightly greater than
1.5 atm separately in each of the three 30 m long primary pipes, 2.3. Analytical methods
from where the plants of the three inorganic fertilization
treatments were fertigated by 5 m long secondary dripper- Just after harvest the fresh weight (f.w.) of the plants was
pipes. Plants of control and organic fertilization treatments determined; then four external leaves from each plant were
were irrigated through separate pipes with water only. Every removed and their f.w. was also determined. The leaves were
plant was irrigated by a labyrinth type dripper (Netafim) then washed off with tap water and blotted dry on absorbing
supplying about 3 l h1 at a pressure of 1 atm. paper. Their dry weight (d.w.) was subsequently determined
The irrigation water used had the following characteristics: following drying in a drying chamber at 70 8C for 6070 h. Dry
electrical conductivity 686 mS/cm (at 25 8C), pH 7.29, S.A.R. leaves were ground to 40-mesh size by a centrifugal mill
2.7, HCO3 409 mg l1, Ca2+ 52.1 mg l1 and Mg2+ (TecatorSweden). Homogenized leaf powder was reinserted
12.2 mg l1. to the drying chamber at 70 8C for about 24 h, and 0.225 g were
Table 4
Cultivation and irrigation practices and climatic conditions during the three successive crop seasons: late-spring (first), late-autumn (second) and late-winter (third)
First crop season Second crop season Third crop season
Lettuce transplanting 6 April 2000 26 October 2000 6 February 2001
Date of harvesting 17 May 2000 5 December 2000 28 March 2001
Crop duration (days) 41 39 50
Irrigation water (l plant1) total 12.2 6.9 7.7
Total number of irrigations 26 12 17
Number of fertigations 13 8 11
Sunshine duration (h) total 354 229 329
Average photoperiod (h) 13.53 10.27 11.47
Average air min temperature (8C) 13.3 11.8 8.8
Average air max temperature (8C) 32.7 29.5 27.7
322 G.C. Pavlou et al. / Scientia Horticulturae 111 (2007) 319325

Table 5
Yield of lettuce (fresh weight) grown under different organic (O) or inorganic (I) fertilization schemes during three successive crop seasons: late-spring (first), late-
autumn (second) and late-winter (third)
Fertilization treatment First crop season (g plant1) Second crop season (g plant1) Third crop season (g plant1)
I-max 841 a 439 a 625 a
I-mid 827 a 445 a 624 a
I-min 689 b 441 a 534 ab
O-max 701 b 443 a 569 ab
O-mid 670 bc 432 a 536 ab
O-min 561 cd 409 a 504 b
Control 541 d 335 b 262 c
Means in the same column (crop season) followed by different letters denote significant differences according to Duncans multiple range test ( p = 0.05).

used for colorimetric determination of nitrate-N (NO3-N) at concentration in the external leaves of lettuce. Treatments were
410 nm by a Hitachi U-2001 spectrophotometer following the thereafter compared by Duncans multiple range test
nitration by salycilic acid method described by Cataldo et al. ( p = 0.05). Simple linear regression was also applied using
(1975). Five replicate subsamples were measured to obtain each the SPSS v.10 for Windows OS.
plot-sample value. Randomly selected samples were also cross-
checked for NO3-N content with the copperized-Cd reduction 3. Results
method (Keeney and Nelson, 1982) to confirm methodological
accuracy. 3.1. Lettuce plant growth
Length and width of the biggest leaf on each of the 12 plants
under harvest were measured the day before harvest. During the first crop season (late-spring) characterized by
Air temperature at plants height was recorded by a the greatest temperatures, photoperiod, and sunshine duration
Lambrecht thermograph, while sunshine duration was obtained (Table 4) high yields were observed in all treatments (Table 5).
from the Kalamata Meteorological Station of the National Lettuce biomass yield in the maximum and medium inorganic
Meteorological Service. Photoperiod was calculated using the fertilization treatments (I-max, I-mid) was significantly greater
approximate local sunrise and sunset hours. than in all other treatments (Table 5). The respective organic
Soil available P was estimated by extraction with NaHCO3 fertilization treatments (O-max, O-mid) did not differ
(Olsen-P) and measured colorimetrically at 880 nm (Hitachi U- significantly from the minimum inorganic fertilization treat-
2001) following the ascorbic acid molybdate reduction method ment (I-min) whereas the control resulted in the lowest yield
(Olsen and Sommers, 1982). Soil available K (exchangeable which was similar to the minimum organic fertilization (O-min)
plus water soluble) was estimated following extraction with treatment only.
NH4OAc (Thomas, 1982) and determined by flame photometry During the second crop season (late-autumn), characterized
(Coring 410). The anaerobic incubation index of soil-N by intermediate temperatures and the shortest photoperiod and
availability was estimated by measuring the NH4-N production sunshine duration (Table 4), reduced and uniform yields were
from soil samples incubated anaerobically for 1 week at 40 8C observed among treatments (Table 5). Similar lettuce biomass
according to Keeney (1982). yield was obtained in all treatments apart from the significantly
smaller yield of the control (Table 5).
2.4. Statistical analysis During the third crop season (late-winter) characterized by
the lowest temperatures and intermediate photoperiod and
Analysis of variance was performed to evaluate differences sunshine duration (Table 4) plant yields were also increased,
in leaf length, leaf width, plant fresh weight, and nitrate but did not reach the yields observed in the first (late-spring)

Table 6
Maximum leaf length and width of lettuce plants grown under different organic (O) or inorganic (I) fertilization schemes during three successive crop seasons: late-
spring (first), late-autumn (second) and late-winter (third)
Fertilization treatment First crop season Second crop season Third crop season
Length (cm) Width (cm) Length (cm) Width (cm) Length (cm) Width (cm)
I-max 32.92 a 21.85 a 31.49 a 16.63 abc 32.30 a 19.40 a
I-mid 32.33 a 21.23 a 32.10 a 17.48 a 32.28 a 19.35 a
I-min 30.41 b 19.42 b 31.53 a 16.94 ab 31.02 ab 17.69 bc
O-max 30.65 b 19.77 b 31.87 a 17.18 ab 31.94 ab 18.59 ab
O-mid 30.16 bc 19.10 bc 31.17 a 16.30 bc 31.22 ab 17.82 bc
O-min 29.24 cd 18.56 cd 30.92 a 15.83 c 30.90 b 17.28 c
Control 29.00 d 18.12 d 29.44 b 14.15 d 28.14 c 12.72 d
Means in the same column (length or width) followed by different letters denote significant differences according to Duncans multiple range test ( p = 0.05).
G.C. Pavlou et al. / Scientia Horticulturae 111 (2007) 319325 323

Table 7
Nitrate concentration, on a fresh weight basis, in the external leaves of lettuce plants grown under different organic (O) or inorganic (I) fertilization during three
successive crop seasons: late-spring (first), late-autumn (second) and late-winter (third)
Fertilization treatment First crop season (mg kg1) Second crop season (mg kg1) Third crop season (mg kg1)
I-max 623 a 664 a 662 a
I-mid 572 a 588 a 623 a
I-min 314 b 363 bc 372 b
O-max 311 b 435 b 295 b
O-mid 253 bc 412 b 277 b
O-min 186 c 282 c 148 c
Control 71 d 92 d 25 d
Means in the same column (crop season) followed by different letters denote significant differences according to Duncans multiple range test ( p = 0.05).

season (Table 5). Similar lettuce biomass yield was obtained in maximum and medium inorganic fertilization treatments (I-
all treatments apart from the minimum organic fertilization (O- max, I-mid) (Table 7); the two respective organic fertilization
min) treatment which resulted in significantly lower yield treatments (O-max, O-mid) followed, together with the
compared to the maximum and medium inorganic fertilization minimum inorganic fertilization treatment (I-min). The
treatments (I-max, I-mid) but greater yield compared to the concentration of nitrates was even lower in the minimum
control (Table 5). organic fertilization treatment (O-min) except for the O-mid
The width and length of the largest leaf in each lettuce plant treatment in the first crop season and the I-min treatment in the
were measured, as indicators of plant growth. Statistical second crop season (Table 7). Finally the control (non-
differences in leaf length among treatments (Table 6) were fertilized) treatment showed the lowest nitrate concentration
identical to the lettuce biomass differences (Table 5). compared to all fertilization treatments in each season
Differences in leaf width were analogous to the lettuce biomass (Table 7).
differences for the first crop season, but small differentiations The same nitrate accumulation patterns and treatment
were observed in the following seasons (Table 6). differences were obtained by expressing nitrate concentration
on a dry weight basis and the data were highly correlated to the
3.2. Nitrate accumulation in leaves per fresh weight data (r2 = 0.892, p < 0.01) apparently due to
the small variability of the d.w.-to-f.w. ratio at harvest time,
Nitrate concentration in the external leaves did not exceed indicating that both expressions were valid for analyzing nitrate
the 664 mg kg1 f.w. (Table 7) in all three cultivation periods, accumulation data.
even under the maximum fertilization treatments. Similar levels
and patterns of nitrate accumulation in the external leaves were 3.3. Residual fertilization effects
observed in all three crop seasons (Table 7). A remarkable
increase in nitrate accumulation was observed in the organic Following the three successive crop seasons the residual
fertilization treatments during the late autumn period, availability of N, P and K in the soil was estimated. Residual N,
characterized by short photoperiod and sunshine duration P and K availability in the soil was generally higher and tended
(Table 4), but specific experimentation is needed to confirm this to increase with organic fertilization treatment dose (O-min, O-
trend. In all three crop seasons nitrate accumulation in the mid, O-max) compared to control (non-fertilized) plots, but did
external leaves of lettuce plants was significantly greater in the not respond to inorganic fertilization dose (Fig. 1AC,

Fig. 1. Residual effect of three different doses of inorganic fertilization (I-max, I-mid, I-min) and organic fertilization (O-max, O-mid, O-min) applied to lettuce
plants in three successive crop seasons on the soil-N availability (A), on the soil-P availability (B) and on the soil-K availability (C). Bars with different letters denote
significant differences according to Duncans multiple range test, p = 0.05.
324 G.C. Pavlou et al. / Scientia Horticulturae 111 (2007) 319325

respectively). Specifically, the O-max treatment resulted in a conditions led to adequate photosynthetic activity and nitrate
significantly greater index of soil-N availability compared to all reduction rates in planta during all seasons and therefore the
the inorganic fertilization treatments (Fig. 1A), the treatments accumulation of nitrates in the lettuce leaves were far bellow
O-mid and O-max resulted in a significantly higher P the maximum limits set by the E.C. 563/2002 Regulation for
availability compared to all the inorganic fertilization treat- lettuce fresh product (3500 and 4500 mg kg1 for the
ments (Fig. 1B), whereas all the organic fertilization treatments cultivation periods April-to-September and October-to-March,
significantly exceeded the exchangeable-K values observed in respectively). Indeed raised nitrate reductase activity has been
the inorganic fertilization treatments (Fig. 1C). observed at increased light levels (Gaudreau et al., 1995) and a
close negative correlation between photosynthetic activity and
4. Discussion nitrate content has been reported for various lettuce cultivars
(Behr and Wiebe, 1992) and was used for modeling plant nitrate
Comparisons between inorganic and organic fertilizer-N accumulation (Scaife and Schloemer, 1994).
sources are hard to perform since there is usually a dramatic Nitrate accumulation in lettuce leaves was roughly doubled
difference in N availability from these two sources of N. in all crop seasons in the maximum and medium inorganic
Comparisons on the basis of similar amounts of total N applied fertilization treatments compared to the following treatments
are therefore of limited relevance to agricultural practice, (maximum and medium organic, minimum inorganic). This is
whereas comparisons on the basis of similar N availability are despite the higher total N in the respective sheep manure
hindered by the lack of reliable nitrogen release estimates for treatments and is apparently explained by the higher N
organic fertilizer sources derived from animal manures (Van availability in the inorganic fertilizer applications. However, a
Kessel and Reeves, 2002). A general rule of thump assuming significant growth advantage was obtained for those two
35% of total-N mineralization in the first year following manure inorganic fertilization treatments in the late-spring crop season
application to soil is occasionally used (Klausner, 1997; Van only, with the highest sunlight intensity and duration (20% and
Kessel and Reeves, 2002), but deviations are large and manure- 23% yield increase compared to the respective maximum and
N mineralization may also significantly depend on soil medium organic fertilization treatments). Sheep manure or low-
temperatures and moisture (Agehara and Warncke, 2005). dose inorganic N fertilizer applications may therefore be
Our main objective, however, was to compare nitrate successfully used under suboptimal environmental conditions
accumulation in lettuce cultivated in different seasons under to avoid unnecessary costs and nitrate accumulation in lettuce
typical organic and inorganic N fertilization regimes. A leaves.
standard fertilization by inorganic fertilizers or sheep manure Reduced nitrate accumulation results have been reported for
used in the cultivation area was therefore chosen as the standard farm yard manure fertilized lettuce for one crop season only
fertilization treatment, increased by 50% in N content to make (Gianquinto et al., 1992; Stopes et al., 1989). Our results show
the maximum dose treatment and decreased by 50% in N that composted sheep manure applications were particularly
content to make the minimum dose treatment. As sheep manure safe in terms of nitrate accumulation in lettuce leaves in the
had a standard elemental synthesis, all other nutrients applied long-term. They remained significantly lower than in the
varied in the same order (+50% and 50% for the O-min and respective inorganic fertilization treatments, even after sub-
O-max, respectively); however, treatment effects on lettuce sequent high level applications in different cultivation seasons
growth should be attributed mainly to N availability since other (for example, I-max caused 100%, 53% and 124% increase in
nutrients in soil were adequately available and no deficiencies nitrate concentration compared to O-max in the respective first,
were observed in randomly checked plants (data not presented). second and third crop season, but a significant yield benefit was
General changes in lettuce growth between crop seasons observed in the first season only). Moreover, sheep manure
were in line with changes in photoperiod and sunshine duration. applications contributed to the built up of soil fertility at the end
This is expected, since photoperiod and sunshine duration of the experiment, since they resulted in significantly increased
differed widely, whereas minimum temperatures differed only N, P and K availability, contrary to the inorganic fertilization
slightly and were near optimum during all seasons. High lettuce treatments that showed residual fertilizer effects in soil not
plant biomass production occurred mainly in the late-spring different from the control.
season associated with long sunshine duration, which is in line The effects of fertilization treatments on lettuce yield were
with the positive effects that have been observed on the biomass similar to their effects on lettuce growth indicators (largest leaf
of lettuce by supplementary light (McCall and Willumsen, length and width). However, leaf length showed limited
1999) and long photoperiod (Chadjaa et al., 1999). Fertilizer variability between treatments (2833 cm) and was not a
effects on growth were most clearly differentiated in this spring sensitive indicator of lettuce growth (r2 = 0.218, p = 0.03); on
season; on the contrary a poor response of plants to either the contrary leaf width varied widely (1322 cm) and was
organic or inorganic N fertilization was observed in the late strongly correlated to lettuce growth (r2 = 0.938, p < 0.01).
autumn season indicating that suboptimal environmental
conditions (short photoperiod and sunshine duration) limited 5. Conclusions
plant growth independent of N availability.
Seasonality affected plant growth more than nitrate Nitrate concentration in romaine lettuce leaves depends on
concentration in leaves. Apparently, Mediterranean climatic fertilizer type and dose rather than on the crop season and may
G.C. Pavlou et al. / Scientia Horticulturae 111 (2007) 319325 325

remain in Southern Greece far below the upper limits set by Demsar, J., Osvald, J., 2003. Influence of NO3:NH4+ ratio on growth and
nitrate accumulation in lettuce (Lactuca sativa var. capitata L.) in an
the European Commission Regulation, even under high and
aeropinic system. Agrochimica 47, 112121.
repeated fertilization treatments, a result attributed mainly to Drews, M., Schonhof, I., Krumbein, A., 1995. Influence of growth on the
the sufficient sunlight intensity and duration throughout the content of nitrate, vitamin-C, beta-carotin, and sugar of head lettuce under
year in this region. High application rates of sheep manure greenhouse conditions. Gartenbauwissenschaft 60, 180187.
may be needed to reach the lettuce growth obtained by Escobar-Gutierrez, A.J., Burns, I.G., Lee, A., Edmondson, R.N., 2002. Screen-
inorganic fertilizers under optimal climatic conditions only. ing lettuce cultivars for low nitrate content during summer and winter
production. J. Hortic. Sci. Biotechnol. 77, 232237.
Increased inorganic fertilizer doses should be avoided since, Gaudreau, L., Charbonneau, J., Vezina, L.P., Gosselin, A., 1995. Effects of
especially under suboptimal light conditions, they might photoperiod and photosynthetic photon flux on nitrate content and nitrate
result in significant rise in nitrate accumulation in leaves but reductase-activity in greenhouse-grown lettuce. J. Plant Nutr. 18, 437453.
marginal yield increase; on the contrary composted sheep Gianquinto, G.P., Borin, M., Scaife, A., 1992. Nitrate content in vegetable crops
as affected by soil characteristics, rate and type of fertilization. In:
manure may be safely applied even at high doses. High
Proceedings of the 2nd Congress of the European Society for
residual availability of N, P and K may be obtained in the soil, Agronomy. pp. 256257.
following the sheep manure applications, which relates to Gunes, A., Post, W.H.K., Aktas, M., 1995. Effect of partial replacement of
manure dose; on the contrary no residual availability of N, P nitrate by NH4-N, urea-N and amino acid-N in nutrient solution on nitrate
and K was obtained by inorganic fertilization applied via accumulation in lettuce (Lactuca sativa L.). Agrochimica 39, 326333.
fertigation. Keeney, D.R., 1982. Nitrogen availability indices. In: Page, A.L. (Ed.), Methods
of Soil Analysis Part 2, Agron. Monogr. 9, 2nd ed. American Society of
Agronomy, Soil Science Society of America, Madison, Wisconsin, pp. 711
Acknowledgement 733.
Keeney, D.R., Nelson, D.W., 1982. Nitrogeninorganic forms. In: Page, A.L.
This work was financially supported by the National (Ed.), Methods of Soil Analysis Part 2, Agron. Monogr. 9, 2nd ed. American
Agricultural Research Foundation. Society of Agronomy, Soil Science Society of America, Madison, Wis-
consin, pp. 643698.
Klausner, S.D., 1997. Nutrient Management: Crop Production and Water
References Quality. Northeast Regional Agricultural Engineering Service, Ithaca, NY.
Lundberg, J.O., Weitzberg, E., Cole, J.A., Benjamin, N., 2004. Nitrate, bacteria
Abu-Rayyan, A., Kharawish, B.H., Al-Ismail, K., 2004. Nitrate content in and human health. Nat. Rev. Microbiol. 2 (8), 681.
lettuce (Lactuca sativa L.) heads in relation to plant spacing, nitrogen form Maynard, D.N., Barker, A.V., Minotti, P.L., Peck, N.H., 1976. Nitrate accu-
and irrigation level. J. Sci. Food Agric. 84, 931936. mulation in vegetables. Adv. Agron. 28, 71118.
Addiscott, T.M., Benjamin, N., 2004. Nitrate and human health. Soil Use McCall, D., Willumsen, J., 1998. Effects of nitrate, ammonium and chloride
Manage. 20, 98104. application on the yield and nitrate content of soil-grown lettuce. J. Hortic.
Agehara, S., Warncke, D.D., 2005. Soil moisture and temperature effects on Sci. Biotechnol. 73, 698703.
nitrogen release from organic nitrogen sources. Soil Sci. Soc. Am. J. 69 (6), McCall, D., Willumsen, J., 1999. Effects of nitrogen availability and supple-
18441855. mentary light on the nitrate content of soil-grown lettuce. J. Hortic. Sci.
Behr, U., Wiebe, H.J., 1988. Relations between nitrate content and other Biotechnol. 74, 458463.
osmotica in the cell sap of lettuce cultivars (Lactuca sativa L.). Garten- Olsen, S.R., Sommers, L.E., 1982. Phosphorous. In: Page, A.L. (Ed.), Methods
bauwissenschaft 53, 206210. of Soil Analysis Part 2, Agron. Monogr. 9, 2nd ed. American Society of
Behr, U., Wiebe, H.J., 1992. Relation between photosynthesis and nitrate Agronomy, Soil Science Society of America, Madison, Wisconsin, pp.
content of lettuce cultivars. Scientia Hortic. 49, 175179. 403430.
Blom-Zandstra, M., 1989. Nitrate accumulation in vegetables and its relation- Santamaria, P., Elia, A., Serio, F., Todaro, E., 1999. A survey of nitrate and
ship to quality. Ann. Appl. Biol. 115, 553561. oxalate content in fresh vegetables. J. Sci. Food Agric. 79, 18821888.
Blom-Zandstra, M., Eenink, A.H., 1986. Nitrate concentration and reduction Scaife, A., 1989. A pump/leak/buffer model for plant nitrate uptake. Plant Soil
in different genotypes of lettuce. J. Am. Soc. Hortic. Sci. 111, 908 114, 139141.
911. Scaife, A., Ferreira, M.E.S., Turner, M.K., 1986. Effect of nitrogen form on the
Blom-Zandstra, M., Lampe, J.E.M., 1985. The role of nitrate in the osmor- growth and nitrate concentration of lettuce. Plant Soil 94, 316.
egulation of lettuce (Lactuca sativa L.) grown at different light intensities. J. Scaife, A., Schloemer, S., 1994. The diurnal pattern of nitrate uptake and
Exp. Bot. 36, 10431052. reduction by spinach (Spinacia oleracea L.). Ann. Bot. 73, 337343.
Bruning-Fann, C.S., Kaneene, J.B., 1993. The effects of nitrate, nitrite and N- Siomos, A., 2000. Nitrate levels in lettuce at three times during a diurnal period.
nitroso compounds on human health: a review. Vet. Human Toxicol. 35, J. Veget. Crop Product. 6 (2), 3742.
521538. Stopes, C., Woodward, L., Forde, G., Vogtmann, H., 1989. Effects of composted
Buwalda, F., Warmenhoven, M., 1999. Growth-limiting phosphate nutrition FYM and a compound fertilizer on yield and nitrate accumulation in three
suppresses nitrate accumulation in greenhouse lettuce. J. Exp. Bot. 50, 813 summer lettuce cultivars grown in an organic system. Agric. Ecosyst.
821. Environ. 27, 555559.
Cardenas-Navarro, R., Adamowicz, S., Gojon, A., Robin, P., 1998. Diurnal Tesi, R., Lenzi, A., 1998. Controlled-released fertilizers and nitrate accumula-
nitrate uptake in young tomato (Lycopersicon esculentun Mill.) plants: test tion in lettuce (Lactuca sativa L.). Agric. Mediterranea 128 (4), 313320.
of a feedback based model. J. Exp. Bot. 49, 21730. Thomas, G., 1982. Exchangeable catios. In: Page, A.L. (Ed.), Methods of Soil
Cataldo, D.A., Haroon, M., Schrader, L.E., Youngs, V.L., 1975. Rapid colori- Analysis Part 2, Agron. Monogr. 9, 2nd ed. American Society of Agronomy,
metric determination of nitrate in plant tissue by nitration of salicylic acid. Soil Science Society of America, Madison, Wisconsin, pp. 159165.
Commun. Soil Sci. Plant Anal. 6 (1), 7180. Van Kessel, J.S., Reeves, J.B., 2002. Nitrogen mineralization potential of dairy
Chadjaa, H., Vezina, L.P., Gosselin, A., 1999. Effect of supplementary lighting manures and its relationship to composition. Biol. Fertility Soils 36 (2),
on growth and primary nitrogen metabolism of greenhouse lambs lettuce 118123.
and spinach. Can. J. Plant Sci. 79, 421426. Walker, R., 1990. Nitrates, nitrites and N-nitroso compounds: a review of the
Corre, W.J., Breimer, I., 1979. Nitrate and Nitrite in Vegetables. Centre for occurrence in food and diet and the toxicological implications. Food Addit.
Agricultural Publishing and Documentation, Wagheningen, p. 85. Contam. 7, 717768.