[advances in agronomy] volume 28 || nitrogen stress in plants

NITROGEN STRESS IN PLANTS

E.A.N. Greenwood Division of Land Resources Management. CSIRO. Floreat Park. Western Australia

I . Introduction .................................................. I1 . Quantitative Concepts of Nutrient Deficiency .........................

A . Classic Approach to Nutrient Response ........................... B . Nutrientstress ..............................................

111 . Nitrogen Stress ................................................ A . Definition .................................................. B . Measurement ...............................................

IV . Factors Affecting Nitrogen Stress .................................. A . Supply .................................................... B . Demand ...................................................

V . Alternative Evaluators of Nitrogen Stress ............................. A . LeafNitrogenFractions ....................................... B . LeafElongation ............................................. C . LeafArea .................................................. E . Conclusions ................................................

VI . Applications ................................................... A . AgronomyandEcology .......................................

C . Modeling ................................................... VII . Conclusions and Aspirations ......................................

References ....................................................

D . Carbon Dioxide Exchange Rate .................................

B . Stress Physiology ............................................

1 2 3 6 7 7 a

14 14 16 18 18 21 25 27 28 28 29 32 33 34 34

I . Introduction

This article is about the quantitative measurement of nitrogen deficiency in plants . It is not a general review of the extensive field of nitrogen nutrition .

It must occur to many agronomists to ask why water stress can appear to be so precisely measurable. whereas for nutrient stress we are limited to such broad terms as clinical. subclinical. severe. and mild . Our dependence on these qualita- tive terms reflects an extraordinary weakness within the discipline of plant nutrition in quantifying nutrient deficit . You will say at once that there are valid reasons why water deficit must be

easier than nutrient deficit to determine . And I will agree . My criticism is not directed to the greater challenge. but to the meager evolution within the discipline to deal with it . To some extent the cause may be historical . This

1

2 E. A. N. GREENWOOD

century has seen classic plant nutrition engrossed in the search for essential elements, the physiological role, availability in the soil, the clinical symptoms of deficiency and toxicity, and the response of plants to the quantity and chemical composition of nutrient supply. Plant water research was, of course, not concerned with essentiality and diagnostic critera. Whatever the reason, water deficit is commonly defined in terms of the physical stress of the water, whereas nutrient deficiency has come to be defined in terms of the plant’s response to the added nutrient. Consider the succinct definition proposed by Goodall and Gregory (1947): “a plant is deficient in a certain element if supplying that element to the plant in a suitable form causes an increase in yieM [my italics] , this effect being specific to the element in question.” It follows from this definition that in order to quantify the degree of deficiency one can measure the magnitude of the response of the plant. Hence the universal methods by which agronomists seek an estimate of nutrient deficit are based on plant response. If the increase in yield itself after application of fertilizer is not measured, then the soil or plant tissue might be analyzed in order to predict the response, the predictor having been previously calibrated against a response curve.

Although the term nutrient stress has been in limited use for a number of years, I am not aware of any exposition of definitions and philosophy. Thus, an attempt is made in Section I1 to state in agronomic terms some basic considerations that agronomists may well accept as axiomatic in the field of water stress, but which have been ignored in the field of nutrient stress. They are my current, personal viewpoints and all of them are arguable (by author as well as reader). Regardless of whether you agree, your constructive participation will certainly assist the advancement of this aspect of agronomy.

II. Quantitative Concepts of Nutrient Deficiency

The term “stress” as used in plant science needs some clarification. It is a broad term that can be defined from more than one point of view; that is, as the status of the stress factor or as the effect of that status on the growth of the plant. For example, Taylor (1968) considered water stress to be the physiological condition of water in the plant, although a plant is often said to be under water stress whenever the conditions of water are unfavorable to plant growth. Because of this ambivalence of meaning, Taylor considered water stress too broad a term to quantify numerically. Hsiao (1973) confines the meaning of water stress to deficit, which is clearly separate from the plant responses which it induces. Central to the issue of evaluating nutrient stress, then, is to decide whether it is to be defined as deficit or as induced response.

My current attitude (I have not always held this view) is that nutrient stress should remain a quantitative concept of nutrient deficiency-like growth, it is not measurable. Our efforts, as with water stress, should be directed toward

NITROGEN STRESS IN PLANTS 3

developing measurable parameters of stress which can be used for its evaluation. It would be no departure from convention to call such evaluations “nutrient stress,” for we do not hesitate to invest increments in biomass with the name of “growth rate.” A vital rule must be to indicate which evaluator of nutrient stress is being used.

If we allow these first steps to be taken, some difficult problems in estimating nutrient stress can be resolved. But, as these steps are only preliminary, they can be readily retraced should the ground rules be found unacceptable.

If the evaluation of nutrient stress is to be analogous to water stress, then we would have to measure such parameters as relative nutrient content and the chemical potential of the nutrient in the plant in some way. Assuming, for the argument, that it is feasible to do this, it would seem that the situation is rather different to what it is for water stress. We take for granted that the upper limit to the water content of a plant cell (full turgor) closely coincides with the water con- tents at which zero water potential and optimum plant growth occur. But, as is evident from the ability of plants to take upluxury and toxic quantities of nutrients, the upper limit to the nutrient content of a plant cell is well above that required for optimum plant growth. While the lack of a reference point is a major constraint to the concept of relative nutrient content, it is not an objection to the use of chemical potential. It simply implies that the relationship between plant performance and chemical potential would not be so simple, or perhaps as general, as it is in the special case for water stress. The main barrier to the use of chemical potential for estimation of nutrient stress seems to be that it is not yet a feasible proposition to obtain a meaningful measurement of activity of specific ions in plant tissue. And so, for the present, it becomes necessary to fall back to suitable parameters of current plant response. How appropriate to this specific purpose are the classic approaches to the evaluation of nutrient response, viz., nutrient response curves and critical concentrations?

A. CLASSIC APPROACH TO NUTRIENT RESPONSE

This section starts with response curves because the concept dominates the thinking of agronomists about the severity of nutrient deficiency.

The only critical criterion of the severity of a deficiency which can be derived from a response curve is the magnitude of the reduction in yield below the maximum, This implies that, for the evaluation of a deficiency, it is unnecessary to know the form of the response curve. The practical significance of this point will be taken up again later in this section.

With respect to our present purpose, the main weakness of the nutrient response curve is that it fails to deal with changes in response which inevitably occur in the field with time. The curve is the resultant of all the curves that might have been constructed between sowing and harvesting. Maybe an agron-


omist does not always want to know more than the final outcome. But sometimes he does need to know what has happened in order to understand how the final response developed-particularly if the results were unexpected. A moment’s consideration shows that responses do vary with time, even to the point of changing sign. These variations in response arise from changes in the supply of the nutrient and the demand for it by the plant.

The nutrient supply to the plant may change with time because of uptake by the plant, or as a result of gaseous or leaching losses, chemical reactions, or microbial activity. When fluctuations in nutrient supply are large, the original levels of fertilizer applied inaccurately represent nutrient supply. Further, efficient uptake of fertilizer by the plant implies that the supply of applied fertilizer must approach zero with time, and so response might be expected to decline with this depreciation in treatment strength.

The demand by the plant for a given nutrient also changes with time since it is influenced by the changes in all other environmental factors that control plant growth-other nutrients, water, radiation, etc. For example, the amount of nitrogen required by a plant now may be quite large if the environment is favorable for growth, whereas it may be small if, say, the temperature or the supply of available phosphorus is low. In addition to these influences on demand there is the compensating effect of plant size. Plants that response positively to the higher initial levels of the nutrient will, because they are bigger sinks, make larger demands on other nutrients or water. These may in turn become limiting, thereby curtailing the initial response. More certainly, the large plants will intercept so much light that the lower leaves will receive suboptimal illumina- tion.

Another important influence on the response of deficient plants is age. The responsivity of plants to a deficient nutrient declines with ontogenetic drift, particuarly in annuals where response approaches zero with maturity.

Two conclusions follow from these remarks: nutrient response curves give very little information about the current intensity of deficiency, and in some cases they may also be quite misleading. From the foregoing discussion it is clear there will have to be some integration of supply and demand.

A familiar approach has been to determine the concentration of the nutrient in the plant, arguing that the plant itself is the actual integrator of supply and demand. The graphic expression of this approach is to plot yield against nutrient concentration in the tissue (Fig. 1). Refinements are made by selecting the yield of a specific product and the chemical determination may be of some sensitive compound in sensitive tissue. In Fig. la the yield is beet weight, the compound determined is nitrate nitrogen, and the tissue is petiole.

A frequent, but not universal, characteristic of the relationship between yield and chemical composition is that, for severe deficiency, yield increases linearly with increase in concentration with a well-defined steep slope, whereas at sufficiency, the curve flattens and becomes poorly defined (Fig. 1). If the change

"Mature" Petioles

20

A A

i N0.25 -

I I 1 I 1 I 1 1

A 0

0

b 100

80

16,000 24,000 8.000

Nitrate Nitrogen (ppm)

' 0 128 8

8 256

A 96 0 0512

88

0

0

A

A B A

8

0

0 0 4 2,500 5,000 7,500 1 0 , ~ 12,500 15,000 17,500 20,OOO

~ L ~ l l l ~ ~ l l l l l 1 1 1 1 1

1 .m Nitrate-N In Blade 1 (ppm)

FIG. 1. Relation of yield and nitrate nitrogen in plant tissue. (a) Weight of sugar beet and nitrate in petioles (after Ulrich, 1950). (b) Dry weight of Italian ryegrass and nitrate in leaf blade 1. dy/dx = 1 at approximately 1000 ppm nitrate-N. After Hylton etal. (1964).

6 NITROGEN STRESS IN PLANTS

in slope is abrupt, a “critical concentration” is discernible at the discontinuity, above which there is little increase in yield and below which yield is greatly reduced. It is not always easy to locate the critical point because of the variability of the data or because the curve may be broad and continuous. In the latter case, a numerical definition of the critical concentration can be based on the slope of the curve. For example in Fig. lb a slope of dy/dx = 1 has been adopted by Hylton et al. (1964) as critical. It is a step toward numerically quantifying the degree of deficiency, for a continuous change in slope can provide a continuous numerical scale.

There are two serious weaknesses in the use of the slope of the yield concentration curve as a continuous indicator of the intensity of deficiency, as distinct from a single critical point. First, as seen in Fig. 1, the deficient arm of the curve shows little change in slope. Second, it is well known that the critical concentration declines with age and that in annuals this decline is precipitous after flowering. As for the response curve, a whole suite of curves like Fig. l a can be obtained for successive ages. Nevertheless it can be a most useful technique for estimating deficiencies in highly standardized crops (e.g., Ulrich, 1950).

The critical concentrations of nutrients in plants is the main form of reference to nutrient status to be found in the literature. Extensive use is made of the reference tables compiled by Chapman (1966). For more specific information, agronomists turn to their colleagues in plant nutrition for interpretation of “spot” chemical analyses. The persistent attraction of such information lies in the certainty that below an accepted concentration in the plant a nutrient will be severely limiting, and that usually there is no better information available. It seems paradoxical to make such a claim in face of the vast literature on the use of plant analyses as a tool for assessing the nutritional status of plants. The problem is that although the concepts seem simple, the results must be qualified, as with response curves, by strong interactions with time. This aspect is discussed by Smith (1962) in his comprehensive and lucid review of tissue analysis.

B. NUTRIENT STRESS

It is evident that response curves and tissue analyses will not provide a satisfactory basis for using plant response as an indicator of nutrient status. We ought now to return to the definition of deficiency proposed by Goodall and Gregory (see Section I), and develop a proposition for nutrient stress. For it seems reasonable to argue that if it is acceptable to base a definition of nutrient deficiency on the yield response to a dose of the nutrient, then a quantification of this response should provide an acceptable basis for nutrient stress. Goodall and Gregory used yield (presumably of dry matter) as the criterion of response. But other criteria can be envisaged such as size (e.g., height, leaf area), or a

E. A. N. GREENWOOD 7

process (e.g., carbon dioxide exchange rate), or any other partial expression of plant growth which can be measured readily. The problem with this choice is that each parameter of growth has its own functional relationship with deficiency which would in turn give different estimates of nutrient stress. The use of the full expression of growth itself for these purposes is not possible since growth is not’ measurable, as pointed out by Arnott et al. (1974) in their introduction on the measurements of “growth.”

Setting aside the choice of growth parameter until Section V, let me show what can be done with dry weight. The first task will have to be the development of a reference point to represent zero stress.

It is implicit in the nutrient response curve and in the tissue concentration curve that the highest yield obtained under the circumstances represents the complete absence of the deficiency. This circumstantial maximum is the standard against which the degree of deficiency at lesser yields is judged. It was pointed out in Section 11, A that one does not need to know the form of the response curve in order to evalute a deficiency. What is required is the relationship of the yield of the deficient plant to the yield of the plant at the circumstantial maximum. It is necessary also to establish that maximum yield has been obtained. In other words, a numerical value for deficiency can be derived from the shortfall in yield relative to the circumstantial maximum yield. This is the crucial point on which the whole of the remainder of th is article is based.

There is a further point to recall from the criticism of yield response curves in Section 11, A-an inability to indicate current response. This shortcoming can be easily dealt with by measuring current growth rate instead of the accumulated yield of dry matter.

If all these considerations are adopted, the current intensity of deficiency of a nutrient-nutrient stress-can be evaluated as the proportion by which the growth rate of the plant or crop is limited by that nutrient under the prevailing conditions. This definition needs to be qualified by the parameter of growth rate used-in this case, dry weight. The transformation of these ideas into a workable technique will be the objective of Section I11 which will be confined to the specific case of nitrogen stress. At this point also there will be a change in emphasis from a critique to a review of the subject.

I l l . Nitrogen Stress

A. DEFINITION

Nitrogen stress is a quantitative estimate of the intensity of current nitrogen deficiency in a plant or crop. It can be evaluated as the proportion by which the growth rate of the plant falls short of maximum growth rate attained with a


nonlimiting supply of nitrogen over the period when stress is being measured. For this representation of nitrogen stress, Greenwood, Goodall, and Titrnanis (1965) used the symbol SN when it was made on a biomass basis. The relative shortfall can be expressed as a percentage, i.e.,

(prowth rate at maximum N response)-(growth rate at deficiency) SN = 100

growth rate at maximum N response (1) For example, if the growth rate of the deficient crop is 7 g/m2/day and 10 g/m2/day when it is given a nonlimiting dose of nitrogen, then nitrogen stress would be 100[(10-7)/10] = 30%. SN has some broad similarities with relative water content, for which the

weight of a leaf deficient in water is compared with its weight after it has been brought to a standard, nonlimiting water content.

In order to put this expression into practice, it is necessary to decide precisely what is meant by growth rate and how to find the current limitation of it by nitrogen.

B. MEASUREMENT

The split-plot technique is basic to all methods so far devised for estimating SN. At the time SN is to be measured, one subplot is left untreated and the other is given sufficient nitrogen to make nitrogen nonlimiting. Current growth rate is then measured in the untreated subplot, and the maximum growth rate that can be attained by adding nitrogen is measured in the other subplot.

1. Growth Response

There are two conventional ways of expressing growth rate on a dry weight basis.

Symbol Working formula Units

Crop growth rate

1 dW Relative growth rate - - Wdt

C

R In W2-ln W 1 t 2 - t 1

g/g/day

where W1 and W 2 are the dry weights at time tl and t 2 , respectively. The numerical value of SN obtained may be influenced by which expression is used. Broadly speaking, C is appropriate for crops or swards with a closed canopy and R is appropriate for spaced plants. In order to distinguish which form of growth rate has been used for estimating SN, the appropriate symbol can be used as a


subscript, i.e., SNC or SNR . The expressions for SN then become:

SNC = 100 [(cM-C)/cMl (2)

SNR = 100 [ ( R M - R ) / ~ V I (3)

where C or R is the growth rate in the control subplot and CM or RM is the growth rate in the subplot to which nitrogen has been added.

Equation (3), the one originally proposed by Greenwood et LIZ. (1969, has been used consistently in publications on nitrogen stress. Nevertheless, as indi- cated above, it is not necessarily the most appropriate equation. Relative growth rates were originally adopted as a compromise in the interests of general application. The problem, briefly, is the exponential character of plant growth in unclosed canopies. Under steady conditions, the daily increment in weight increases with plant size, with the implication that the magnitude of the response to nitrogen is confounded with the weight of the plant. More specif- ically, when one wishes to compare the responsivity of two plants of different size due to different treatments or age, a bias is introduced. The bigger plant will have the bigger potential response in absolute terms. If the exponent of growth were constant then the bias could be exactly overcome by using relative growth rate. Although R does change less than C over the life of a crop by an order of magnitude, its change is appreciable.

Operationally there is no difference between using SNC and SNR, for they both require the same primary data, W 1 and W2. The real dilemma is that both C and R are nonideal over the whole life of the crop. And there seems no way of deciding which of them is to be preferred, for young crops behave as a community of spaced plants until the canopy is closed. Even if a working rule is adopted such that R is appropriate for crops with LA1 < 1 and C for crops with LA1 > 1 (LA1 = leaf area index), we would be well aware of a very broad transition zone in which neither R nor C would be fully appropriate.

We must conclude that the approach to plant stress through dry weight response cannot be taken without some bias. The importance of the bias can be gauged by comparing SNC and SNR derived from the same dry weight data. This can be seen in Fig. 2c for a situation supposedly favoring SNR . The difference between the two curves is small at the extremes but large in the middle portion.

In the case of wheat, the real situation does not seem to be as bad as it sounds from these considerations. In Fig. 3, SNR is plotted against SNC from two sets of data. One is the pot data used for Fig. 2c. The other is a reworking of the field data of Halse e ta l . (1969) in which SN was estimated at four stages from 4 to 16 weeks (ear emergence) with LAI ranging from about 0.1 to 2.5. The one curve fits both sets of data (R2 = 0.995). Three important points follow: age or LA1 has not affected the relationship between SNR and SNC; SNR is neither more nor less appropriate than SNC ; and it is a simple matter to convert SNR to SNC or vice versa. It can be seen from Fig. 3 that because the intercept is close

.02 u 0 2 4 6 8 1 0 0 2 4 6 8 10 0 2 4 6 8 10

N Supply (mM1 N Supply (mM1 N Supply (mM)

FIG. 2. Four expressions of response by wheat to nitrogen supply between the third and fiith week after emergence. (a) Yield of dry matter; (b) relative growth rate (R) and crop growth rate (0; (c) nitrogen stress based on R and on C. Data are from Greenwood (1966).


80

40

20

0

0 20 40 60 80 100

sNC(%)

FIG. 3. Relationship of nitrogen stress in wheat based on relative growth rate, Sm, to nitrogen stress based on crop growth rate, SNC. Points denoted as are from Fig. 2(c). Other points are derived from Halse et 01. (1969) from a field crop sampled between 4 and 7 weeks (A), 6 and 9 weeks (A), 10 and 13 weeks (+) and 13 and 16 weeks (x) after sowing. The curve y = 3.55 + 0.25% + 0 . 0 0 6 0 6 ~ ~ was derived from all points and accounts for 99.5% of the variance.

to zero, and, for subclinical levels of nitrogen deficiency only, the quadratic term can be ignored, the relationship of SNR to SNC can be simplified to SNR = 0.75~~. These remarks apply to wheat; they may not hold for other species, particularly dicotyledons.

A useful reference point is the value of SN at which nitrogen deficiency symptoms begin to develop. In all published work with grasses and cereals this

This section has dealt with growth response only in terms of dry weight. Other Point Occurs at SNR = 40% (SNC = 60%).

parameters of growth rate are considered in Section V.

2. Response Interval

Ideally, growth rates on the control and the plus-nitrogen subplots should be measured as quickly as possible after the plants have responded to the addition of nitrogen. This is because current response is required and SN may be changing rapidly. In practice there is a lag period between application of the nonlimiting dose of nitrogen and its entry into the roots and leaves even in solution culture


where immediate uptake of nitrogen is ensured. Bouma (1970a) measuring both leaf area and carbon dioxide exchange rate (CER) on subterranean clover at 2-day intervals could clearly detect a nitrogen response after 2 days. Wolf and Greenwood (unpublished data) used a much shorter time interval and measured the leaf elongation rate (L), and CER of expanding and mature leaves of wheat seedlings in light and in the dark. They found that dark respiration responded to added nitrogen in 2-7 hours but the response was small and insensitive. The CER of mature and expanding leaves responded after 22 hours, and a similar time lag was taken by L for elongating leaves. Full response by AL occurred after 48 hours, whereas CER required more than 72 hours. There was no evidence of any temporary toxicity when the nitrogen supply (as ammonium nitrate) was raised from 1 'to 20 mEq N/liter. An example of one of the several response runs is given in Fig. 4. From the foregoing evidence, it seems that at least 2-3 days should elapse between applying nitrogen and commencing to measure the response.

In practice, as agronomists will appreciate, it is difficult to get accurate estimates of dry weight increments in less than a week. This is because field sampling is imprecise and because the variance of the increment W2-W, is about twice the variance of a single dry weight measurement. In addition to this we are measuring differences between increments on one subplot and increments on another. Thus the variance of SN is high.

80

I 60 8 - f G

e 8

40

2 20

0

0 0

t i Dark Dark

I I 1 1

0 20 40 60

Hours

80

FIG. 4. Apparent change in nitrogen stress in deficient wheat seedlings immediately after the nonlimiting dose of nitrogen was given to alternate matched plants. Successive values of rates of elongation (L) and carbon dioxide exchange rate Q of the emerging second leaf were substituted into Eqs. (4) and (6) in the text. Developing response is indicateli by rising slopes and the attainment of full response by flat slopes. Replication was X4. From unpublished data by Wolf and Greenwood.


My experience is that a growth interval of at least 10-14 days is required to get meaningful and reliable differences between growth rates on the subplots when these are expressed as dry weights. This is far from being instantaneous. If SN is changing rapidly, only an average value over the time interval can be used. Rapid changes in SN do occur naturally. Power (1971) reported an increase in SNR from 4 to 73% in bromegrass within 3 weeks. On the other hand, there is an advantage in the field in having a fairly long time interval over which SN is being estimated, for it helps to integrate the day-to-day variations in weather which may be of little interest in themselves. Of course, where one wishes to establish relationships between stress and the environment a short-term response would be welcomed. Alternative, short-term, nondestructive methods for estimating stress are described and evaluated in Section V.

3. The Nonlimiting Supply of Nitrogen

SN can be estimated without invoking any assumptions as to the form of the response surface. It requires only the values for the actual growth rate and the growth rate with nitrogen nonlimiting. Since the response curve (i.e., the section of the response surface at prevailing levels of factors other than nitrogen supply) generally has an extensive plateau around the optimum, high precision in the choice of nonlimiting nitrogen levels is not necessary.

In cases where there is adequate information available (Power, 1971), one estimate of a nonlimiting nitrogen supply level is sufficient. Otherwise, at least two different levels should be used in order to judge whether the deficiency has been completely removed and whether toxicity has been avoided. The following working rules are useful: when the two “nonlimiting” levels of nitrogen do not produce growth increments that are significantly different from each other, then the increments are averaged to give a best estimate of growth rate with nitrogen nonlimiting; when the difference between them is significant but small, the larger value is taken for nitrogen nonlimiting; when the difference between them is large, then the data are abandoned. Significant growth differences for the two levels of nitrogen selected can be easily avoided provided that some precautions are taken. These precautions can be generalized: very young seedlings and heavily defoliated plants require much lower levels of nitrogen for maximum response than do older and intact plants; for the former, temporary toxicity may occur, particularly if nitrogen is supplied in the ammonium form.

4. Operational Procedures

For the estimate of SN by destructive dry weight harvest, a multi-split-plot technique is required. A quartet of matching subplots or quadrat areas is selected. Each of the four subplots is assigned at random to one of the following


procedures. Dry weight, W1 ,

is determined at the beginning of the test interval t l and, similarly, Wz is obtained at the end of the interval tz . The actual growth increment of the crop is computed from these two values. Meanwhile, at r , each of the remaining two subplots is given a different “nonlimiting” application of nitrogen and is harvested at tz . From the two dry weights for these subplots the value for WM is obtained. The growth increment of the crop with nitrogen nonlimiting is obtained from W l and WM.

In the field, if the soil is wet throughout the root zone, the nonlimiting dose of nitrogen can be applied in solution using the equivalent of say only 2 mm of artifical rain in order to avoid disturbing the water regime. A similar amount of nitrogen-free water must also be added to the Wz subplot. Where the surface of the soil is dry due to a short and perhaps unimportant absence of rain, then a decision must be made either to wait for rain or to add sufficient water with the nitrogen to simulate rain which could be expected to fall. Again, if water is added it should include Wz. In either case the value for SN would apply to situations where water is not an important limiting factor. In cases where it is realized that water is an important limiting factor, albeit unevaluated, but where rain falls during the growing season, another technique is available. Since it is unlikely that in these circumstances an argonomist would want to know the importance of nitrogen as a limiting factor without similar information for water (though few have sought it), the technique used by Power (1971) can be most effectively employed. This involves the use of supplementary water as well as nitrogen, and it will be discussed in Section VI.

IV. Factors Affecting Nitrogen Stress

Nitrogen stress can be considered as a concept that integrates the rate of nitrogen supply with all the other factors essential to growth: genetic, ontogenetic, nutritional, environmental, symbiotic, and other factors. It follows that a change in nitrogen stress may be brought about through variation in either the supply of nitrogen or in any of these other determinants of growth. The following is a review of the limited experimental evidence on factors affecting nitrogen stress.

A. SUPPLY

Curves relating SNR and SNC to nitrogen supply for wheat between weeks 3 and 5 are given in Fig. 2c. In this example, both curves lead to a credible


extrapolation to 100% stress (no net increment in dry weight) at zero introgen supply, and, of course, they both reach zero stress (no response to nitrogen) as the nitrogen supply approaches the nonlimiting level.

In nutrient culture work, it is usual to make some attempt to keep the concentration of nitrogen in the solution fairly steady by periodic or by continuous replacement. But where only a single application is given, say at the beginning of a pot experiment, the progressive depletion of the applied nitrogen must result in an increase in SN , other factors being held unchanged. In the field, marked fluctuations in nitrogen supply in both directions may occur. On the one hand, these may be leaching and gaseous losses and, on the other hand, all those factors such as temperature and wetting and drying which may control the rate of microbial production of available nitrogen in the soil.

Some practical perspective is given by the work of Halse et ul. (1969) who grew a wheat crop in a nitrogen-deficient sandy soil (average annual rainfall, 390 mm) and applied nitrogen at three rates at sowing: nil, 56 kg/ha, and 112 kg/ha, the last also receiving two further applications of 112 kg/ha. These treatments gave grain yields of 900, 1800, and 3000 kg/ha, respectively. SNR was determined on the nil and 56 kg/ha treatments. On the nil treatment SNR remained at 48% for several weeks and then fell to 14% at the late boot stage. The application of 56 kg (kglha) almost eliminated stress during the first few weeks, but by floral initiation it had reached 23% before falling to 5% (Table I). These results were obtained in the Mediterranean climatic zone of southern Australia during the growing seasons of autumn, winter, and spring.

TABLE I Effect of Nitrogen Fertilizer and Plant

Age on Nitrogen Stress (SNR) in a Wlieat Crop'

Nitrogen applied

Weeks after sowing Nil 56 kg/ha

% i SE

4-7 48i1 6 i 2 6-9 47il 2 3 i 3 10-13 26i5 11i4 13-16 14i7 5i8

'Adapted from Hake e l al. (1969).


B. DEMAND

The age and ontogeny of a plant can greatly influence the magnitude of SN. Greenwood and Titmanis (1968) found that for annual ryegrass grown on a constant nitrogen concentration of 1 mM, S N ~ increased from 10% at 2 weeks after emergence to 11% at week 3, to 17% at week 4, and to 32% at week 5. The explanation could be that, as the daily increment in dry weight increases with age, the demand for nitrogen must increase. If the supply is fixed in concentration and inadequate, the shortfall in supply, whence stress, must also increase. At a much later stage, individual axes of the plant produce flowers and become less capable of response to nitrogen in the sense of net increment of dry weight-at least in more determinate species. This implies, particularly for annuals, that SN must approach zero as the plant approaches maturity. These trends are evident in Tables I and 111. Light will increase SN provided that light intensities are below the optimum

for plant growth. Table I1 records some unpublished results for wheat seedlings. When the potential growth rate of a plant is limited by other nutrients as well

as nitrogen, then an increase in the supply of those nutrients ought to increase SN. A very clear demonstration of th is point with respect to sulfur and phosphorus can be inferred from the data of Bouma and Dowling (1967). They showed increasing responses (whence stress) of leaf area to nitrogen in TrifoZium subferraneum. For example, on a very low supply of inorganic nitrogen, values of stress are 25%, SO%, and 70% for sulfur supplies of 0.125,1.0, and 8.0 ppm. In computing these values, I have taken the highest supply of nitrogen as being nonlimiting, which is good enough for this exercise.

TABLE I1 Effect of Shading on Nitrogen Stress

(SNC) at lS"/lO"C in Wheat

N fllpply (mM) Unshaded Shaded

2 42 31 4 17 11 6 13 4

NOTE: Plants were grown in a naturally lit glasshouse with noon light intensities of 45,000 (unshaded) and 18,000 lx (shaded). Replication was X 7.


Power (1971) studied the interaction between wafer sfress and nitrogen stress on bromegrass in the northern Great Plains of the United States. He found, over a range of conditions, that the plant stress caused by each of these two limiting factors was roughly additive. To give one instance, the value of nitrogen stress (SNR) as obtained by making nitrogen nonlimiting was 24%. The value of water stress (SWR) as obtained by making water nonlimiting was 32%. But when nitrogen and water were both made nonlimiting, the value of plant stress obtained was 54%. This implies that as water becomes more limiting the value of SN declines. Further results are displayed in Table VI.

Defoliation, whether by cutting or by grazing, might be expected to reduce SN at low values of LAI when a reduction in photosynthetic area might be expected to limit potential growth during the recovery period. Greenwood and Titmanis (1968) established this point experimentally with young swards of annual ryegrass in pots with clipping, and in the field with sheep. The particular defoliation regimes used reduced SNR from 32 to 19% in the pot experiment and from 11 to 3% in the grazing experiment. These examples probably under- estimate the immediate effect of defoliation on SN since the latter was, of necessity, estimated during the ungrazed recovery period.

One of the general, and important, effects of cultivation is to increase the supply of available nitrogen to a crop. This should result in a reduction of stress provided that there is a deficiency of nitrogen and that cultivation does not affect other limiting factors. The situation is likely to vary with soil type and other circumstances. The integration of all these factors by the crop can be expressed, with reference to nitrogen, as SN. Table I11 shows that the effect of cultivating sandy loam was to reduce the subsequent values of SNR in a wheat crop. This wil l be discussed further in Section VI, A.

TABLE 111 Effect of Age and Cultivation on Nitrogen Stress (SNR) in Wheata

Nitrogen stress (%)

Cultivation treatment Wk after emergence: 3-6 6-9 9-12

Nil

Conventional

13 26, 13 * *** ns. 5 17 10

Adapted from Greenwood e? al. (1970).


V. Alternative Evaluators of Nitrogen Stress

The evaluation of nitrogen stress by dry weight increment (SN) as described in Section IV is a simple procedure, but it has three important disadvantages in that it requires destructive sampling, a high replication for precision, and a lengthy period for the response to manifest. Obviously it would be a great advantage if nondestructive or more rapid methods could be found. Alternatives are available and they fall into two classes: parameters of plant nitrogen status which are used as indices of SN, and parameters of plant growth which are used as direct alternatives to dry weight.

A. LEAF NITROGEN FRACTIONS

When attempting to relate leaf nitrogen to nitrogen stress, similar specifica- tions to those which usually apply to the establishment of “critical levels” in tissue analysis must be considered. Decisions must be made as to the choice of nitrogen fraction to determine, organ or tissue to sample, and the time of sampling. Insight into these aspects can be gained first from reference tables such as in Goodall and Gregory (1947) and Chapman (1966) as well as individual research papers. For example, Ulrich (1950) gives critical values of nitrate nitrogen for leaves and for petioles at three physiological ages and at different dates of sampling of Beta vulguns; Hylton et al. (1964) give the value of nitrate nitrogen in several plant parts at which dy/& = 1 for Lolium multijlorum; and Rauschkolb el al. (1974a, b) evaluate the nitrogen status of maize and sorghum in terms of the total nitrogen and nitrate-nitrogen concentrations in the whole leaf, midrib, and basal section of the stem.

We have examined the relationship of SN to the concentration of total nitrogen and of ninhydrin (mainly a-amino) nitrogen in the youngest fully expanded leaf of the tillers of annual ryegrass (Lolium rigidum) and in several genotypes of wheat. We used the youngest fully expanded leaf on the tiller at the time of sampling because its physiological age is constant and because it is easy to identify and sample. Total nitrogen was chosen because of its common use. Nitrate nitrogen was rejected, mainly because it does not accumulate in measurable quantities over the whole range of deficiency (e.g., Hylton et al., 1964), but also because it is highly variable in concentration, and it is sensitive to the form of nitrogen supplied and to time of day (Allen et al., 1961). Ninhydrin nitrogen was chosen as being intermediate between nitrate (unmetab- olized) nitrogen, and total (mainly protein and therefore historic) nitrogen. Typical curves for total nitrogen and ninhydrin nitrogen in the youngest fully expanded leaves of wheat tillers are given in Fig. 5 .


K z *. 60 a i b lJY

40

b .- 20

0

80

0

- \o Total Nitrogen 60

1 \o 40

:\

20

\ 0 1 I I 0

80

2 3 4 5 6 7 0.1 0.2 0.3 0.4

Leaf Nitrogen (%I FIG. 5 . The relation between nitrogen stress (SNR) and the concentration of nitrogen in

the youngest expanded leaf of wheat between the third and fgth week after emergence. After Greenwood (1966).

To test the general applicability of leaf nitrogen fractions as estimators of SN, the constancy of the relationship must be investigated under a range of conditions. The results of some investigations are reviewed below.

A direct comparison of the relationship of SN to leaf nitrogen was made between four commercial genotypes of wheat between the third and fifth week after emergence. Whereas some genotypes had similar calibration curves others differed (Titmanis and Greenwood, 1969), which leads to the conclusion that, in practice, it would be necessary to calibrate each genotype separately regardless of whether total nitrogen or ninhydrin nitrogen was to be used. Table IV gives the predicted value of SNR for a given value of the estimator in each of the four

TABLE IV Comparison of Estimates of Nitrogen Stress (SNR) Given by Set Values

of Leaf Nitrogen Fractions in Several Genotypes of Wheat'

Nitrogen stress (%)

Value of estimator Mendos Gamenya Emblem Olympic Gab0

Total N 4% 25 25 30 45 21 5% 8 9 - 17 9

1200 ppm 23 23 20 35 33 1500 ppm 10 10 - 11 20

Ninhydrin N

'Adapted from Titmanis and Greenwood (1969) and Greenwood (1966).


60

50

40

- ap

genotypes, and also for Gabo at the same age but from another experiment (Greenwood, 1966).

Since the relationship between SNR and leaf nitrogen vanes between close genotypes of the one species, it can also be expected to vary between closely related species. This certainly holds for the two species that have been investigated-annual ryegrass and wheat. At 28 days after seedling emergence the predicted value of SNR at 4% N content is 46% for ryegrass (cf. Fig. 6), which is a much higher value than four out of the five wheat genotypes at the same age just mentioned (Table IV).

Work on the effect of age was conducted in two separate experiments by Greenwood and Titmanis (1966); the results have been brought together and reexpressed in Fig. 6. Over the first 5 weeks after emergence, a given concentration of total nitrogen in the youngest fully expanded leaf of the tiller gives a fairly constant estimate of SNR . Thereafter the predicted value of stress falls. For ninhydrin nitrogen, the two experiments gave inconclusive results.

-

-

-

-

0 ,

3.5%N Total Nitrogen -0

0-0-0

\

- I 1

Spring Experiment Winter Experiment I 1 I 1 I 1

6 . M N

0

0-0-0-

DO 10


Insofar as one can generalize from two species, the period over which the concentration of total nitrogen in the youngest fully expanded leaf of the tiller can be used as a stable estimator of SN is short, i.e., 5-6 weeks at the most.

No precise work has been recorded on the effects of either the composition of the nitrogen source or the relative supply of other nutrients on the regression of SN on leaf nitrogen.

In experiments with young annual ryegrass, we examined the effects of defoliation on the relation of SN to leaf nitrogen (Greenwood and Titmanis, 1968). The various defoliation treatments used had very little effect on the relationship except at high concentrations of plant nitrogen (cf. Fig. 6). Total nitrogen and ninhydrin nitrogen performed simiiarly as estimators of stress.

Light exerted a marked effect on the relationship between SNC and the total nitrogen concentration in the youngest fully expanded leaf of wheat tillers in an unpublished experiment by Greenwood. The treatments were full daylight and shading. Average noon light intensities for the unshaded and shaded treatments were 45,000 and 18,000 lx, respectively. Figure 7 and the accompanying statistics show that the estimation of SNC by total leaf nitrogen was good (R2 > 0.98) and that the effect of shading was to produce greater slopes and intercepts than the unshaded treatments.

If leaf nitrogen is to be used, then which fraction of nitrogen is to be preferred? There is usually little to choose between total nitrogen and ninhydrin nitrogen. The former is easier to deal with in the field and this may be the deciding factor. Nitrate nitrogen is unsatisfactory because it is not present in detectable amounts in severely deficient plants and because it shows diurnal and other variations. My own conclusion now is that any fraction of leaf nitrogen is rather unsatisfactory for general use as a quantitative estimator of SN.

B. LEAF ELONGATION

The use of the rate of leaf or tiller elongation as an estimator of current growth rate in Gramineae has been briefly reviewed by Williams and Biddis- combe (1965). This reference also includes an excellent photograph of both continuous-recording and multi-point auxanometers, which are instruments used for the automatic measurement of tiller elongation rate. Leaf elongation has turned out to be the most useful of the estimators of SN so far evaluated for Gramineae. Leaf elongation rate is sensitive to nitrogen stress and, because of the morphology and pattern of leaf development in Gramineae it is simple and cheap to measure. Under steady conditions the daily rate of elongation of a given leaf of a Gramineae is constant from its first appearance until just before its full expansion. Since the latter coincides with the emergence of the next leaf this period approximates the leaf appearnce interval. Leaf elongation is obtained as


70

60

50

40 - 8 0 z

v)

- 30

20

10

0 2

X

Unshaded R2=O!

R2=0.995)

\ X

I I 1 I 1 1

3 4

Leaf Nitrogen(%)

5

FIG. 7 . Effect of shading on the estimation of nitrogen stress (SNC) by total nitrogen in the leaf.

the difference between successive measurements of leaf length (in Gramineae this can be from the base of the ligule of the older leaf to the tip of the emerging leaf). Tiller elongation can be measured from a bench mark on the ground to the tip with a rule, or by an awanometer attached to the emerging leaf tip. More elaborate devices may be used for measuring elongation, such as the one used by Hsiao el al. (1970) for maize.

The use of leaf elongation rate as an estimator of SN has not been evaluated in dicotyledons. It may well be suitable, for Wadleigh and Gauch (1948), for example, found leaf elongation to be a sensitive estimator of water stress in cotton plants.

Leaf elongation rate, L (cm/day), can be used to estimate SN by substituting L for R or Cin the nitrogen stress equation

SNL = 100 [(LM-L)/LM] (4)

The ability of SNL to estimate SN in Lolium is seen in Fig. 8. For subclinical


100

80

60

- s? z

v)

40

20

0

28-42 Days SNR 0 44-58Days

A 28-42 Days sNC A ~ - 5 8 D a y r

0 20 40 60 80 100

SNL(%)

FIG. 8. Linear regressions of SNR on SNL and of SNC on SNL in Lolium rigidum. Points derived from Greenwood and Titmanis (1966) for plants between 2 8 4 2 days and 44-56 days after emergence. Values from plants with symptoms are not included in the regression.

levels of nitrogen deficiency, over 97% of variation in either SNR or SNC can be accounted for by SNL . Where deficiency symptoms are present there is a marked increase in slope. This seems to be caused by a rapid recycling of nitrogen from the older leaves to the emerging leaf. Consequently, even when SN approaches 100% (zero growth on a dry weight basis), leaf elongation continues.

Unfortunately, annual ryegrass is the only species to have been studied with respect to leaf elongation and nitrogen stress. Some unpublished data of Power show that leaf elongation is sensitive to the effect of nitrogen supply on certain growth rate in a wide range of grasses from May to July in North Dakota (Table V) and therefore might also be useful as an estimator of stress. Power compared leaf elongation rate in swards that received either no nitrogen fertilizer or 220 kg Nlha in early spring. It is tempting to calculate SNL values from this array but to do so would be far from rigorous. This is because at each time of comparison,


TABLE V Rate of Leaf Extension of Various Grass Species Prior to

Inflorescence as Affected by Nitrogen Fertilization (Values Are Means of 6 Tiller@

Date

N rate

Species (kdha) 5/27

Reed canary

Smooth brome grass

Western wheatgrass

Russian wild we

Crested wheatgrass

Green needle grass

Garrison creeping foxtail

Intermediate wheatgrass

0 180

0 180

0 180

0 180

0 180

0 180

0 180

0 180

8.7 14.0 10.1 8.2 7.2 8.1 4.8 8.5 7.0 3.8 5.2 4.4 5.5 2.5

10.6 8.5

Average SE 6/2 6/9 6/17 (%of mean)

mm/day

13.5 14.0 17.8 10 18.2 25.3 22.0 12 7.8 7.5 7.2 17

14.5 18.2 11.3 24 9.7 9.3 10.5 18

12.5 21.2 25.6 19 7.3 10.2 11.3 15

18.2 25.0 32.3 20 11.0 7.6 6.0 26 20.0 10.7 2.7 27 9.0 8.4 6.7 17

10.0 16.0 12.5 18 6.8 6.3 9.0 14

21.5 36.2 32.2 17 12.3 11.3 9.7 14 19.2 18.8 9.5 19

‘Unpublished data of J. F. Power.

the No and Nzzo plots were not at all comparable, the latter having responded extensively to nitrogen applied in early April; consequently a split-plot design that is required for SN did not apply. The data have been presented here because of the rare insight they give into the sensitivity of leaf elongation under a variety of seasonal conditions. For example, soil water measurements showed that the fertilized plots contained 2-8 cm less water in the root zone in late May than did the check plots. This could account for the negative response to nitrogen obtained at that period. The dry matter responses obtained (to be published by Power elsewhere) reflected the variation in leaf elongation rates. The day-to-day change in temperature also produced a noticeable effect on elongation (Power, personal communication), an observation that supports the elegant results with Phalaris tuberosa L., P. arundinacea L., and Festuca arundinacea Schreb. obtained by Williams and Biddiscombe (1965).

The standard errors in Table V give some idea of the adequacy of the fourfold replication used by Power. More comprehensive information on the number of


replicates required for a given level of precision in L is provided by Scott (1961) for tussock grasses.

At the present stage of evaluation it appears that leaf elongation is a simply measured and promising estimator of SN, and its use should be encouraged. Accordingly, it would be profitable to investigate its performance early in any program where it might be applicable.

Even in the case of annual ryegrass it has been shown that the relation of SNL and SNC does not hold indefinitely. From Fig. 8 it can be seen that a steady relationship holds at least for the first 8 weeks. But Greenwood et al. (1965) have shown that values of SNR begin to rise in relation to SNL shortly after 8 weeks.

The application of leaf elongation is taken up again in Section VI, A.

C . LEAFAREA

Since leaf elongation rate is sensitive to nitrogen status, it follows that the rate of increase in leaf area should also reflect accurately the influence of deficiency on growth. Furthermore, lead area is both an expression of size and a partial expression of photosynthetic potential. In this context leaf area is taken to be total leaf area present either per plant or per unit ground area (LAI) as distinct from that of a selected expanding leaf as is used for leaf length.

Nutrition can influence photosynthesis (whence growth) through affecting leaf area itself or through changes in photosynthetic rate per unit leaf area (net assimilation rate). These aspects of crop nutrition have been reviewed by Watson (1963) who concluded that, for nitrogen, the main effect on growth is through leaf area rather than through net assimilation rate.

More specific evidence of the close relationship between leaf area and the influence of nitrogen on growth can be derived from the data of Bouma and Dowling (1966) and of Halse et al. (1969). Bouma and Dowling measured the dry weight and leaf area response of subterranean clover to nitrogen supply in water culture. I have computed the linear regression of the dry weight data on the leaf area data for each nitrogen level. It shows that 98% of the variation in dry weight is accounted for by the regression on leaf area. Halse measured photosynthetic area (green leaf plus stem area) and SNR in a wheat crop grown at three levels of nitrogen fertilizer: nil, 56 kg N/ha at sowing, and 336 kg N/ha split over three applications. Figure 9 shows my plotting of the change in leaf area against SNR for each period when stress was measured. The exercise shows a close relation between LA1 and nitrogen stress and a strong interaction with time.

What is required for the estimation of stress is a set of leaf area data (derived from appropriate plots split for nitrogen) from a range of nitrogen levels and

26

2.5

2.0

1.5 - 4

i i?l

C -

- c 1.0

0.5

0 -

E. A. N. GREENWOOD

-

-

-

-

.

4-7Weeks

0 6-9Weeks

A 10-13Weeks

X 13-16W~ks

1 1 L I I 00,

0 10 20 30 40 50

SN R (%I

FIG. 9. Relationship between increase in leaf area index (LAI) and nitrogen stress (Sm) in a crop of wheat over successive 3-weekly intervals. Nitrogen fertilizer treatments were nil, 56 kg N/ha at sowing, and 112 kg N/ha applied at sowing at 5 and at 10 weeks (taken here as producing zero stress). Data are derived from Halse et al. (1969).

which can be substituted into the expression

SNA = 100 [(AM-A)/AMI ( 5 )

where A is the change in leaf area of the deficient plant over a given time interval and A M is the corresponding change in leaf area for a plant given a nonlimiting dose of nitrogen at the beginning of the interval. The adequacy of leaf area as an estimator of SN could then be tested by plotting SNA against SNC which would be derived from the corresponding dry weight values.

The author is not aware of any set of data that completely fulfill these requirements other than a fragment from Bouma (1970a). In this instance, subterranean clover was grown in sterile culture solutions containing 4, 16, or 64 ppm N for 27 days. Among other treatments the 16 ppm plants were split into two groups: (1) the nitrogen level remained at 16 ppm; (2) the nitrogen level was raised to 64 ppm (assumed by me to be nonlimiting). Leaf area was estimated frequently. From Bouma’s Fig. 1 , at day 36, leaf area was 34.5 cm for 16 ppm


and 55.9 cm for 66 ppm. Substituting into Eq. (9, SNA = 38%. This value is very close to that for wheat at the same age and nitrogen supply (Greenwood, 1966) as also is the value of 59% derived by my extensive extrapolation from Bouma’s Fig. 1 for plants on 4 ppm N raised to 64 ppm.

The technique for assessing the nutrient status of plants used extensively by Bouma in Canberra over the last decade has all the essentials of the split-plot approach for estimating nutrient stress. It is unfortunate for this review that most of his research has been concerned with nutrients other than nitrogen, for it has led to a more specific understanding of the physiology of nutrient response. Because of the importance of the work to the field of nutrient stress, the essence of the technique and results will be given.

Bouma’s procedure is to grow subterranean clover seedlings in a nutrient solution containing all essential elements except the nutrient to be studied. The latter is provided as a pretreatment over a range of deficient levels. At a certain time some of the plants on each pretreatment are transferred to complete solutions, this time containing the previously deficient nutrient at a nonlimiting level. The treatment for the remainder is unchanged. On several occasions the leaf area of each plant is estimated using the photographic standards of Williams et al. (1964), and the carbon dioxide exchange rate (CER) is measured under standard conditions with an infrared gas analyzer. The results demonstrate the speed of response of leaf area and CER, and the relative importance of the contribution of leaf area and net assimilation rate. With these and other measurements, Bouma has studied what might be called the physiology of recovery from nutrient deficiencies such as nitrogen (Bouma, 1970a, b), phosphorus (Bouma, 1967a, b, 1969a, 1971, 1975; Bouma and Dowling, 1969a, b), sulfur (Bouma, 1967a, c, 1970c, 1971; Bouma et al., 1972), potassium and magnesium (Bouma 1970c), and boron (Bouma, 1969b). For most of these references it would be possible to compute nutrient stress in terms of leaf area, CER, and dry weight.

With the advent of bench and portable photometric devices the measurement of leaf area has become a rapid operation. If the area of intact leaves is measured, by either photographic standards or, better, electronic scanner, then it is feasible to consider using the expansion rate of selected individual leaves. This would be much quicker than measuring all leaves.

In conclusion, leaf area increment must rank as one of the simplest and most meaningful parameters of growth for estimating nitrogen stress of plants.

D. CARBON DIOXIDE EXCHANGE RATE (CER)

Photosynthesis being the major process for accumulation of dry matter in the plant, its rate is likely to be closely correlated with growth rate as controlled by nutritional deficiencies. The rate of CER may be considered as the most


comprehensive single measurement that can be taken to indicate instantaneous growth rate of the plant. So it would seem to be an ideal way of obtaining a rapid assessment of nitrogen stress, or any other nutritional stress. In practice it has some limitations. First, the initial cost of the apparatus for determining carbon dioxide concentration (infrared gas analyzer) is great, its use in the field is a little cumbersome, and it has a high maintenance requirement. Second, the rate of CER varies diurnally and between days and seasons. Hence, a near instantaneous determination of CER is not integrated over these variations and will give a biased result. Of course, a more representative result can be obtained by taking several CER readings but this is tedious.

If the value of CER of the deficient plant is E , and the corresponding value for a plant given a nonlimiting dose of nitrogen is EM, then the expression for nitrogen stress becomes

SNE = 100 [(EM-~IEMI (6)

It seems to me that the use of CER in studying nutritional stress has its greatest application in laboratory or controlled environment studies when CER for a large number of treatments can be compared under a standard environment. Bouma (Section V, C) has made great use of this approach. Wolf and Greenwood (unpublished) found that CER measurements in the laboratory gave very satisfactory results in the estimation of nitrogen stress in wheat seedlings. The application of these techniques to physiological studies in the laboratory is taken further in Section VI.

E. CONCLUSIONS

Five plant parameters have been proposed for estimating nitrogen stress-leaf nitrogen, dry weight, leaf elongation, leaf area, and CER. What are the criteria for choosing which one of these parameters (or any other which might be proposed) should be used? It has been argued that it is not feasible to measure nitrogen stress in a way similar to water stress, e.g., chemical potential, and that there is no single measurement that can be made which can be called “growth.” Therefore, for the present, practical considerations should be given high priority. The following criteria are helpful: (1) the parameter that has the greatest meaning for the objectives of the project (what measurements are being made irrespective of the measurement of stress); (2) the equipment and expertise that are available; and (3) the measurement that uses the least resources.

VI. Applications

It might be said of agronomists in general that they tend to apply treatments, obtain an end result, and then speculate as to how the measured outcome


occurred. Those who rigorously examine processes and their interactions come to be called crop physiologists! The foregoing remarks are indeed an oversimpli- fication and are not intended as a criticism insofar as there are obvious and compelling reasons why agricultural research does concentrate on yield of products.

But it must also be said that there is an aversion to studying the processes underlying yield, which is largely due to the daunting complexities and the lack of practical techniques to resolve them. The previous sections of this article have been directed to developing concepts and techniques that promise some easing of these constraints with respect to nutritional aspects of agronomy. This section deals with their practical application with the aim of suggesting the easiest way of doing the job in a variety of circumstances.

A. AGRONOMY AND ECOLOGY

Consider the basic operation of cultivation. Let us say that in certain situations it has been found to improve crop yield. And let us suppose that appropriate research showed that cultivation controlled weeds, thereby reducing competition for light, water, nitrogen, and other nutrients. On the other hand, the supply of available nitrogen and other nutrients was increased through microbial activity and through greater root exploration, . . . , and so on. In the event, how much of the response to cultivation can be ascribed to the nitrogen status of the plant during successive stages of growth? And from this, what deductions can be made about the mechanism and the timing of the nitrogen effect on final yield? Could we avoid cultivation by substituting an appropriately timed application of nitrogen and weedicide, and, if so, which would be the better solution? A comprehensive way of obtaining answers to such questions would be to have a large number of plots allocated to cultivation treatments and rates and times of application of nitrogen fertilizer. A shortcut to answering some of the questions posed, though certainly not all of them, would be to restrict the experiment to the two cultivation treatments upon which SN would be determined over successive periods. If, in addition to nitrogen stress, one were to determine other stresses such as water and phosphorus, then the agronomist would be in a strong position not only to answer some of the questions about nitrogen but also to comment on the importance of nitrogen vis-A-vis phosphorus and water stress.

Cultivation has been chosen as an example because data already presented (Table 111) as a partial illustration can be used.

A knowledge of nitrogen stress of a crop at a particular time does not provide a direct and accurate answer to the practical question: How much fertilizer is required to reduce stress to an acceptable level? This is the price to be paid for avoiding the response curve. Naturally it provides a partial answer; that is, whether the amount of fertilizer required is zero, a little, or a lot. In most cases


this may be good enough unless the agronomist has available an integrating model that qualifies the current biological requirement by economic and market- ing factors and also accounts for change in requirement with time.

A decade ago most agronomists worked exclusively in agricultural ecosystems. They could rely on a strong background of research literature and unpublished knowledge on which to base their future research policies. In the seventies, with increasing emphasis on nonagricultural ecology many agronomists have been assigned to work on ecosystems with which they are unfamiliar and for which little “hard” data exist. When one is confronted with a new ecosystem to be studied, the first questions to be asked are, What are the important limiting factors? How do they interact? The answers provide such penetrating insight into an ecosystem that they are almost a prerequisite for rationalizing research priorities. For example, there seems little point in embarking early, if at all, on a research program of plant nutrition or nutrient cycling in an ecosystem if it can be shown that nutrients are not important limiting factors. Similarly, if nutrition is limiting plant growth then which element is the most limiting?

The technique of evaluating nutrient stress is an efficient way of rating nutrients as limiting factors. The techniques described here for estimating nitrogen stress can also be adapted for other nutrients (e.g., Bouma er al., 1969), for temperature (Greenwood er aL, 1976), and for water (Power, 1971).

The most elegant example of this approach is the study by Power (1971) on the northern Great Plains of the United States. In these grassland ecosystems both nitrogen and water were known to be limiting plant growth. Since rainfall declined to the west it was considered that water stress would increase in that direction and that nitrogen stress would increase to the east. Further, as soil water declined during the growing season, water stress was expected to increase. At any one location and at any one time, what were the respective limitations to plant growth by the two factors? Power estimated SNR by using nitrogen fertilizer. Concurrently, he estimated water stress, SWR (Sw in his article), by a comparable technique in which water stress was removed by irrigation. An extract of the results is given in Table VI. There was a strong interaction with time, but the two stresses were, in general, additive. It became clear that, for the particular situation studied, nitrogen was more limiting than water.

The dry weight approach to stress used by Power is obviously appropriate for the arable grasslands of North Dakota. It would not be satisfactory for arid rangelands where the spatial distribution of plants is exceedingly variable and sparse. Here it would seem better to use a nondestructive method such as SNL , which has the real advantage for remote areas that there is no equipment to break down.

M. A. Ross and M. Friedel (personal communication) are currently evaluating leaf elongation rate of grasses in arid rangelands of Central Australia with a view to estimating SN and S, as did Power. Ross is studying the selection of


TABLE VI Percent Stress on Top Growth of Bromegrass Due to Nitrogen and Water

Deficiencies'

Sampling interval

Stress due to 4/28 to 5/22 5/22 to 6/10 6/10 to 6/30 6/30 to 7/22

56 stress

A. Low basal N (0-N)

N 4 73 61 104 Water 1 57 22 61 N + Water 8 83 73 103

B. Medium b a d N (9@N)

N -1 3 24 35 Water 2 32 32 10 N + Water 4 49 54 58

C. High basal N (270-N)

N 0 -7 7 13 Water 4 31 24 32 N + Water 5 23 33 59 LSD (0.5) ns. 18 17 25

'Reproduced from Power (1971).

suitable tillers relating leaf elongation rate to dry weight increments, and developing meaningful ways of applying nutrients and water in remote and and locations. He has observed an initial surge in elongation rate for a few days following a subsurface application of water to plants under long-term water stress. A similar study of leaf area increment is envisaged for dicotyledons. If these techniques are successful they will be used to estimate the changes in SN and S, which occur after effective rainfall.

A technique for estimating phosphorus status using the dry weight response of detached leaves when placed in solutions with and without the element, has been developed by Bouma and Dowling (1976). It should be adaptable to evaluating nitrogen stress. The approach seems appropriate to those situations where water is nonlimiting and where the standard conditions of response imposed match the cultural conditions of the plant, as in the laboratory. In the field the technique would be most useful where the nutrient in question is the major limiting factor, otherwise some bias in the magnitude of response may develop. Further work on the development of this technique is proceeding (Bouma, personal communication).


B. STRESS PHYSIOLOGY

If functional relationships between nutrient deficiency and specific plant processes are to be established, then two conditions must be met. First, the deficiency must be expressed in terms that arise from the plant itself as distinct from some external supply term. Second, the stress must be expressed numerically. The general failure of nutritional physiologists to meet these two conditions has led to a crippling weakness in research in, and also an avoidance of, this field. It is indicative that the abstracting journal Current Advances in Plant Science cites very few papers dealing with nutrient stress in Section 21 on Stress physiology.

It must be admitted that some of the techniques of estimating nitrogen stress, as have been reviewed here, are barely adequate to meet the precision and speed often required by physiologists, particularly in the laboratory. Three suggestions follow.

The most suitable technique for physiologists so far discussed is that by Bouma for the determination of CER using the infrared gas analyzer. Either the whole plant or a selected leaf can be used. CER determinations are made in the deficient plant and on a comparable plant for which nitrogen stress has been removed by an adequate addition to the nitrogen supply about 2 days prior to the measurements. The major requirements are a controlled environment chamber, an infrared gas analyzer, and an air-sealed photosynthesis chamber of appropriate geometry (Wolf et aL, 1969).

Wolf and Greenwood (unpublished) have designed an extremely simple and sensitive arrangement for determining SNE in grasses and cereals on a relatively large scale. In this system the whole controlled environment chamber becomes the equivalent of the mixing chamber of the above-mentioned air-sealed device in which pots can be placed. The air in the large chamber is homogenized with one or more fans. The leaf is inserted in a glass tube through which the homogenized air is continuously drawn. Reference air is similarly sampled and both streams are led to the external analyzer which determines CER by the difference method. A large battery of plants can be harnessed prior to a run and the tubes may be left on the leaves indefinitely, provided that the leaf does not expand beyond the dimensions of the tube. Speed and precision can be obtained with this arrangement. A manifold of 2-way taps is installed outside the growth chamber so that when the air from a particular leaf is not being analyzed it wil l still be drawn over the leaf by another pump at the same rate. This reduces equilibration time within the tubing almost to zero and allows successive determinations of CER to be made at about 2-minute intervals. Some simple precautions must be taken to isolate the air inside the growth chamber from the massive fluctuations in carbon dioxide which may occur outside. The resolution


and precision of this technique is demonstrated indirectly in Fig. 4, and, of course, it is nondestructive. Wolf and Greenwood used the technique only for one type of situation, but it seems capable of wide adaptation within a controlled environment.

A further nondestructive laboratory technique is available for obtaining short- term weight changes such as would be required by whole-plant physiologists. This is the weighmg technique of Amott et al. (1974), which permits separate live weighings of tops and roots of plants to be made at frequent intervals and with changes in nitrogen supply. Very good estimates of SN on a fresh weight basis can be obtained with this device while the plants themselves are available for other physiological measurements. Two units would be required for each estimate of stress.

C . MODELING

The concepts of nutrient stress can be of direct use to modelers of plant growth in at least two ways. First, a prior knowledge of the stress values for several nutrients allows the modeler to rank the elements in order of importance. He can then filter out the unimportant elements. Second, if the model first generates a potential growth rate which in turn is successively reduced according to the constraints from each important element, then the stress values themselves will be appropriate terms to govern the magnitude of those reductions without necessarily calling on a nutrient supply subroutine. Where the supply of the nutrient, say nitrogen, is also being generated then stress can be estimated through the following steps. F;-iGT-ty/ _(,,,,,J {-iziq

= supply controller increment CO, , etc.

Actual I increment

Nitrogen demand is estimated by computing the product of the potential dry matter increment and the nitrogen concentration in the plant. The concentration is derived from a curve of the time course of nitrogen for a plant grown on a nonlimiting supply of nitrogen. This information requires minimal experimentation. The supply of nitrogen is generated by the supply subroutine. The final step is to reduce the potential dry matter increment by a proportion given by the ratio N supply/N demand, which is, of course, a term similar to nitrogen stress.


VII. Conclusions and Aspirations

In this article the opinion has been expressed that the discipline of plant nutrition has badly neglected the quantification of deficiency. It has been suggested that the conventional approaches to deficiency through response curves and tissue analyses are inadequate bases on which to quantify deficiency rigorously except in special circumstances. By analogy with water stress, an attempt has been made to establish the basic requirements for a concept of nutrient stress and a proposal has been offered on how they might be put into practice. But the latter has proved too difficult to accomplish without some compromise and there has been insufficient experimentation to evaluate fully the several techniques available. It is hoped that this article will stimulate the evolution of plant nutrition whether it be through the development of the ideas presented or through more fruitful ideas arising from them.

ACKNOWLEDGMENTS

The author is grateful to Dr. J. F. Power, Dr. M. A. Ross, and Dr. D. D. Wolf for supplying unpublished data and to Dr. N. J. Barrow, Dr. D. Bouma, and Mr. G. B. Taylor for their help with the manuscript. The use of leaf elongation was suggested by Dr. R. F. Williams. The work with Dr. Wolf was done while the author was a guest of Virginia Polytechnic Institute. Special thanks go to Dr. R. C. Rossiter for the many enjoyable arguments over the concepts of nutrient stress.

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[advances in agronomy] volume 28 || nitrogen stress in plants

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