DOI: 10.

1007/s11099-011-0019-x PHOTOSYNTHETICA 49 (1): 149-153, 2011

BRIEF COMMUNICATION

Nondestructive determination of nitrogen and chlorophyll content

in olive tree leaves and the relation with photosynthesis
and fluorescence parameters

O. BOUSSADIA1,+, K. STEPPE2, H. ZGALLAI4, S. BEN EL HADJ3, M. BRAHAM1, R. LEMEUR2,

and M.C. VAN LABEKE5

Institute of the Olive Tree station of Sousse, 40 Rue Ibn Khouldoun 4061 Sousse, Tunisia1
Faculty of Bioscience Engineering, Laboratory of Plant Ecology, Ghent University, Coupure links 653,
B-9000 Ghent, Belgium2
Agronomic National Institute of Tunisia: 43 Avenue Charlnicol 1082 Cité El Mahrajen, Tunisia3
Bayer BioScience N.V. Nazarethsesteenweg 77 B-9800 Astene (Deinze), Belgium4
Faculty of Bioscience Engineering, Department of Plant Production, Ghent University, Coupure links 653,
B-9000 Ghent, Belgium5

Abstract

For Tunisian olive tree orchards, nitrogen deficiency is an important nutritional problem, in addition to the availability of
water. Establishment of relationships between nutrients such as nitrogen and ecophysiological parameters is a promising
method to manage fertilisation at orchard level. Therefore, a nitrogen stress experiment with one-year-old olive trees
(Olea europaea L. ‘Koroneiki’ and ‘Meski’) was conducted with trees respectively subjected to four nitrogen supply
regimes (23.96 meq l–1, 9.58 meq l–1, 4.79 meq l–1and 0 meq l–1 NO3–).
The current paper focuses on the use of the SPAD-502 portable chlorophyll (Chl) meter, a nondestructive method for
fertilisation management under nitrogen stress conditions of olive trees. Maximum net photosynthetic assimilation rates,
chlorophyll fluorescence parameters and the SPAD Chl index were therefore measured simultaneously and the Chl and
nitrogen content of the leaves were analysed. Significant correlations were established in the olive tree leaves between
SPAD-502 readings on the one hand and Chl content, nitrogen content, photosynthetic assimilation rate, and Chl
fluorescence parameters (ΦPSII and ETR) on the other hand.

Additional key words: electron flow; nitrogen deficiency; olive tree; photosynthesis; pigments; SPAD-502; quantum yield.

Introduction

For Tunisian olive tree orchards, nitrogen (N) deficiency For certain crops and terrestrial ecosystems, good
is an important nutritional problem, in addition to the correlations between Chl content and photosynthesis have
availability of water. The SPAD-502 portable Chl meter been established (Brougham 1960, Dawson et al. 2003).
allows rapid, nondestructive measurements of the N In addition, significant correlations between leaf N and
status and the Chl content of leaves and has been tested in photosynthesis have been reported (Field and Mooney
some major crops (Smeal and Zhang 1994, Neilsen et al. 1986). The SPAD-502 Chl meter has therefore not only
1995, Balasubra-Manian et al. 2000, Porro et al. 2001). the potential to detect N deficiency, but might also

———
Received 17 August 2009, accepted 25 November 2010.
+
Corresponding author; tel: ++21673236135, fax: ++21673236135, e-mail: [email protected]
Abbreviations: Chl – chlorophyll; Ft – steady-state fluorescence in the light-adapted leaves; Fm’ – maximum fluorescence in the light;
ETR – total electron flow, N – nitrogen; PN – maximum net photosynthetic assimilation rate; ΦPSII – quantum yield of PSII electron
transport.
Acknowledgements: The authors also wish to thank the Vlaamse Interuniversitaire Raad (VLIR) for funding the doctoral research of
the first author and the Research Foundation – Flanders (FWO-Vlaanderen) for the Postdoctoral Fellow funding granted to the second
author.

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provide additional information about the photosynthesis lations among SPAD-502 readings and destructively
process. In this study, we investigated olive trees growing determined Chl a and leaf N content, maximum
under different N-deficiency levels, searching for corre- photosynthetic rates, and Chl fluorescence parameters.

Materials and methods

One-year-old olive trees (Olea europaea L. ‘Koroneiki’ According to Krall and Edwards (1992) the total
and ’Meski’) were grown in vermiculite (2-L containers) electron flow (ETR) can be derived from ΦPSII, the light
under greenhouse conditions from 22 January till intensity incident on the leaf (PAR), the fractional
21 April 2008. The air temperature fluctuated between 20 absorption of light by the leaf (a) and the absorptance of
and 32°C and the relative humidity of the air ranged PSI and PSII (f), ETR = ΦPSII × PAR × a × f, where ‘a’
between 60 and 70%. Plants were fertigated with a full- equals 0.84 and ‘f’ equals 0.5 (Schreiber 1997).
strength modified Hoagland’s solution (EC = 2.5, pH = Measurements of PN, ΦPSII, and ETR were made on
6.5). After 34 days of culture, plants were randomly the 8th fully expanded leaf counted from the top of the
allocated to four groups and each group consisted of olive tree and for three plants per cultivar per treatment.
16 plants (8 plants for ‘Koroneiki’ and 8 plants for Measurements took place between 09:00 and 17:00 h and
‘Meski’) Four N levels were provided for 58 days: were done in 3 replications. The replications were
(1) 100N received a full-strength nutrient solution measured respectively at 09:00, 12:00 and 15:00 for each
throughout the entire experiment (NO3– = 23.96 meq l–1); treatment and each cultivar in order to cover as such the
(2) 40N reduced N to 40% N (mild N stress with NO3– = existing daily variability. The SPAD-502 readings
9.58 meq l–1); (3) 20N reduced N to 20% N (moderate N (Konica Minolta Sensing, Inc., Osaka, Japan) were
stress with NO3-= 4.79 meq l–1) and (4) 0N reduced N to determined for the 3rd and the 8th fully expanded leaf (two
0% N (severe N stress with NO3– = 0 meq l–1). The four leaf-age classes) counted from the top of the tree (three
treatments aimed to induce different leaf N levels. measurements per leaf). Each measurement was done in
After 58 days of N stress, maximum net photosyn- seven replications. After the SPAD readings, Chl a for
thetic assimilation rate (PN) was measured simultaneously both leaf-age classes was extracted according to Moran
with several Chl fluorescence parameters using an open (1982). Leaf discs (7 × 19.6 mm2 plant–1) were extracted
gas exchange system (LI-6400; LI-COR, Lincoln, NE, with N, N dimethylformamide (DMF) and absorbance
USA) with an integrated fluorescence chamber head was measured at 664 and 647 nm (UV-VIS, Biotek Uvikon
(LI-6400-40 leaf chamber fluorometer). Photosyntheti- XL, USA).
cally active radiation, air temperature and CO2 concen- Leaf N content was determined on the 8th fully
tration inside the sensor head were set at 1,500 µmol expanded leaf counted from the top. Leaves from eight
m–2 s–1, 25°C and 450 µmol mol–1, respectively, when plants in each treatment were combined and analysed
measuring PN, quantum yield of PSII electron transport using the Kjeldahl method.
(ΦPSII) and total electron flow (ETR). Data were analysed by polynomial regression analysis
The steady-state (Ft) and the maximal (Fm’) values using statistical software Version 16.0 (SPSS, Chicago,
of fluorescence are used to determine the ΦPSII IL, USA).
[(Fm’ – Ft)/Fm’].

Results and discussion

Recent research indicates a close link between leaf Chl the N content (‘Meski’ R2 = 0.84, p<0.001; ‘Koroneiki’
concentration and leaf N content, which makes sense R2 = 0.93 p<0.001 for the 8th leaf from the top) (Fig. 1).
because the majority of leaf N is contained in Chl mole- Cultivar differences were observed: lower SPAD values
cules (Peterson et al. 1993). Chl concentration or leaf were found for ‘Koroneiki’ for a given N content
greenness is affected by a number of factors, one being N compared to ‘Meski’ (Fig. 1). The Chl index (using the
status of the plant. Since the Chl meter has the potential SPAD-502 meter) has been successfully used to assess
to detect N deficiencies, it also shows promise as a tool the N status in apple trees (Neilsen et al. 1995),
for improving N management (Peterson et al. 1993, grapevines (Porro et al. 2001), hardwood tree species
Smeal and Zhang 1994, Balasubramanian et al. 2000). (Chang and Robinson 2003) and woody ornamental
SPAD values ranged from 45 to 80 for the 3rd leaf plants (Demotes-Mainard et al. 2008). However, in most
from the top compared to 70 to 95 for the 8th leaf (Fig. 1). of such studies a simple linear regression was fitted to the
A positive curvilinear relationship between the SPAD data, which is different from our results. In coffee leaves,
readings and the Chl a content was found (‘Meski’ a polynomial quadratic fitting between SPAD readings
R2 = 0.72 and 0.90, p<0.001; ‘Koroneiki’ R2 = 0.96 and and photosynthetic pigments was found (Netto et al.
0.92, p<0.001 for 3rd and 8th leaf from the top, 2005). polynomial relationship gives good correlations
respectively) (Fig. 1) and among the SPAD readings and for leaves of evergreen trees which are more leatherly and

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Fig. 1. Relationship between chlorophyll a (Chl a) concentration and SPAD-502 readings (A: 3rd fully developed leaf from the top; B:
8th fully developed leaf from the top) and between nitrogen (N) content and SPAD-502 readings (C: 3rd fully developed leaf from the
top; D: 8th fully developed leaf from the top); this for two olive tree cultivars under nitrogen deficiency. (▲ ‘Koroneiki’, □ ‘Meski’,
means n = 12). Dotted and solid lines indicate significant polynomial regressions for ‘Koroneiki’ and ‘Meski’, respectively. The
coefficient of determination (R2) is also shown.

often coated with wax. content is strongly related to the photosynthetic process
In the present study, the Chl index was strongly in olive trees as was also shown for other crops
correlated with Chl a (Fig. 1). Also a strong correlation (Brougham 1960, Dawson et al. 2003). In both cultivars,
between the SPAD readings and the N content was found, a Chl a content of at least 80 µg cm–2 seemed necessary
which supports the potential of the SPAD-502 Chl meter for optimal CO2 gas exchange. When N deficiency is
to diagnose the N status in olive trees (Fig. 1). Leaf Chl imposed, gas exchange will be affected (Field and
meters (SPAD-502) have been used with various crops as Mooney 1986). In this experiment, the significant
an indirect indicator of plant N status (Turner and Jund correlation between SPAD readings, Chl a content and
1991, Schepers et al. 1992, Chapman and Barreto 1997, PN showed that SPAD measurements can predict PN in
Denuit et al. 2002, Gianquinto et al. 2003). olive trees. The relationship between ΦPSII and SPAD
A wide SPAD-reading range was found for the 3rd readings (Fig. 2) showed that leaf ΦPSII reached its
leaf from the top, a correlation was made with the N maximum SPAD values above 83 for both cultivars.
content of the 8th leaf (‘Meski’ R2 = 0.90, p<0.001; Lower values indicate a decrease in the quantum yield of
‘Koroneiki’ R2 = 0.98, p<0.001) (Fig. 1). The high PSII and, hence, an induced disturbance in the electron
correlations supported the idea that the higher colour transport or even possible damage to PSII causing
sensitivity of younger leaves might be successfully used reducing power of the biochemical processes. The
to judge changes in the whole-tree N status. relationship between ETR and SPAD readings was non-
More than half of the N in leaves is used in linear for ‘Meski’ and showed the same trend as was
photosynthetic proteins (Evans 1989), so it is also observed among SPAD and ΦPSII. ‘Koroneiki’, however,
interesting to investigate the correlation between the showed a more linear relation among SPAD values
destructively determined Chl a content and PN and ranging from 75 to 90 and ETR, which made it difficult
between SPAD readings and PN (Fig. 2). Indeed, positive to propose a threshold SPAD reading for this parameter.
though nonlinear relations between Chl a content and PN In the same context, Netto et al. (2005) showed for coffee
(‘Koroneiki’: R2 = 0.84 p<0.001; ‘Meski’: R2 = 0.91, leaves an exponential relation between the SPAD
p<0.004) and between SPAD readings and PN readings and the initial fluorescence, maximum fluores-
(‘Koroneiki’: R2 = 0.85, p<0.001; ‘Meski’: R2 = 0.87, cence and maximum quantum efficiency of PSII centres
p<0.004) were found. These results confirm that Chl a (Fv/Fm). Our results showed a polynomial relationship

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Fig. 2. Relationship between chlorophyll a (Chl a) concentration and maximum net assimilation rate (PN) (A) and between SPAD-502
readings and PN (B), quantum yield of PSII (ΦPSII) (C) and total electron flow (ETR) (D) in the 8th fully expanded leaves of the olive
tree cultivars ‘Koroneiki’ and ‘Meski’(▲‘Koroneiki’, □ ‘Meski’). Dotted and solid lines indicate significant polynomial regressions
for ‘Koroneiki’ and ‘Meski’, respectively. The coefficient of determination (R2) is also shown.

between SPAD readings and ΦPSII and ETR, but low required. The SPAD-502 Chl meter has a potential to be
correlations were found with Fv/Fm (data not shown). used as a rapid tool to support site-specific N manage-
In conclusion, SPAD-502 measurements were useful ment in olive orchards. Furthermore, this study indicates
to determine N deficiency and Chl a content in olive that SPAD readings can be used to assess certain
trees. Differences in cultivar responses and leaf age parameters describing the photosynthetic capacity of the
classes suggest that calibration for each cultivar is olive leaves under N stress.

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