Detection of Nutrient Deficiencies of Maize by Laser Induced Fluorescence Imaging

Detection of Nutrient Deficiencies of Maize by Laser Induced Fluorescence Imaging

FRANCINE HEISEL!, MALGORZATA SOWINSKA1

, JOSEPH ALBERT MIEHl~^{1, MICHAELUNG2,}

and

HARMUT

K.

LICHTENTHALER2

1 Groupe d'Optique Appliquee, Centre de Recherches Nucleaires, IN2P3, CNRS, 23, rue du Loess, F 67037 Strasbourg Cedex 2, France

2 Botanisches Institut II der Universitat Karlsruhe, Kaisersrr. 12, D-76128, Germany

Received Augusr 3, 1995 . Accepred October 20, 1995

Summary

Laser-induced fluorescence is an active method of sensing the state of health of the plants and the pho- tosynthetic apparatus, as it is related not only to the pigment concentrations but also to the physiological activity. A high gain and high spatial resolution fluorescence imaging set up, consisting of a pulsed Nd:YAG laser for the excitation (355 nm) and of an intensified gated CCO numerical camera, has been used for monitoring various nutrient deficiencies of maize(Zea maysL.) by recording fluorescence images of the leaves at 440, 520, 690 and 740 nm. The plant status was characterised by the fluorescence ratios F440/F520, F440/F690, F440/F740 and F690/F740. The experiments performed on field maize sup- plied with various amounts of nitrogen and on greenhouse maize with defined mineral deficiencies showed that all the deficiencies could be monitored by the fluorescence ratios and in some cases directly on the fluorescence images by considering the spatial distribution of the emission on the leaf surface. From this work it appeared that the efficiency of detection depended on the period of measurements and on the age of the leaves. The fluorescence ratios F440/F690 and F440/F740 were found more sensitive to the growth conditions than the most frequently used chlorophyll fluorescence ratio F690/F740.

Key

words: Zea mays L.,fluorescence ratios, laser-induced fluorescence imaging, leaffluorescence, mineral deficiencies: Fe, Mg, Zn, nitrogen fertilisation.

Abbreviations: CCO= charge coupled device; F440= blue fluorescence intensity at 440 nm; F520 = green fluorescence intensity at 520 nm; F690= red chlorophyll fluorescence intensity at 690 nm; F740= far-red chlorophyll fluorescence intensity at 740 nm.

Introduction

Remote sensing of the vegetation health status, based on the optical properties of the plants, is a very attractive tool since the method is non destructive and gives immediate results that allows, in the case of stress detection due to nutri- ent deficiencies, timely correction of the fertilisation. If far field reflectance measurements, which are at the present time technologically well developed (Slater, 1980; Brunel et al., 1991), have application for example in green biomass evalua-

© 1996 by Gustav Fischer Verlag, Stuttgart

tion, they are insensitive to physiological variations of the plant status.

The fluorescence emission, which for plant leaves excited by UV-A radiation has a spectrum with maxima/shoulders near to 440, 520, 690 and 740 nm (Chappelle et al., 1984 a;

Lang and Lichtenthaler, 1991; Lang et al., 1992, 1994 a), depends not only on the pigment concentration but also on the efficiency of the photosynthesis and other biochemical reactions. Much work has been performed to investigate the relation between the plant health and the fluorescence inten-

IMAGE PROCESSING

SYSTEM

smes (Kautsky and Hirsch. 1934; Buschmann et al., 1991;

Lichtenthaler et al., 1991; Scober et Lichtenthaler, 1992.

1993;Gunther et al., 1994;Morales et al., 1994)especially in what concerns the chlorophyll red emission (690 and 740nm) (Krause and Weis, 1984;Lichtenthaler and Rinderle, 1988;Lichtenthaler, 1987, 1990;Snel and Van Kooten, 1990;

Lippucci di Paola. 1992; Methy et al.• 1994;Valentini et al., 1994). Indications came out that laser induced fluorescence signatures seem co be useful for remote detection of certain nutrient deficiencies (Chappelle et al .• 1984b; Kochubey et al., 1986;Hagg et al., 1992;Hak et al., 1993;McMurtrey et al., 1994;Subhash and Mohanan, 1994), in spite of the fact that the blue-green fluorescence is a very complex signal from various plant phenolics (Goulas et al., 1990;Lang et aI., 1991;

Lichtenthaler et al., 1991; Stober et al., 1994) and that the physiological significance of this blue-green fluorescence is not yet fully clear.

The aim of this work wascoassess the possibility of nutri- ent deficiencies detection and characterisation by laser-in- duced fluorescence imaging (Edner et al., 1994; Balachand- ran et al., 1994; Lichtenthaler et aI., 1995)with a newly de- veloped high spatial resolution numerical camera used in conjunction with a high gain gated image intensifier that al- lows measurements in the presence of the ambient light (Lang et al., 1994a, b, 1995). Experiments have been per- formed on maize(Zea maysL.)grown in the field with differ- ent nitrogen fertilisation degrees, or grown in a greenhouse with defined mineral deficiencies.

Since further investigations will be devotedtoremote sens- ing at long distances in the fields. we were interested in char- acterising the plant status by various fluorescence ratios, namely F440/F520, F440/F690, F440/F740 and F6901 F740, and not by the fluorescence intensities which strongly depend on external parameters like distance, geometry of the foliage subjected tothe excitation beam, etc.

Material and Methods P!4nt growth

Nitrogen deficiency has been studied on the D6l variety maize (Zea maysL.) planted at a density of 90,000 plants per hectare, on week 16 in 1994, in alluvial argillaceous land parcels cultivated by the Lycee Agricole d'Obernai (Bas-Rhin, France). These parcels were used since three years for testing the influence of the nitrification on the plant productivity. The ground contains all the other essential nutrients, in particular it is well furnished in K andP. The nitrogen was supplied at the 3-4 leaf stage in the ammonium-nitrate form at various amounts from 0 to 160 kg/ha, for a recommended supply of 120 kg/ha. For each N level, there were four randomly situated repli- cate areas: 6 m x 10 m, with 8 maize ranks spaced by 75 cm. The ex- periments which began at the 8-leaf stage after eight weeks of plant growth. were carried out during five weeks. Measurements were per- formed on the same leaf storeys for 0, 60 and 160 kg N/ha parcels.

The leaves were cut from the maize plants of the four replicate par- cels and the fluorescence of the upper leaf-side quickly measured in the laboratory (Strasbourg). The leaves were numbered from the bottom: as when becoming old the first leaves could fall down. a mark has been made on the stems at the beginning of the experi- ments to have a reference.

Fluorescence imaging of nutrient deficiencies

623

The Volga variety of maize has been used to srudy the effect of deficiency in Mg, Zn or Fe. The maize was grown in the greenhouse of the Centre de Recherches de la Societe Commerciale des Potasses et de I'Azote (Aspach Ie Bas, Haut-Rhin, France), and planted in pots containing quartz sand, previously leached during four days with a HCl +oxalic acid solution and with doubly demineralised water during two days, to eliminate the endogenous elements. After rhree days of growth in a seed-bed, four seeds per pot were planted out and the pots were supplied with a complete nutririve solution of type COIc-Lesaint (pH 6.2) (Lesaint and COIc, 1983) for the control plants, and deficient in one element for the others: respectively Fe:

0, Mg: 1/10 of the full nutritive solution, Zn:Y2duting 2 weeks and

o

after (a low amount of magnesium and zinc is necessary for the growth of maize). The temperature in the greenhouse was main- tained between 20'C and 28 'C with a relative humidity of 40- 80 % and the plants were illuminated (= 30,000 lux) 15 hours per day, with 400Whigh pressure natrium vapor lamps. There were two identical series of pots, one for measurements three weeks after the seed planting, the other for experiments two weeks later.

Fluorescence imaging

The laser induced fluorescence imaging arrangement is repre- sented in Fig. 1.The excitation source was acwQ-switched, mode locked and cavity dumped Nd:YAG laser emitting. after third har- monic generation, 355 nm light pulses at I kHz repetition rate, with an energy of 10

III

and a width of 100 psec. The laser beam was di- rected onto the sample at right angle and shaped by a divergent lens to a 20 cm diameter spot on the leaf level.

The fluorescence imaging system (RAGM6+Animater VI,ARP, Strasbourg, France) was composed ofi) a gated intensified digital CCD camera operating at SO frames per second and including all the electronic CCD's readout with 8 bits digitisation per pixel of the image due to one frame; the CCD array (Thomson TH 7863) is a 384 x 288 elements; the gated intensifier (Philips XX1414M/E image intensifier tube) has an adjustable gain (up to 10³⁾ and the width of the laser synchronous gating can be varied from 10 ns till to 100 ms ii) an interface card for the PC microcomputer where the images are stored iii)an image analysis software. After each digitisa- tion, the result is transferred to the interface card where an addition of successive readouts can be made till to 16 bits (0-65,535), fol- lowed (or not) by the subtraction of the noise due to the camera it- self and to the ambient light. The resulting image is then automati- cally sent in the extended memory of the Pc. Besides the real-time visualisation and the storage of the images, the software allows fast recall and treatment of images, their number (maximum 99) de-

~Tm(:FR

I~M~~~/ ~. ~ ^j l!j!1

!~

I

I

L .

Fig.I:Scheme of the laser induced Huorescence imaging set up.

pending on the size of the free extended memory ('" 0.23 Mbyte per image).

Before and during the measurements, additional white light was applied on the leaves to simulate solar illumination so that the meas- ured intensity of the chlorophyll fluorescence emission corre- sponded to the light adapted state of the chlorophyll fluorescence induction kinetic (Kautsky effect),

Fluorescence images were acquired, at an angle of'" 30°, through bandpass filters (bandwidth of 10 nm) in a distance of 50 cm via a focusing lens on the entrance of the intensifier. For this distance the height of the image corresponded to 20 cm at the leaf level. The in- tensifier was working with a 20 ns gate width, time long enough to collect the totality of the fluorescence and shon enough to allow measurements in the presence of the white light. The filters were centred around the four characteristic emission wavelengths 440 nm (blue), 520 nm (green), 690 nm (red) and 740 nm (far-red). All im- ages were corrected for the spectral sensitivity of the camera, includ- ing the attenuation factor of the bandpass filters and of the focusing lens.

Table 1: Description of the leaves taken for the measurements on the field Dea maize planted on week 16 in 1994, The numbers (No.) of the leaves represent the leaf storeys labelled from the bottom of the stems. For each leaf number and each treatment the sample size was 8, except for the last week where it was 8 for the leaf No, 8 and 40 for the others.

Week No. in 1994 24 25 26 27 28

Leaves No, 4^a 5^a 6" 7^a 8^a 9^{a lOa} 11a 8^a 11"

(0-60-160 kgN/ha) 6^a 7^b 8^a 9^{b lOa llb} 12^b 13^b 12^a Number

ofleaveslplant

o

^{kg N/ha 7-8 9-10 10-11 12-13 12-13}

60 kg N/ha 8-9 10-11 11-12 13-14 13-14

160 kg N/ha 8-9 11-12 12-13 13-14 13-14

At a given week the leaves with different superscripts showed fluores- cence ratio means significantly different (p<0,05) in the 0 kg N/ha par- cels,

Spectrofluorimetry

For having an overall view of the spectral distribution of the fluo- rescence, several additional emission spectra were recorded with a Spex Fluorolog 2 fluorimeter Oobin Yvon, Longjumeau, France), which allows front face detection and provides spectra corrected for lamp intensity variations, spectral sensitivity of the monochromator- photomultipliet, etc. The excitation wavelength (355 nm) was the same as that used for imaging.

gard to those coming from the fields. Our results could be re- lated to the visual observation that the epidermis is thicker for the outdoor maize plants: i) the epidermis is responsible for a major part of the blue-green fluorescence emission, ii) the excitation of the chloroplasts in the mesophyll cells is re- duced by partial absorption of the excitation beam in the epi- dermis and in addition part of the emitted chlorophyll fluo- rescence is reabsorbed by the leaf chlorophyll.

Results and Discussion

Nitrogen fertilisation effect

A general feature of the laser induced fluorescence imaging results was that an increase of the soil nitrogen concentration did not significantly influence the intensity of the blue-green fluorescence emission, whereas, except for the first series of measurements, it enhanced the chlorophyll fluorescence (see also Fig. 2). The chlorophyll concentration has not been de- termined but for the 0 kg N/ha supply a chlorosis of some leaves was visible from the second week of measurements, and this chlorosis was associated with a weaker red fluores- cence. It was also observed on all fluorescence images of maize leaves that the fluorescence intensity was roughly uni- formly distributed on the leaf surface, with the main leaf vein fluorescence not markedly different from that of the other leaf parts. So, for each leaf the fluorescence ratio values were determined by averaging on the whole illuminated area.

In Table 1 are indicated, for each week of fluorescence imaging experiments, the storeys of the cut leaves as well as the number of leaves per plant for the studied nitrogen amounts. For each treatment and fluorescence ratio we have compared the leaves each to the others.Asa general rule, less difference between the leaves of different ages was apparent for the 60 and 160 kg N/ha amounts than for the 0 kg N/ha parcels, that could be attributed to the fact that the latest cut leaves are younger in the absence of N. For the last week sam- pling (week 28), it has been observed that for the same ex- citation intensity, the fluorescence intensities of the leaves No. 11 and 12 were much higher than those of leaves No.8, but that the fluorescence ratios were the same. For compar- ison between the different nitrogen fertilised plants, the mean OkgN

160kgN

\

800nm

.£ 5

^0.5

.5

,----.

" "

0,0

Wavelength600

Fig. 2: Normalised fluorescence emission spectra of leaves of the Volga greenhouse maize and of the Dea field maize (excitation 355 nm, 400 nm long wave pass edge filter). The field maize was grown without (0kg N per hectare) or with nitrogen fertilizer (160kgN per hectare).

The first result to note is that the greenhouse Volga maize and the field Dea maize presented very different emission spectra, as illustrated in Fig. 2 where typical fluorescence curves are reported: the chlorophyll emission relative to the blue one was always more intense for the Volga maize than for the Dea maize. This behaviour is comparable to the en- hancement of the red fluorescence observed by Stober et al.

(1994) for various plants grown in the greenhouse with re- LO

Fluorescence imaging of nutrient deficiencies

625

Week in N/ha F440/F520 F440/F690 F440/F740 F690/F740 1994

Table 3: Mean values (with standard deviation) at different times in the year, of the fluorescence ratios found for the field maize with dif- ferent nitrogen fertilisation amounts.

Means for one ratio at a given week followed by the same superscript were not significantly different at the level of 5% of probability (p>0.05). The sample size was 24 leaves for weeks 24, 25 and 26, 16 leaves for week 27 and 88 leaves for the last week 28.

Okg 2.14' (0.28) 7.48'b (2.8) 5.20,b (1.7) o.n'(0.14) 60 kg 2.18^a (0.27) 8.12' (3.2) 5.52' (0.8) 0.73' (0.17) 160 kg 2.24' (0.41) 6.48b (1.8) 4.55b (1.3) 0.71' (0.14) Okg 2.54' (0.38) 9.22' (7.1) 5.71' (2.4) 0.70' (0.16) 60 kg 2.n' (0.34) 6.92' (2.0) 4.97' (2.1) 0.71' (0.15) 160 kg 2.62' (0.35) 4.74b (1.9) 3.33b (1.0) 0.73' (0.13) Okg 2.49' (0.30) 11.87' (3.4) 6.74' (1.5) 0.58' (0.10) 60 kg 1.78b (0.48) 8.46b (3.4) 5.07b (1.9) 0.61' (0.61) 160 kg 2.84b (0.37) 5.62^c (2.4) 3.4l^c (1.2) 0.63' (0.1I) Okg 2.55'b (0.34) 7.14' (2.6) 4.88' (1.6) 0.69' (0.06) 60 kg 2.78' (0.32) 3.93b(2.3) 1.78b(1.3) 0.76' (0.16) 160 kg 2.55b (0.24) 3.15b (1.3) 2.33b (0.8) 0.79' (0.19) Okg 2.54' (0.34) 6.81' (3.2) 4.78' (2.0) 0.74' (0.20) 160 kg 2.51' (0.34) 3.37b (1.6) 2.47b (1.1) 0.80' (0.44) 27(leaves

10+I I)

by a factor near to two when the nitrogen supply decreased from 160

kg

to 0 kg/ha. This feature is promising for nitrogen lack detection by fluorescence imaging. It should be noted that inside a treatment, the standard deviations associated to the mean values were much more important for the fluores- cence ratiosF440/F690andF440/F740 than for the fluores- cence ratios F440/F520 and F690/F740. Aconsequence of these large standard deviations was that in some cases, a no- ticeable variation of the mean of the bluelred fluorescence ra- tio with the nitrogen amount was found not significant by using a robust statistical treatment.

In Fig.

4

all results on the dependence of the fluorescence ratios on the nitrogen supply are presented, upon which the following comments can be made:

1) The absence of a nitrogen deficiency effect for the first set of experiments can be understood by the fact that this pe- riod of growth corresponded to the beginning of the nitrogen consumption by the plants.

2) Since the blue fluorescence emission was constant, the increase of the ratios F440/F690 and F440/F740 for nitro- gen deficient leaves corresponded to a decrease of the chloro- phyll emission at 690 and 740 nm. This decrease is not sur- prising in view of the importance of the nitrogen nutrient for the chlorophyll production and accumulation (corroborated by the chlorosis of the0kgN/ha^leaves).Asthe photosynthe- sis and the fluorescence are competitive reactions, and conse- quently have opposite effects on the red fluorescence emis- sion intensities, the observed decrease of the chlorophyll fluo- rescence emission with decreasing nitrogen supply seems to indicate that the lack of nitrogen did not strongly inhibit the photosynthetic activity of the lower amounts of chlorophyll being present in these leaves.

3) In spite of the fact that the 690 and 740 nm intensities and hence the chlorophyll concentration increased with the

24(leaves 4+5+6) 25(leaves 6+7+8) 26(leaves 8+9+ 10)

28(leaves 8+11+12) 10

• Leaf IO

• Leaf II

+

Leaf 12

• Leaf 13

=

0\'C

'- ₅

~

.,.

.,.

'-

o

kgN/ha

Leaf 10 2.67' (0.28) 7.88^a (2.87) 5.35^a (1.56) 0.70^a(0.08) 11 2.43^a (0.36) 6.40^a (2.16) 4.41^a (1.51) 0.69^a(0.04) 12 2.56^a (0.19) ·2.90^b (1.26) 2.46^b (0.77) 0.90^b(0.16) 13 2.60^a (0.32) 1.39^c (0.40) 1.25^c (0.36) 0.90^b(0.06) 60 kgN/ha

Leaf 10 2.86^a (0.30) 3.84^a (1.85) 2.88^a (1.41) O.77^a(0.14) 11 2.70^a (0.35) 4.01a (2.78) 2.68^a (1.21) 0.76^a(0.19) 12 2.83^a (0.31) 2.31ab(0.97) 2.06^a (0.91) 0.94^a(0.34) 13 2.87' (0.29) 1.34^b (0.76) U5^b (0.62) 0.87' (0.06) 160kgN/ha

2.94^ab(1.46) 2.12^ab(0.69)

Leaf 10 2.58^ab(0.21) 0.79^a(0.19)

11 2.51' (0.27) 3.36^a (1.23) 2.54^a (0.79) 0.79^a(0.20) 12 2.98^b (0.25) 2.12^b (0.75) 1.59^b (0.34) 0.80^a(0.24) 13 2.79^b (0.30) 1.34^b (0.83) 1.12^c (0.51) 0.89^a(0.12)

o

^{100 200}

N concentration (kg/ha)

Fig. 3: Influence of the nitrogen supply on the fluorescence ratio F440/F690 measured for different leaves of the e1even-week-old field grown Dea maize (see Table 2).

O+----r--'"'"T---r---,

F440/F520 F440/F690 F440/F740 F690/F740 Table 2: Mean values (with standard deviation) of the fluorescence ratios for the field Dea maize measured after eleven weeks of growth (week 27 in 1994). Each value is the mean of8leaves.

For one fluorescence ratio and one nitrogen amount the means fol- lowed by the same superscript were not significantly different (p>0.05).

values of the ratios have been calculated by taking into ac- count only the leaves with the superscript a in Table1.

The results of the experiments performed for e1even-week- old plants (Table 2 and Fig. 3), clearly show that the effect of nitrogen on the fluorescence ratios depended on the age of the leaves. The fluorescence properties of the youngest leaves had not yet been affected by the nitrogen shortage.

The overall results on the fluorescence ratios of maize leaves, are summarised in Table 3 which shows that the only fluorescence ratios which significantly varied with the nitro- gen concentration were the bluelred F440/F690 and the blue/far-red F440/F740 (the blue emission was nearly con- stant). These ratios were increased (except for the first week of experiments when no nitrogen deficiency effect was detected)

2.0 +---r---.---":--~+--~-r---,.---._L0.50

Nconcentration (kWha)

Fig.4: Variation of the fluorescence ratiosF440/F520, F440/F690, F440/F740andF690/F740versus the amount of nitrogen fertilisa- tion at different periods of growth. Results for the groups of leaves indicated in Table 3. Week 240;25

e;

26

+;

27+;and 28 • in

1994. .

not be made on the stems for the oldest leaves, the data will be presented and discussed for cut leaves sensed directly after cutting.

The three-week-old maize plants had six leaves and two weeks later there were nine leaves for the control maize and only eight in an average for the deficient plants. Fluorescence images were recorded for all the leaves except the last week (in particular for magnesium lack) when the leaves number 1 and 2 were too much stunted or tumbled down (numbering from the bottom).

A first observation which has been made is that independ- ently of the growth conditions, for the oldest leaves (in an average, number 1to 4 in the first series and up to 5 in the second series) the blue and green emissions were always higher in the main leaf vein than in the neighbouring tissue, whereas for the youngest leaves the contrast was opposite (Fig. 5A). For the red emission, as expected from the chloro- phylilocalisation, the tissue aside from the main leaf vein was always more fluorescent (Fig. 5 B). Consequently, the fluores- cence ratios values were calculated only on the leaf tissue aside the main leaf vein.

- three-week-old maize

The overall results showed that within one treatment, the fluorescence ratios could be considered as identical for leaves 3, 4, 5 and 6, and different from the ratios for leaves 1 and 2, particularly for the magnesium deficiency and in a lesser ex- tent in the other cases. So we have analysed the data in con- sidering two groups of leaves. The results are summarised in Table 4 and Fig. 6 which demonstrate that the fluorescence ratios allowed the detection of differences between control and deficient plants. For the group of leaves 3, 4, 5 and 6 the fluorescence ratios F440/F690 and F440/F740 were found to be enhanced by each deficiency, but the fluorescence ratio F440/F520 did not present any significant difference be- tween the control and the other plants. For the chlorophyll fluorescence ratioF690/F740, a small variation was observed only in the case of zinc deficiency. For the older magnesium deprived leaves (number 1 and 2), an important increase of all the fluorescence ratios was apparent: the values ofF4401 F690 and F440/F740 were ten times greater than for the other plants. For these senescent, magnesium deprived leaves we observed an increase of the blue emission and a strong de- crease of the red fluorescence which could be related to a de- crease of the chlorophyll concentration as suggested by the si- multaneous increase of the value ofF690/F740.That magne- sium deficiency essentially appeared on the first leaves of the maize plants is not surprising since it is known that the upper leaves can import their magnesium from the lower and older leaves. On the contrary, the lack of the oligoelements zinc and iron affects primarily the health of the upper leaves. It has to be noted that the magnesium deficient old maize leaves presented visible symptoms, which was not the case for the other deficiencies.

- five-week-old plants

At this stage of growth, besides the central leaf vein, alter- nation between the leaf tissue and the lateral veins could be clearly seen by eye on all the leaves. In addition, the upper iron deficient leaves are clearer than the others.Asbefore, the fluorescence ratio values were calculated in excluding only the main leaf vein.

1.00

0.75 12.0 9.0

6.0

3.0

200

F690!F740

100 200 0

100

F4401Fi40 F4401F520

o

6.0

4.0 2.5

2.0 8.0 3.0

nitrogen supply, the chlorophyll fluorescence ratio F6901 F740 of the maize leaves was almost constant. This is not necessarily in disagreement with the inverse relationship be- tween the ratio F690/F740 and the chlorophyll concentra- tion (Lichtenthaler, 1987; Lichtenthaler and Buschmann, 1987; Lichtenthaler and Rinderle, 1988; Hak et al., 1990;

D'Ambrosio et al., 1992) since this fluorescence ratio does not much change at high chlorophyll concentrations and that it was established for bifacial or equifacial leaves, whereas maize is a C₄-plant with a particular arrangement of cells and leaf veins (<<Kranz anatomy») (cf. Lichtenthaler and Pfister, 1978). Taking into account that the red/far-red ratio was con- stant, the variations ofF440/F690 and F440/F740 gave re- dundant results for these measurements.

4) Finally it should be remarked that in previous work, Chappelle et al. (1984 b, 1994) have studied the influence of the nitrogen supply on the fluorescence spectra of maize. Our results are in good agreement with their finding of the ab- sence of a nitrogen influence on the blue intensity, but for the red emission they found at once (1984 b) a decrease in the intensity for nitrogen deficient maize and later for other growth conditions (1994) a higher intensity at 690 nm for the

Okg

N/ha samples.

Mineral nutrients deficiencies

Asthe maize plants were transferred in their pots from the greenhouse to the laboratory, images have also been recorded for some leaves still attached to their stem. Results obtained for cut leaves measured immediately after cutting or after one hour waiting showed in the case of maize practically no stress effect. Since, due to the ordering of the leaves, images could

Fig. 5: Fluorescence images recorded in the blue region at 440 nm (Fig. 5 A) and in the chlorophyll fluorescence emission spectrum at 690 nm (Fig. 5 B) for leaf No.4 (right) and leaf No.6 (left) of the five-week-old greenhouse control maize. False-colour coding: in- creasing fluorescence intensity from blue via green, yellow to red.

For the analysis of the measurements, three groups of leaves were considered: the youngest (number 7 and 8, with also 9 for the control), the mature (number 5 and 6) and the senescent (number 3 and 4). Indeed, it was found that within each treatment the leaves of one group had the same fluores- cence ratios, except for the first group in the case of zinc and iron deficiencies where all the ratios were higher in leaves 8 than in leaves 7, but with a difference much weaker than the difference to the other groups. When measured (excluded for magnesium deficient plants) leaves I and 2 were not different in their fluorescence ratios from leaves 3 and 4.

Fluorescence imaging of nutrient deficiencies

627

The results are collected in Table 5 and illustrated in Fig. 7.

General findings were that (except for the magnesium deficient senescent maize leaves), i) both fluorescence ratios blue/red F440/F690 and blue/far-red F440/F740 strongly decreased from the top to the bottom of the plants, ii) in case of defi- ciency effect, these blue/red and blue/far-red fluorescence ratios were lower than for the control plants which is in contrast to what has been observed for the three-week-old plants, iii) a comparison with the data in Table 4 showed that for the con- trol, magnesium and zinc deficient leaves 5 and 6, the fluores- cence ratio F440/F690 markedly increased during growth while the ratio F690/F740 decreased. This could be due to the thickening ofthe epidermis or the accumulation ofUV-absorb- ing compounds in the epidermis from which the major part of the blue emission originates and which hinders the penetration Table 4: Greenhouse grown Volga maize. Mean values (with stand- ard deviation) of the fluorescence ratios for two groups of leaves and for different treatments. Measurements were performed three weeks after planting.

F440/F520 F440/F690 F440/F740 F690/F740 Leaves

3+4+5+6

Control 1.95'b (0.29) 0.21' (0.05) 0.17' (0.04) 0.82' (0.05) Minus Zn 2.12b (0.16) 0.29b (0.06) 0.23b (0.04) 0.78b (0.05) MinusMg 2.03bc (0.46) 0.32b (0.07) 0.26bc (0.06) 0.81'b (0.05) Minus Fe 1.98'c (0.13) 0.33b (0.07) 0.29c (0.07) 0.87' (0.10) Leaves

1+2

Control 1.68' (0.21) 0.24' (0.15) 0.20' (0.14) 0.79' (0.05)

Minus Zn 2.17 0.26 0.18 0.70

MinusMg 2.61^b (0.19) 2.09^b(1.06) 1.94^b (0.92) 0.94b (0.09)

Minus Fe 2.08 0.15 0.11 0.77

For the first group the sample size was 16 and for the second 6 for control and 4 for magnesium deficient leaves. For zinc and iron lack only 2 leaves have been measured. Inside one leaves group and one ratio, the means with the same superscript were not significantly different at the 5%level of probability (p>0.05).

ratio

Fig. 6: Mineral nutrient deficiencies effect on the fluorescence ratios for the greenhouse grown Volga maize, investigated three weeks after planting. Mean values for the group of leaves 3, 4, 5 and 6 (see Ta- ble 4). For one fluorescence ratio, different letters indicate signifi- cant differences (p<0.05).

628

FRANCINE HEISEL, MALGORZATA SOWINSKA, JOSEPH ALBERT MIEHE, MICHAEL LANG, and HARTMUTK. LICHTENTHALER

Fig.7: Histogram of the different fluores- cence ratios measured for different nutrient deficiencies of the five-week-old greenhouse Volga maize (see Table 5). The mean values of the fluorescence ratios have been deter- mined for three groups of leaves: leaves 7+ 8 (+9 when available), leaves 5+6 and leaves 3+4. For one fluorescence ratio and one leaf group different letters indicate signifi- cant differences (p<O.05).

o o

D

0.0~

I.

I

~ .oj

~ 0.5

o

• Cootr01

l ^I_

^ontrol

^:l

~ MinusZn ^f:i:l MmusZn

o

^{\nUS g} 2.0

o

^{Minus g}

mus Fe Minus Fe

I

8: ^1.0

A

2.0 0.0

s.Of

~

~B

!

^1.0

Table5: Mean values (with standard deviation) of the fluorescence ratios for the greenhouse maize leaves measured after five weeks of growth at different nutritional conditions. For the first and second groups of leaves there were four repetitions of each leaf number, for the last group the sample size was 8 for control and minus Fe, and 7 and 6 for the zinc and magnesium deficiencies, respectively.

Leaves 7+S (+9)

Control 2.94" (O.lS) O.Wb (0.19) 0.52"b (0.09) 0.5S" (0.06) Minus Zn 2.S7" (0.19) 0.95" (O.lS) 0.60" (0.11) 0.62"b (0.06) MinusMg 2.62b (0.12)

o.n

^{b (0.22)} 0.45b (0.12) 0.64^b (0.04) Minus Fe 1.Slc(0.06) 0.32^c (0.17) 0.3Sb (0.19) 1.20^c (0.09) Leaves

Control5+6 2.59" (0.09) 0.57" (0.07) 0.30" (0.05) 0.53" (0.05) Minus Zn 2.51" (0.12) 0.42b (0.16) 0.25"C (0.11) 0.59^b (0.03) MinusMg 2.2S^b(0.19) 0.63" (0.10) o.40b (0.05) 0.64^c (0.03) Minus Fe 1.65^c(0.16) 0.17^c (0.03) 0.19^c (0.02) 1.11^d (0.10) Leaves

3+4

Control 2.44" (0.20) 0.21" (0.04) 0.14" (0.02) 0.64" (0.05) Minus Zn 2.36" (0.10) 0.23" (0.07) 0.14" (0.03) 0.60" (0.04) MinusMg 2.39" (0.23) 2.41 b (1.07) 2.37b (1.11) 0.9Sb (O.OS) Minus Fe 1.73b (0.30) 0.16^c (0.04) 0.13" (0.02) 0.S5b (O.lS) For one leaves group and one ratio the means followed by the same superscript were not significantly different at the 5% level of prob- ability (p>O.05).

of the UV-A excitation light so that less red and far-red chloro- phyll fluorescence is excited in the leaf mesophyll cells.

F440/F520 F440/F690 F440/F740 F690/F740

For the two first groups of leaves, Table 5 and Fig. 7 dis- play that the most striking changes in the fluorescence ratios resulted from the iron absence which led to variations of all the ratios: the F440/F690 ratios were about three times lower and F690/F740 twice higher than in the control maize. The intensities at 440 nm were roughly the same for both treat- ments and at 690 nm the fluorescence was strongly enhanced by the iron deficiency. The higher values of the chlorophyll fluorescence ratio F690/F740 indicate a lower concentration of the chlorophyll. These results would signify an important decrease in the photosynthetic capacity that is expected for iron absence during the plant growth. A small effect of zinc deficiency could be detected only for leaves 5 and 6 and as seen in the variations of the ratios F440/F690 and F 690/

F740. For the magnesium deficient plants, a decrease of the blue/green fluorescence ratio and an increase of F690/F740 with respect ^to the values measured for the control has been observed with an amount of only 10-15%.

For the old leaves (3 and 4), the iron deprived leaves pre- sented also a clear effect on the fluorescence ratios F4401 F520 andF690/F740,but the most important variation was observed for the magnesium deficient plants for which an in- crease of the ratiosF440/F690andF440/F740by a factor of more than ten was observed with practically the same values as those measured in the first series of experiments. The zinc deficiency, which was already difficultto detect in the earlier growth stage of maize plants did, in the senescent leaves, not lead to changes in the fluorescence ratios as compared^to the control plants.

Table 5 and Fig. 7 also show that the deficiencies could al- ways, with more difficulty for zinc, be distinguished by con-

sidering the amount of the variation of the ratios, which var- ied in a differential way.

Some of our results have to be compared to those obtained by Chappelle et aI., 1984 b, who also studied some nutrient deficiencies on greenhouse maize, although their plants were seven weeks old. In case of iron absence they found, as dem- onstrated here for three-week-old maize plants, a considerable increase of the ratio F440/F690. For magnesium deficiency they did not observe any effect.

Finally, it has to be emphasized that the mean values of the fluorescence ratios, determined for practically the whole tis- sue surface, were only little sensitive to the zinc deficiency;

the latter could only be detected by making use of the spatial distribution of the fluorescence intensities on the images. In- deed, the fluorescence was roughly uniformly distributed on the leaf tissue of the control and magnesium deficient leaves, which was not the case for the zinc deficient two penultimate leaves. For the latter the fluorescence images showed the pres- ence of oblong area practically parallel to the main vein, with more blue and less red fluorescence emissions than on the average of the leaf surface (there was no visible symptom for the three-week-old plants). These fluorescence intensity inho- mogeneities were enhanced in the images resulting from the fluorescence ratio pixel by pixel of the images recorded at 440 and 690 nm. An example is given in Fig. 8A. Another out- standing feature to note is that for the five-week-old iron deprived plants, showing practically uniform blue emission, apparent streaks were observed on the images for the red fluorescence. These corresponded to the alternation between the lateral veins and the tissue with less intensity for the veins, while the blue emission was more uniform. This is il- lustrated by Fig. 8 B which represents the F440/F690 image for iron deficient leaves number 6 and 7.

Conclusion

This work has demonstrated in the case of maize plants, that the detection of laser-induced fluorescence of leaves with a very sensitive and high spatially resolved imaging system al- lows to make evident all the nutrient deficiencies we have in- vestigated.

The difference in nitrogen fertilisation amounts was de- tected only by the blue/red and blue/far-red fluorescence ra- tios and our results emphasized the influence of the growth stage on the fluorescence ratios values and the importance of the age of the leaves.

Concerning the mineral deficiencies for the non-senescent leaves, the iron deficiency resulted in an important difference in the fluorescence ratio values between deficient and control plants. The magnesium and zinc deficiencies were also appar- ent in the change of the fluorescence ratios, but to a lesser ex- tent and these could be discriminated only with difficulty.

Complementary results allowing discrimination between the deficiencies were obtained by taking advantage of the spa- tial resolution of the fluorescence imaging system. The con- trol and magnesium deficient leaves showed uniform distri- bution of the emitted fluorescence over the leaf surface. For zinc deficiency the fluorescence images of mature leaves pre- sented characteristic oblong area with fluorescence intensities

Fluorescence imaging of nutrient deficiencies 629

Fig.8:Fluorescence rario imagesF440/F690offive-week-old green- house maize plants: A: control (leEr) and zinc deficient (righr) leaves No.6, B: iron deficient leaves No.6 and 7 (from leEr ro righr). False- colour coding: increasing fluorescence intensity from blue via green, yellow ro red.

different from those observed on a average of the leaf surface.

In the case of iron absence, regularly distributed streaks ap- peared on the red fluorescence images of leaves of five-week- old plants.

These results are encouraging for future work on remote sensing of nutrient deficiencies by field measurements. It should be noted that with this new fluorescence imaging sys- tem, equipped with a telescope in front of the camera, well spatially resolved images can be and have been obtained at distance of up to 100 m applying a more powerful laser than the one described here.

Acknowledgements

The authors are grateful to Mrs. V. Lombaert of the Centre de Recherches de la Societe Commerciale des Potasses et de l'Azote (SCPA, Aspach Ie Bas, France) for growth and care of the green- house Volga maize, and to Mrs.V. Gourbeau and Mr. F. Merkling of the Lycee Agricole d'Obernai (France) for placing at disposal the land parcels used for nitrogen supply testing, as well as for fruitful discussions and help in defining the experimental procedure.

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