SPIE Proceedings [SPIE AeroSense '97 - Orlando, FL (Monday 21 April 1997)] Advances in Laser Remote Sensing for Terrestrial and Oceanographic Applications - Laser-induced fluorescence

Laser-induced fluorescence imaging for monitoring nitrogen fertilisingtreatments of wheat

Francine Heisel", Malgorzata Sowinskaa, Elisabeth Khalilib, Caroline Eckert",Joseph A. Miehé" and Hartmut K. Lichtenthaler"

aGroupe d'Optique Appliquée, Centre de Recherches Nucléaires, 1N2P3/CNRS, 23 rue du Loess,F 67200 Strasbourg, France

de Recherche Mathématique Avancée, Université Louis Pasteur et CNRS (URA 01),7 rue René Descartes, F 67000 Strasbourg, France

Institut II, Universität Karisruhe, Kaiserstr.12, D 76128 Karisruhe, Germany

ABSTRACT

The laser-induced fluorescence imaging system allows the recording of spectrally selected fluorescence images ofthe whole leaves or plants which is better and in contrast to the so far applied spot spectrofluorometer measurements. Thefluorescence images of leaves of winter wheat (Soissons variety, Alsace) have been recorded at the four characteristicemission bands (440, 520, 690 and 740 nm) with a high resolution imaging device consisting of a frequency triplet ordoubled Nd:YAG source for 355 nm or 532 nm excitation and ofan intensified and gated CCD digitized camera. The effectof four different nitrogen treatments (0, 100, 140 and 180 kg/ha) on the fluorescence parameters (intensities F440, F520,F690, F740 and ratios F4401F520, F440/F690, F4401F740 and F690/F740) obtained by image processing has been analysedby statistical treatment, in a randomised blocks experiment. The measurements have been carried out on two leaf storeysweekly gathered during two months (May and June 1 996). For 355 nm excitation, a significant decrease of the fluorescenceratios F4401F690 and F4401F740 was observed for increasing nitrogen concentration: the blue and green mean fluorescenceintensities remained much the same, while the red and far-red chlorophyll fluorescence emissions were enhanced by thefertilisation. The fluorescence results are in excellent correlation with the crop yields.

Keywords: wheat, plant stress, nitrogen fertilisation, fluorescence imaging

1. INTRODUCTION

When illuminated by 1W-light, green plants emit a broad fluorescence emission with maxima or shoulders in the blue(F440) and green (F520) spectral region as well as the red and far-red chlorophyll fluorescence with maxima near 690 nm(F690) and between 730 and 740 nm (F740) 17 The blue and green fluorescence emissions of green leaves primarilyemanate from the cell walls ofepidermis cells and leafveins and is due to ferulic and p-coumaric acids which are covalentlybound to the cellulose 6,8 and possibly to flavonols and other phenolic or non-phenolic compounds 2in the vacuole. Thechlorophyll fluorescence, in turn is emitted by chlorophyll a in the chloroplasts ofthe green mesophyll cells .The examination of the various forms of plant stress by applying different chlorophyll fluorescence techniques is wellestablished 9,11,12 The chlorophyll fluorescence ratio redlfar-red (F690/F740) proved to be a non-destructive indicator ofthe in situ chlorophyll content, whereby this fluorescence ratio is inversely correlated to the chlorophyll content 9,13,14 jalso changes during nitrogen deficiency, e.g. in barley leaves when the fluorescence was excited by a Ne/He laser of 632.8nm j recent years the blue (F440) and green (F520) fluorescence have been included in the fluorescence spectroscopicinvestigations 4,1617 or corn grain 18•Here we report on the application of a UV-laser-induced fluorescence imaging system, which allows to screen fluorescenceimages ofleaves or plants 1922, the investigation ofwheat plants grown with different amounts ofnitrogen fertiliser. Theaim of this research was to check whether N-deficiency as medium or high N-supply can be differentiated via the highresolution fluorescence imaging technique.

F.H. (correspondence), M.S., C.E., J.A.M.: Email: Eckertcrn.in2p3.fr; Phone: 33 3 88 10 66 51; Fax: 33 3 88 10 65 48E.K.; Email: Khalilimath.u-strasbg.fr; Phone: 33 3 88 41 63 27; Fax: 33 3 88 61 90 90;H.K.L.; Email: [email protected]; Phone: 49 721 608 3833; Fax: 49 721 608 4874

10 SPIE Vol. 3059 • 0277-786X1971$10.00

Downloaded From: http://proceedings.spiedigitallibrary.org/ on 06/12/2013 Terms of Use: http://spiedl.org/terms

2. MATEfflAL AND METHOD

2.1 Conditions of plant growthField winter wheat (Triticum aestivum L, var. Soissons), growing in experimental parcels cultivated by the ITCF (InstitutTechnique des Céréales et des Fourrages) for nitrogen fertilisation tests, was planted in October 1995, at a density of 400plants/rn2 jj alluvial argillaceous soil situated at Obernai (Alsace, France). There were four replicate areas for each mode ofnilrogen supply in a randomised block design (Fig. 1). LW experiments were devoted to the comparison ofthe spectroscopiccharacteristics ofthe leaves from wheat growing with a total added amount of 0, 100, 140 and 180 kg N/ha (ashen zones).The supply of 140 kg N/ha is considered optimal for satisfying the needs of wheat in the Obernai fields which contained aresidue near 35 kg N/ha. Nitrogen was supplied in the ammonitrate form according to the time schedule given in Table I.No other nutrient was furnished, the natural mineralisation in the parent soil was considered as sufficient.

Block IV

Block III

Block II

Block I

Fig. 1: Schema of the wheat field. Each parcel is arectangle of 9.80 m x 3.15 m. The dose X is therecommended supply equal to 140 kg N/ha for this field.

2.2 Laser induced fluorescence imaging set-upThe laser induced fluorescence imaging system

(already described 1922 ) which allows scanning of theemission intensity distribution over the whole surfaceof the sample under study and due to its highresolution to detect small inhomogeneities or gradientsin the emission, is represented in Fig. 2. The excitationsource was a Q-switched Nd:YAG laser (Spectra-Physics Bl0-355Q) pumped by a diode laser andemitting, after third harmonic generation, 355 nm lightpulses of 10 ns width and with a repetition rateadjustable up to 15 kHz. At 10 kHz, the commonlyused frequency, the pulse energy was equal to 1 5 pJ.Excitation at 532 nm with a pulse energy of 45 iJ canalso be provided by using the second harmonic of thelaser. The excitation beam was directed onto thesample at right angle and shaped by a divergent lens toirradiate the complete sample surface.

Table I: Dates of the nitrate supplies. These datescorresponded respectively to the stages 21 and 30 onthe Zadoks scale.

kg of N

applied

NITROGEN SUPPLY (kg/ha)

February 27, 1996

Tilling

April 16, 1996

0 0

X-40 50 50

X 50 90

X+40 50 130

Fig. 2: Scheme of the laser-induced fluorescence imagingsystem (LIF system).

11

HEHHERHEHEHH


12

The fluorescence imaging system (RAGM6 + Animater Vi , ARP, Strasbourg, France) was composed of i) a gatedintensified digital CCD camera operating at 50 frames per second and including all the electronic CCD's readout with 8bits digitisation per pixel of the image due to one frame; the CCD array (Thomson TH 7863) is composed of 288 x 384elements; the gated intensifier (Philips XX1414MIE image intensifier tube) has an adjustable gain (up to iO) and thewidth of the laser synchronous gating can be varied from 10 ns to 1 00 ms. The gating is necessary for experiments in thepresence of ambient light to avoid a possible saturation of the camera by this light and to increase the signal (fluorescence)to noise ratio ii) an interface card for the PC microcomputer where the images are stored iii) an image analysis software.After each digitisation, the result is transferred to the interface card where an addition of successive readouts can be madeto 16 bits (0-65,535), followed (or not) by the subtraction ofthe noise due to the camera itselfand to the ambient light. Theresulting image is then automatically sent in the extended memory of the PC. Besides the real-time visualisation and thestorage of the images, the software allows fast recall and treatment of images, their number (maximum 99) depending onthe size ofthe free extended memory (0.23 Mbyte per image).A filter wheel is situated in front of the intensifier with the possibility ofusing six different filters (filter diameter equal to25 mm).

2.3 ExperimentalMeasurements were performed in the laboratory on the two upper completely developed leaves, the same day as thecollection in the field, and once weekly from beginning of May to mid June 1996 (a week after the flowering). At eachdate, 20 ofthe last formed leaves (labelled No 1) and 20 leaves next to them (labelled No 2) were randomly plucked fromeach ofthe four blocks for each nitrogen concentration.LIF measurements were performed using a 355 nm excitation. The fluorescence images of the leaf adaxial side weresystematically recorded through interference filters (10 nm bandwidth) centred at 440, 520, 690 and 740 urn and wereacquired, at an angle of 300, j a distance of 50 cm via a focusing lens on the entrance of the intensifier. For thisdistance the height of the image corresponded to 20 cm at the leaf level (one pixel represented 0.25 mm2 on the leafsurface). All recordings were made in the presence of an additional white light, which simulates day light so that themeasured intensity of the chlorophyll fluorescence emission corresponded to the light adapted steady state of thechlorophyll fluorescence induction kinetic (Kautsky effect). The intensifier was working with a 40 ns gate width, a timelong enough to collect the complete leaf fluorescence and short enough to allow measurements in the presence of the whitelight. In general 200 (depending on the fluorescence intensity) successive readouts were accumulated followed by asubtraction of the same number of readouts in the absence of the laser beam (subtraction of the ambient light). Thisaccumulation allows to ameliorate the statistics.It has to be noted that the laser intensity at the leaf level was the same for all the measurements so that the variations ofthevarious LIF intensities from one date to another were actually due to variations of the leaf fluorescence yields.For behaviour comparison, chlorophyll fluorescence images have been recorded in some cases for an excitation at 532 nm.

2.4 Images treatment and statistical analysis of the resultsThe images were corrected for the non uniformity of the laser excitation, for the spectral sensitivity of the camera,including the attenuation factor of the interference filters and of the focusing lens. Fluorescence ratios imagesF*(440/520), F*(440/690), F*(440/740) and F*(690/740) were formed by a pixel to pixel division procedure. As thefluorescence images or ratios images did not show distinct zones on the leaf surface (Fig. 3), the fluorescence intensities orintensities ratios values were determined for each leaf at the four emission wavelengths or for the four ratio images byaveraging over the largest leaf area possible. This averaging has been performed by means of the Animater ARP softwarewhich allows selection of rectangular regions of adjustable size or with a more powerftul software (Optimas Imasys). Withthis last one, the contour of the area of each leaf was determined by using a smoothing and threshold procedure toeliminate the noise region in the image. This contour or the rectangular region used with Animater was always selected onthe F440 images and was then applied to all the fluorescence images or ratios images corresponding to the same leaf. Testshave been made to compare the data obtained by both methods and to examine a possible influence of the threshold value.For the LIF ratio values, we have compared the means of the fluorescence ratios F440/F740 determined for each leaf fromthe intensities images F440 and F740 to the means of the ratio values F*(440/740) measured on the ratio images (in thefollowing, the sign means that we consider fluorescence ratio images).Statistical treatments were then applied to the results, like determination of the correlation between the intensitiesmeasured at the various wavelengths, two ways variance analysis for testing the significance of the differences due to thevarious nitrogen supplies or to a block effect.


Fig. 3: Fluorescence images of three leaves recorded at 440, 520, 690 and 740 nm and fluorescence ratio imagesF*(440/520), F*(440/690), F*(440/740) and F*(690/740) calculated by a pixel to pixel division procedure. The gradualblack to white scale color coding corresponds to increasing intensity or intensity ratio. From one image to another thescalar coding is not the same.

3. RESULTS AND DISCUSSION

Fig. 4 shows, for an excitation at 355 nm, the sampling distribution functions of the intensities F440, F740 and of the ratioF440/F740 (with the Animater software) for the first series ofmeasurements (May 2, 1996), for leafNo 1 and leafNo 2. Ascan be seen these distributions are smooth along the whole curves. For both leaf storeys, the F440 distribution curves did notallow a distinction between the four nitrogen fertilisation rates. The distributions of the chlorophyll emission intensity F740and of the fluorescence ratio blue/far-red F4401F740 were continuously and markedly shifted by an increase of the nitrogenconcentration for the upper leaf. For leafNo 2 the results obtained in the case of the three nitrogen supplies were not clearlydistinguishable, only the parcels without nitrogen addition gave different results. As will be shown later, the leaves No 1were not always the most sensitive to the nitrogen fertilisation rate: the leaf behaviour changed during the measurementsperiod.In Fig. 5 is represented an overall view of the results of May 2 obtained for the last formed leaves. This figure which is atypical example of the tendencies observed at each date of measurements and also for leaves No 2, illustrates that the blueand green fluorescence intensities were practically independent of the nitrogen supply, whereas the chlorophyll emissioncontinuously increased with the applied nitrogen concentration. In contrast to the blue/green and the red/far-redfluorescence ratios, which were insensitive to the fertilisation rate, the blue/red and blue/far-red fluorescence ratiosdecreased for increasing nitrogen amount. Since the ratio blue/far-red was found the best nitrogen deficiency indicator, wehave focused our data analysis on the F440, F740 and blue/far-red fluorescence ratio values F440/F740. Yet, thefluorescence ratio blue/red is also a suitable indicator of fertilisation.

3.1 Comparison of results obtained with different image analysis proceduresIn Table II are compared, for two dates of measurements and for the upper leaves, the mean values (80 leaves) of the LIFintensities F440 and F740 obtained with the Animater ARP software (rectangular region selected on the leaf fluorescenceimages) or with the Optimas Imasys software for different threshold values. In all cases the region of interest wasdetermined on the F440 images. As can be seen, the different image analysis methods gave similar results. It is clear thatif the threshold is increased, the means of the blue intensities are also enhanced but in a moderate way, except as it couldbe expected, when the threshold was too high: compare in particular the values obtained on the June 11 with 1800 or 3000as threshold (due to the low F440 intensity at this date, it was not possible to operate with a threshold equal to 5000). Theordering of the values from one treatment to another was maintained and the statistical significance was comparable.

13


14

Fig. 4: May 2, 1996 measurements: experimental distribution functions (cumulative probabilities) of the fluorescenceintensities F440, F740 andofthe fluorescence ratio F4401F740 for leaves No 1 (n =80) andNo 2 (n = 80).

0 4000 8000 12000 16000Fluorescence intensity F440

0 4000 8000 12000 16000Fluorescence intensity F440

1.0

0.8

0E 0.60

0.0.2

0.0

1.0

0.8

00.6

0

0.0.2

0.0

1.0

0.8

00.6

0C0

0.2

0.0

1.0

0 0.8C

00.6

0C

0I.

0.2

0.0

1.0

0 0.8C

00.6

0C0.C

0.2

0.0

1.0

0.8C

00.6

0C

0

0.2

0.0

0 2500 5000Fluorescence intensity F740

7500 0 2500 5000Fluorescence intensity F740

7500

0 2 4F440/F740

6 8 0 2 4F440/F740

6 8


. . . 2000 , C 54OO4G00 A . AR BC

20:I[11"""""1 rTIII 250:1r"r1"""""1

A A A A— _-.% I

jTli:J4. :.r::.:;:::::: B

[Lti"". i .t1_".

Fig. 5: Mean values of the different fluorescence intensities or fluorescence ratios values measured on May 2, 1996 on80 leaves No 1 for each nilrogen supply (20 leaves per block). The nitrogen supply was from left to right equal to 0, 100,140 and 1 80 kg/ha. For one fluorescence intensity or ratio, means with the same letter are not significantly different byNewman-Keuls (a = 0.05).

The results of the far-red fluorescence emissions showed the same behaviour, that means that in the leaf area with ahigher blue emission than in an average over the whole leaf, the chlorophyll emission was also higher. Hence, theblue/far-red ratios should be practically independent ofthe way of determining the leafarea of interest.Table II in which are also given the means (n = 80) of the fluorescence ratio values calculated for each leaf from the F440and F740 intensities and the means determined from the LIF ratio (pixel by pixel division) F*(440/740) images shows thatthe fluorescence ratio means were effectively roughly insensitive to the regions selected on the leaf area, even in the caseofa very high threshold (for example, =3000 for the measurements ofJune 1 1).

Table II: Means (n = 80) of the blue (F440) and far-red (F740) fluorescence intensities and of the blue/far-redfluorescence ratios determined by various image processings, for the upper leaves (No 1) and at two dates. In one cell ofthe table, means with the same letter are not significantly different byNewman-Keuls (a =0.05).

Leaf No 1May 2, 1996

N supply(kg/ha)

Mi-mater

Optimas Threshold1800 3000 5000

Leaf No 1June 11, 1996

N supply(kg/ha)

Ani-mater

Optimas Threshold1800 3000

F440 0

100

140

180

7836a

7496a

7582a

7353a

7080a

7088a

7145a

6659a

75-79a

7246a

7514a

6983a

8691a

8321a

8467a

8231a

F440 0

100

140

180

2668ab

2819a

2697ab

2540b

2601a

2792b

2627a

2549a

3667a

3634a

3611a

3611a

F740 0

100

140

180

2061a

2625b

2810b

3091c

1860a

2384b

2522bc

2679c

1929a

2431b

2647c

2774c

2146a

2710b

2919c

3222d

F740 0

ioo

140

180

2979a

4031b

3904b

4010b

2879a

3894b

3713b

3905b

3975a

5103b

5027bc

5533c

F440/F740

FromLIFIntensities

Images

0

100

140

180

3818a

2919b2711b2485c

3817a

3034b2848b2561c

3955a

3035b2873b2621c

4148a

3166b2970b2699c

F440/F740

FromLIFIntensities

Images

0

100

140

180

0910a0717b0708b0•655c

0939a0739b0741b0686b

0979a0769b0783b0•706b

F*(440/740)FromLIFRatio

Images

0

100

140

180

4285a3446b3204b2914c

4374a3364b3164b2g84c

4501a3419b3196b2899c

F*(440/740)

FromLIFRatio

Images

0100140

180

1074a0835b0.833'

0•761b

1082a0832b0847b0•773b

15

00OO A A A A

°°:

A A A


Another observation was that the mean values of the fluorescence ratio (also the fluorescence ratio value for each leaf)were always a little higher when measured on the fluorescence ratios images than when determined from the intensitiesimages. Similar findings have been established for the different periods of measurements. A general outcome from allthese comparisons was that whatever the procedure used to evaluate the different LIF parameters, comparable results wereobtained concerning the separation between the different nitrogen fertilisation rates.

3.2 Correlation analysisThe fact that the fluorescence ratio values did not depend on the threshold, and were the same in all the leaf zonesindependently of the intensities of the blue emission, suggested that, at least for one leaf storey and one nitrogenfertilisation rate and at one date, there is a strong relation between the blue and the red intensities. To vthfi thisconclusion, we determined the correlation factors between the various characteristic emission intensities. Some examplesare given in Table III and illustrated in Fig. 6.

Table ifi: Values of the correlation factor r between the various fluorescence emissions (n is the number of leaves)

May2,1996 June111996

May2+June11

LeavesNo 1

LeavesNo 1+2

LeavesNo 1+2

LeavesNo 1+2

Correlationfactor r

0 kg N/ha(n = 80)

100 kg N/ha(n 80)

140 kg N/ha(ii 80)

180 kg N/ha(n = 80)

all parcels(n=320)

all parcels(n640)

all parcels(n=640)

all parcels(n1280)

F440 - F520F440 - F690F440 - F740F690 - F740

0.880.700.630.91

0.900.560.560.83

0.900.780.790.87

0.870.680.650.95

0.880.570.520.92

0.910.530.450.93

0.760.470.510.94

0.93-0.26-0.170.95

16

The first result was that for leaves No 1 , leaves No 2 or leaves No 1 + 2 the correlation factor between F440 and F520(r > 0.65) and between F690 and F740 (r > 0.85) was always very high not only inside one treatment but also if the datafrom all the parcels were considered. That is also true even if results from different dates are taken together. Thecorrelation between the blue and far-red (or red) was significant with a factor higher than 0.4, provided that only one dateof measurements was considered.

Fig. 6: Correlation of the far-red fluorescence intensities F740 versus the blue fluorescence F440 and versus the redfluorescence F690. Experimental points are those of all leaves No 1 (4 treatments) taken at two different periods (n =320 ateach date). On May 2, 1996 the correlation factors r were equal to 0.52 between F740 and F440 (linear regression curve A)and 0.92 between F740 and F690. On June 1 1, 1996, these factors were respectively equal to 0.43 (line B) and 0.93.Straight line C is the regression curve for the two series ofmeasurements taken together (n =640, r = 0.93).

x

10000 -0 Experimental May 2, 1996

100000 Experimental May 2, 1996

'1 X x Experimental June 11, 1996 X Experimental June 11, 1996

x

:50

0I

:

10000 20000Fluorescence intensity F440

5000

0 2500Fluorescence intensity

5000F690

0

>K


3.3 Block effectAs for each nitrogen treatment we had four plots, our statistical analysis included tests on the block factor. We have found ablock effect practically at each date of measurements but it was not possible to deduce any general behaviour: at one date,the variations of the blue intensity from one block to another were not related to those of the chlorophyll emission.Furthermore, it was not always the same block which gave results strongly different from the others. Fig. 7 shows someresults illustrating such an unstable behaviour.

I OBlocki t3BIock II IlIBlock Ill OBlock

4 ..U F740 F4401F740

ioo T

I &fl B—coo %

::fl IiftI1[rIi4ay7 May.21 May28

Fig. 7: Influence of the block on the results at various dates of experiments. Each intensity or ratio value is the mean for80 leaves No 1. At one date, means with the same letter are not significantly different by Newman-Keuls (a =0.05).

3.4 Nitrogen fertilisation effectThe results obtained during the whole period of measurements are summarised in Fig. 8 for the blue and far-redfluorescence intensity means and for the blue/far-red fluorescence ratio means. The values reported in this figure aremeans averaged over the four replications. As already noted, no dependence has been found between the fertilisation rateand the blue emission intensities as well for leaf No 1 as for leaf No 2, whatever the date of experiments, whereas thechlorophyll fluorescence was enhanced by the nitrogen supply: the wheat parcels without any nitrogen providing werealways highly different from the others. The F440/F740 ratio appeared as a better indicator of the nitrogen fertilisationrate: even if the differences were not always statistically significant, it continuously decreased for increasing nitrogenconcentration without any inversion for the three added amounts. Comparison of the results for the two different leafstoreys showed that leaf No 1 was more sensitive to the fertilisation at the beginning of our experiments, whereas fromMay 28 the best discrimination was obtained for leafNo 2.With a more powerful laser and with a more sophisticated excitation and collection optics, the LIF imaging system can bedeveloped for in situ remote sensing of the vegetation. In such conditions of image recording, it will be practically notpossible to separate the different upper leaf storeys and it was important to test if in this case the various amounts ofnitrogen led also to significant differences. For field measurements, only evaluation of fluorescence ratios are adequatesince fluorescence intensities are strongly dependent on external parameters (distance, foliage geometry...) difficult tocontrol. The results of the analysis applied to populations with leaf No 1 and leaf No 2 taken together also presented inFig. 8 for the F440/F740 ratio, showed that even in this case the ratio was continuously decreasing for increasing amountsof nitrogen and that the significance of the results was as good as for separate samples of leaves.It is interesting to note that the crop yield was respectively equal to 7265, 9067, 9208 and 9351 kg/ha for the supply of 0,100, 140 and 1 80 kg N/ha, with a significant difference only when there was no nitrogen addition.

17

May7 ay 2.1 May 28 May7 May 21 May28


1GRiOF440

B

Iit 1..iit ñ:in i'rAActoo4H r ti tttt

0H1 Jill Jill I nivrrtr.May2 May7 MyI4 May21 May28 June4 June11

6O) Leaf No I

41m BB

29n:f1 ill J:.

May2 May7 %layl4 May21 May28 June4 June11

F440/F740

:hF440/F740

Leaf No I

F740 Leaf o 26()()O

4Ø)! BBB

2000 jfl1j"fl"

1av2 1ay7 MayW May21 Ma2S June4 Junel I

F440 F740

18

4 AF1!1 k1 1BB

Leaf \o I2

2H M1N-1 t1j MI 11 IBBB(B(:ii ill JI I liii JIi"i ,iri1,

3.5 Comparison of the chlorophyll fluorescence results under 355 and 532nm excitationTill now, currently investigations for plant status characterisation by stationary fluorescence properties have beenperformed by directly exciting the chlorophyll and measuring the fluorescence intensities at the two peak wavelengthsF690 and F740 and their ratio. From a practical point of view, it was interesting to examine the red F690, the far-redF740 intensities and their ratio F690/F740 under 532nm excitation, in order to check if these parameters were moresensitive to the fertilisation rates than for 355nm excitation. Results for May 2 1 are compared in Table IV and show thatthe enhancement of the intensities at both wavelengths for increasing nitrogen amount was the smallest for the 532nmexcitation (same observation could be made at other dates) and that as already indicated for 355 nm excitation there wasno effect of the fertilisation rate on the fluorescence ratio determined for 532 nm excitation. As for green excitation thechlorophyll fluorescence intensities were roughly the same for the different populations (at least for the three ones with anaddition of nitrogen) and since the red/far-red fluorescence ratio was constant, it could be assumed that the chlorophyll

Leaf No I1•440

5000

0

Leaf No 2

AAt.t AAtA—

::: AAAA B

IfIlflhItriL•June4 June11Mzy7 ayi4 y21 May28

4

May2 Ma7 1ay14 May21 May28 June4 June110

ay2 May7 May14 May21 May28 Juue4 Juuel.1

I._ 909!0 140 0 180

i2 May7 May14 tay2i. Mny28 June4 June11Fig. 8: Overall results obtained during the whole period of measurements for leaves No 1 and leaves No 2 (at each date,n = 80 for each leaf storey and treatment: 0, 100, 140, 180 = the kg amounts ofN applied). At one date, means with thesame letter are not significantly different by Newman-Keuls (a =0.05).


concentration (perhaps a little smaller in the absence of fertiliser supply) did practically not depend on the amount ofadded nitrogen and that there was no noticeable inhibition ofthe photosynthetic activity.The inference that the leaf chlorophyll concentration was the same for 100, 140 and 180 kg N/ha supply can be correlatedto the red brightness data from aerial photographs of maize field showing no difference for nitrogen supply equal orgreater than the fertiliser recommendation 23 and to the ear leafrefiectance measurements giving the same values for highor medium level ofnitrogen and a higher reflectance for low N level 24

Table IV: Comparison ofthe F690 and F740 intensities means (n = 80 for each mean) and ofthe fluorescence ratiosF690/F740 evaluated for 355 and 532 nm excitation. Between both excitations, absolute values of the intensities arenot comparable since they are not corrected for the laser beam intensities. For one leaf number and one fluorescenceparameter means with the same letter are not significantly different by Newman-Keuls test (a = 0.05).

May 211996

LeafNo 1 LeafNo 2Excitation 355 nm Excitation_532 nm Excitation 355 nm Excitation 532 nm

N supply(kg/ha)

F690 F740 F690!F740

F690 F740 F690/F740

F690 F740 F690/F740

F690 F740 F690/F740

0100

140

180

1600a

2021b

1996b

2154c

2838a

3359b

3336b

3634c

0566a

0600b0598b0594b

2986a

3131a

3268a

3292a

5311a

6343b

5979b

6098b

0569a0502b0550a0547a

1628a

2275b

2230b

2746c

2687a

3741b

3684b

4357c

0610a0607a0606a0631a

3284a

3471a

3480a

3395a

6362a

7169b

7135b

6996b

0547a

0495a

0495a0550a

The observation of a more pronounced nitrogen influence on the F690 and F740 intensities under 355 nm excitationseemed to indicate that this effect was due to modifications of the absorption properties of the leaves (jenetration depth,chlorophyll fluorescence reabsorption) or of the efficiency of the energy or electron transfer to the chioroplasts, reactionwhich would be inhibited by a nitrogen deficiency.

3.6 Development during the growth periodFig. 9, which visualises the variation with time ofthe fluorescence parameters, shows that on an average the blue intensityregularly decreased from May 14 (the decrease began only in June 1996 for leaf No 2), whereas the chlorophyllfluorescence emission continuously increased up to the end of May 1996. It has to be noted that no new leaf was formedafter May 7. The fluorescence ratio F440/F740 decreased more rapidly from the middle of May 1996 than during the threefirst weeks of the measurement period. Except for the first measurements (May 2) when it was near to 0.5, the F690/F740ratio was practically the same ( 0.6) for all the other measurements and for both leaf storeys. It can be observed that thevariation during growth of the fluorescence yields and of the fluorescence ratio was comparable for the various nitrogensupplies.

0 Ma2 0May' El Ma14 0 May21 0 Ma%28 DJune4 •Juncll1996

Fig. 9: For each treatment, variation during the period of measurements of the mean fluorescence intensities F440 andF740, and ofthe mean fluorescence ratio F440/F740 for leaves No 1 and 2 taken together.

19

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F44WF740

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4. CONCLUSION

The recording of LIF images of wheat leaves in the blue fluorescence emission region and in the chlorophyll (red or far-red) fluorescence peaks allowed to make evident the influence of the nitrogen supply on the plant status. The differencebetween the speciroscopic characteristics due to the various treatments was detectable since the first day of experiments.The best indicator was found to be the fluorescence ratio F440/F740 which decreased for increasing nitrogenconcentration and that without any inversion between the treatments during the whole period of measurements (the ratioF440/F690 showed the same behaviour). This is an important result to assert the reliability ofthe method. It should also benoted that this work corroborates the findings of a preliminary study undertaken in 1995, in an earlier period of plantgrowth, on the same variety ofwheat planted in an other experimental field 25•[i an earlier study on maize leaves, withthe same experimental conditions and set-up, we had already found that nitrogen deficiency did not modify the blueintensity but induced an increase of the fluorescence ratio blue/red 22,jj agreement with the first results published on thisfield by Chappelle et al. 26and opposed to their more recent results 27•The data of the chlorophyll fluorescence obtained at a 355 and a 532 urn excitation showed that the chlorophyllfluorescence emission intensities were little sensitive to the nitrogen deficiency in the case of green irradiation, indicatingthat an other effect than a decrease of the chlorophyll concentration might be responsible for the noticeable variation of thered emission intensity with the nitrogen fertilisation rate observed for UV-A excitation.Continuation of this work is planned but on leaves in situ by remote recording of fluorescence images with a newdeveloped LIF imaging system installed in a mobile vehicle and with UV-A excitation. Experiments will begin at anearlier stage of growth to determine from which plant age the lack of nitrogen can be detected. The ultimate goal of suchwork is to validate the LIF ratios blue/red and blue/far-red as indicators for nitrogen fertilisation as early as possible inorder to optimise the management ofthe nitrogen fertiliser application.

ACKNOLEDGEMENTS

The authors are grateful to M D. Lasserre and M. Remuaux of the Institut Technique des Céréales et Fourrages, Colmar,France, for placing at disposal the land parcels used for nitrogen supply testing, as well for help in defining theexperimental protocol.

REFERENCES

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SPIE Proceedings [SPIE AeroSense '97 - Orlando, FL (Monday 21 April 1997)] Advances in Laser Remote Sensing for Terrestrial and Oceanographic Applications - Laser-induced fluorescence