Laser-induced fluorescence of green plants. 2: LIF caused by nutrient deficiencies in corn

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Laser-induced fluorescence of green plants. 2: LIF caused by nutrient deficiencies in corn

Emmett W. Chappelle, James E. McMurtrey III, Frank M. Wood, Jr., and W. Wayne Newcomb

The effects of nutrient deficiencies on the laser-induced fluorescence spectra of intact corn plants were stud- ied to determine the utility of the LIF technique as a field and remote sensing tool for detection of nutrient deficiencies. A pulsed nitrogen laser emitting at 337 nm was used as the excitation source. The fluorescence maxima in corn were at 440, 690, and 740 nm. A decrease in fluorescence at 690 and 740 nm was observed for those plants deprived of phosphorus, nitrogen, and iron. The absence of nitrogen and iron also caused a small decrease in fluorescence at 440 nm. Plants deprived of calcium, sulfur, and magnesium showed no significant change in fluorescence at any of the bands. The lack of potassium increased the fluorescence at 690 and 740 nm more than threefold along with a small decrease at 440 nm.

1. Introduction

Nutrient deficiencies in plants eventually manifest themselves through impaired and abnormal growth along with other visible symptoms characteristic of the particular deficiency. Plants require at least sixteen elements in large to trace amounts including carbon, hydrogen, oxygen, nitrogen, phosphorus, potassium, calcium, magnesium, boron, sulfur, iron, zinc, chlorine, copper, manganese, and molybdenum. Absence or an insufficient level of any of these essential elements will usually render a plant incapable of normal growth and frequently cause abnormal visible symptoms.¹ It has been pointed out, however, by Krantz and Melsted² that visible symptoms are sometimes difficult to relate to nutrient deficiencies in field conditions due to the effects of environmental conditions such as weather, disease, and insect infestation, which can produce symptoms similar to certain nutrient deficiencies.

A technique which offers promise as a noninvasive in iLvo means of remote sensing detection of certain types of nutrient deficiencies in plants is the use of laser- induced fluorescence (LIF) measurements.

It has been described elsewhere that the level of fluorescence emitted by plants on exposure to light is governed primarily by photosynthetic efficiency and

James McMurtrey is with U.S. Department of Agriculture, Field Crops Laboratory, PGGI ARS, Beltsville, Maryland 20705; Wayne Newcomb is with Republic Management Systems, Inc., 8401 Corpo- rate Drive, Landover, Maryland 20785; the other authors are with NASA Goddard Space Flight Center, Earth Resources Branch, Greenbelt, Maryland 20771.

Received 20 September 1983.

chlorophyll concentration.³-⁵ The primary roles played by certain nutrients in photosynthesis and chlorophyll synthesis suggested that deficiencies in these nutrients in the plant might be detected on the basis of changes in the plant's fluorescence spectra. An initial study of effects of potassium deficiency in corn showed that there was a significant increase in chlorophyll fluorescence when the plant was excited by laser radiation at 337 nm.⁶

Further investigation of the use of LIF measurements as a noninvasive technique for the remote detection of nutrient deficiencies in plants is described here using corn (Zea mays L.) grown in a number of nutrient de- ficiency conditions.

11. Materials and Methods

Nutrient deficient corn plants were grown in the greenhouse in sand culture in the following manner.

Corn seed (cultivar Dekalb XL64A) were planted in 15.24-cm (6 in.) pots and thinned to one plant per pot after emergence. White quartz sand was used as a growth matrix after being leached with distilled water to remove endogenous nutrients. The plants were di- vided into eight groups, omitting one element in each group from the complement of nutrients essential for normal corn growth. The control group contained all the nutrients established as essential using appropriate salts as nutrient sources.⁷ The nutrient deficiencies were produced by withholding from seven different groups the elements nitrogen, phosphorus, potassium, calcium, magnesium, sulfur, and iron, respectively. The potted plants were placed in saucers containing the various nutrient solutions which were allowed to rise to the roots by capillary action.

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720

480

380

240

120

400 500 8o 70D 80

WAVELENGTH IN NANOMETERS

Fig. 1. LIF spectra of corn supplied with all essential nutrients.

The LIF excitation source was a Molectron UV22 pulsed nitrogen laser which emits at 337 nm. It was operated at 30 Hz with an average power output of 450 mW with a pulse energy of 9 mJ. Frequent monitoring of the power during experiments showed a power fluctuation of no more than +3%.

The laboratory configuration used in these experiments was described in the preceeding paper.⁶

Fluorescence intensity is expressed as relative fluorescence intensity (RFI) (see Ref. 6).

Comparative estimates of leaf chlorophyll content were made on the basis of reflectance values taken at 660 nm. The reflectance measurements were made with a Carey model 17D spectrophotometer operating in the reflectance mode.

Ill. Results and Discussion

The results of the lack of nitrogen were visible at the beginning of the third week of plant growth, followed at the beginning of the fourth week by visible symptoms in plants that were deficient in potassium, magnesium, and phosphorus. Calcium and iron symptoms did not develop until the beginning of the sixth week, while sulfur deficiency symptoms had not appeared by the termination of the experiment in the seventh week.

Nitrogen deficient plants showed a yellowing (chlo- rosis) of the older leaves accompanied by necrosis at the tips, while the younger leaves tended to be greener.

The plants quickly became stunted with the older leaves dying.

Phosphorus deficient plants also displayed a retarded rate of growth. The plants had narrow leaf blades with the older lower leaves becoming purple.

The symptoms of potassium deficiency began with the margins of the older lower leaves becoming water soaked and later turning necrotic.

Calcium deficiency symptoms began with a curling or abnormal growth in the whorl (growing meristem of the plants) area. The leaves had ragged margins, and eventually the growing meristem became necrotic. The leaves became hard, stiff, and very green.

Magnesium deficient plant leaves became water soaked on the edges and sometimes in the middle along the veins of the older leaves. Eventually the older lower leaves became necrotic.

In the iron deficient plants, the young upper leaves became chlorotic. The veins remained darker green than the interveinal tissue. In some cases the entire whorl leaf became yellow and almost white.

Fluorescence measurements of the plants were made seven weeks after emergence on three leaves of the five plants in each of the eight nutrient treatment groups.

The fluorescence spectra of corn had maxima at 440, 690, and 740 nm (Fig. 1). Two molecular species. of chlorophyll a are responsible for the fluorescence at 690 and 740 nm,⁸ while the molecule responsible for the fluorescence at 440 nm has not yet been identified.

Changes in fluorescence intensity of the three bands as a function of the different treatments are shown in Figs. 2-4. Informational redundancy among bands was determined by regression analysis (Table I). Changes in fluorescence intensities at 690 nm as the result of nutrient deficiencies strongly correlate with intensity changes at 740 nm, which indicates that the two species of chlorophyll a are similarly affected by the different nutrient deficiencies. The fluorescent intensity at 440 nm is affected differently relative to the effects at 690 and 740 nm as shown by the low correlation values. A high correlation is seen between the intensity ratio of 690 nm/440 nm and the intensity at 690 nm. The consequences of this high correlation are seen in Fig. 5 where the treatment differences are very similar to those seen at 690 nm. In a field situation, the use of the 690/440 ratio should normalize the effects of laser power fluctuation, changes in fluorescence scatter due to changes in leaf geometry, and changes in fluorescence intensity resulting from varying mixes of leaf material and soil. These factors are controlled, however, in the laboratory experiments described here.

At 440 nm, the deficiencies which resulted in intensities significantly different from those of the control were potassium (K), phosphorus (P), nitrogen (N²), and iron (Fe) with a marginal difference seen in the case of magnesium (Mg). In all cases the fluorescence intensity decreased. No significant changes in fluorescence at this band were observed for calcium (Ca), phosphorus (P), and sulfur (S) deficiencies.

The most striking changes in fluorescence resulting from nutrient deficiencies were seen at 690 nm. Ni- trogen, phosphorus, and iron deficiencies caused a decrease in fluorescence. However, a threefold increase in fluorescence resulted from the potassium deficiency.

There were no changes in the fluorescence as the result of deficiencies in calcium, sulfur, and magnesium.

A basic assumption regarding changes in the in vivo fluorescence of chlorophyll is that they bear an inverse relationship to photosynthetic efficiency³ and are di- rectly related to chlorophyll concentration. A signifi- cant correlation between the in vivo reflectance of plants at 660 nm and their chlorophyll concentration has been shown by Bauer,⁹ i.e., the chlorophyll concentration decreases as the reflectance increases. Thus

140 APPLIED OPTICS / Vol. 23, No. 1 / 1 January 1984

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z 0 :

INUS MINUS MINUS MINUS MINUS MINUS MINUS

K Ca P N, Mg S Fe

TREATMENTS

Fig. 2. Nutrient deficiency effects upon mean fluorescence intensity at 440 nm. Means followed by the same letter are not significantly different at the 5% level of probability according to Duncan's Multiple

Range Test. Sample size of each treatment is 120.

350-

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3001 2501

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K Ca P N, Mg S Fe

TREATMENTS

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300

Z 250 US20

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Table 1. Correlation Between Fluorescent Bands and Band Ratios 690 nm/ 690 nm/

440 nm 690 nm 740 nm 440 nm 740 nm

Band R

440 nm 1.00 0.03 0.05 0.00 0.01

690 nm 0.03 1.00 0.96 0.92 0.24

740 nm 0.05 0.96 1.00 0.86 0.12

690 nm/440 nm 0.00 0.92 0.86 1.00 0.26

690 nm/740 nm 0.01 0.23 0.12 0.26 1.00

N = 120

Fig. 5. Nutrient deficiency effects upon the mean ratio of the intensity of fluorescence at 690 nm to that at 440 nm. Means followed by the same letter are not significantly different at the 5% level of probability according to Duncan's Multiple Range Test. Sample size

of each treatment is 120.

the reflectance of corn at 680 nm was used as a relative measure of the chlorophyll concentration. The reflectance values of the plants at 660 nm as a function of treatment are given in Table II, which indicate a rela- tively high decrease in leaf chlorophyll as the result of nitrogen deficiency followed by significant decreases due to deficiencies in potassium, iron, and phosphorus.

There was a marginal decrease due to lack of magnesium and sulfur with no decrease resulting from a deficiency in calcium.

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The significant decrease in chlorophyll concentration as the result of deficiencies in nitrogen, phosphorus, and iron indicates a decrease in the rate of chlorophyll synthesis which is hardly surprising in view of the important roles played by these nutrients in the synthesis of chlorophyll. Nitrogen is a major constituent of the tetrapyrole nucleus of chlorophyll with iron being an important cofactor of enzymes responsible for the synthesis of chlorophyll.'⁰ The phosphate requirement in chlorophyll synthesis is most likely a reflection of its role in the phosphorylation of adenosine diphosphate (ADP) to generate adenosine triphosphate (ATP), a molecule which is both the prime source of energy for biochemical synthesis as well as being required for en- zyme synthesis at the level of transfer ribonucleic acid

(RNA). ⁰

The great increase in chlorophyll fluorescence where the plant is deficient in potassium along with a decreased chlorophyll concentration would indicate a decrease in the efficiency of photosynthesis, thus al- lowing an increase in the amount of the activation energy of the excited chlorophyll molecule dissipated as fluorescence. It is considered that this decrease results from a lowered CO₂ uptake due to the lack of potassium necessary for stomatal opening.'

The plants which were deprived of calcium did not show significant changes in either fluorescence or reflectance, which could mean that the absence of calcium, at least over the duration of this experiment, although manifesting physiological changes did not interfere with photosynthesis or chlorophyll synthesis.

It was surprising that the magnesium deficiency caused only a slight decrease in fluorescence at 440 nm and none at 690 nm and only a small decrease in chlorophyll concentration in view of its being a constituent of the chlorophyll molecule. It may be that there was sufficient residual magnesium in the growth media to support chlorophyll synthesis.

IV. Conclusions

Extreme deficiencies in certain nutrients required for the growth of corn are manifested not only by visible plant changes but also by changes in LIF intensities at specific wavelengths.

Table 11. Corn Leaf Percent Reflectance at 660 nm

Mean percent reflectance at

Treatment 660 nm

Minus nitrogen Minus potassium Minus iron Minus phosphorus Minus magnesium Minus sulfur Minus calcium Complete

8.01 A 6.35 B 6.31 B 6.19 B 5.95 BC 5.35 CD 4.86 D 4.69 D

Means followed by the same letter are not significantly different at the 5% level of probability according to Duncan's Multiple Range Test. Sample size of each treatment is 15.

Deficiencies in phosphorus, nitrogen, and iron are manifested primarily by a decrease in the fluorescence intensity of chlorophyll a at 690 and 740 nm. In con- trast, the fluorescence of chlorophyll increases when the plant is deficient in potassium.

Using the in vivo reflectance of corn at 660 nm as a relative measure of chlorophyll concentration, the low correlation between chlorophyll concentration and chlorophyll fluorescence suggested that changes in chlorophyll fluorescence were due only in part to changes in the chlorophyll concentration. Other factors which can effect chlorophyll fluorescence changes in- clude changes in concentration of quenching agents, e.g., water, and changes in photosynthetic efficiency, i.e., rate of transfer of photon energy into the biochemical reac- tions of photosynthesis.

Although the underlying explanations of the rela- tionships between the fluorescent changes and certain nutrient deficiencies remain to be established, the results of these studies indicate the potential usefulness of LIF measurements in the remote detection of deficiencies in certain nutrients required for the growth of corn. The deficiency in corn which is observed in the most specific and striking fashion is potassium.

If the results reported here are validated using 'other species, a useful technique would be available for the rapid noninvasive remote detection and possible iden- tification of nutrient stress.

The authors gratefully thank Thomas W. Brakke for his invaluable assistance in the computer analysis of the data. Appreciation is expressed to the GSFC Director's Discretionary Funds for the support of these studies.

References

1. D. W. Raines, in Plant Biochemistry, J. Bonner and J. Varner, Eds. (Academic, New York, 1976), p. 561.

2. B. A. Krantz and S. W. Melsted, in Hunger Signs in Crops, H. R.

Sprague, Ed. (David McKay, New York, 1964), p. 25.

3. E. I. Rabinowitch, J. Phys. 61, 870 (1957).

4. L. N. M. Duysens, Science 120, 353 (1954).

5. B. Kok, in Plant Biochemistry, J. Bonner and J. Varner, Eds.

(Academic, New York, 1976), p. 851.

6. E. W. Chappelle, F. M. Wood, Jr., W. W. Newcomb, and J. E.

McMurtrey III, same issue, Appl. Opt. 23, 000 (1 Jan. 1984).

7. J. E. McMurtrey, Jr., US Dep. Agric. Tech. Bull. 340 (1933).

8. J. S. Brown and M. R. Michel-Wolwertz, Biochem. Biophys. Acta 155,288 (1968).

9. M. E. Bauer, in Advances in Agronomy, Vol. 27 (Academic, New York, 1975), p. 271.

10. A. Lehninger, in Plant Biochemistry, J. Bonner and J. Varner, Eds. (Academic, New York, 1976), p. 917.

11. R. A. Fischer and T. C. Hsiao, Plant Physiol. 43, 1953 (1968).

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