FACTORS AFFECTING NITRATE ACCUMULATION IN HERBAGE
Dr Richard Eckard, The Institute of Land and Food Resources,
University of Melbourne & Agriculture Victoria, Ellinbank
TABLE OF CONTENTS
INTRODUCTION
CHAPTER 1 - THE GENERAL METABOLIC ROLE OF NITRATE
CHAPTER 2 - INTERNAL FACTORS
    2.1 Interspecific Differences
    2.2 Localisation
    2.3 Age
    2.4 Nitrate Reducing Enzymes
CHAPTER 3 - EXTERNAL FACTORS
    3.1 Moisture
    3.2 Irradiance
    3.3 Temperature
    3.4 Herbicides
CHAPTER 4 - MANAGEMENT FACTORS
    4.1 Nutrient Supply
         4.1.1 Nitrogen
        4.1.2 Other Nutrients
    4.2 Re-Seeding
    4.3 Cutting Interval and Height of Cut
DISCUSSION AND CONCLUSION
SUMMARY
LITERATURE CITED
INTRODUCTION

The abundant and mobile nitrate ion occupies a position of primary importance in the metabolism of higher plants. It is the major nutrient form of nitrogen in most soils and is often the first factor limiting plant growth. In the great majority of cases it is assimilated so rapidly that it's concentration within plant tissues never rises to critical levels (Wright & Davison 1964).

In 1962, Van Burg (cited by Denium & Sibma 1980) reported that herbage production was not retarded by lack of nitrogen as long as nitrate contents of 0.15% in Italian ryegrass were maintained. Thus, a certain minimum nitrate level exists for maximum growth. On the other hand, nitrate has been observed to accumulate within plants to abnormally high concentrations, with results disastrous to ruminants fed these plants; or, to animals and humans exposed to the gaseous decomposition products of the plants.

Large amounts of nitrogen from chemical fertiliser and from slurry and manure are the main causes of high nitrate in herbage and forages. This leads to feeds high in nitrate, which have been found to cause acute poisoning in cattle, sheep and other livestock (death, nervous collapse, abortion and bloat). Many cases of chronic poisoning have also been reported, as well as significant production losses due to sub-clinical nitrate toxicity (Denium & Sibma 1980).

Over the past few months a number of reports have been received of livestock deaths from nitrate toxicity in the Natal Midlands. Most of these reports are of cattle on ryegrass and in a few cases cattle on kikuyu pastures. In most cases, management malpractice or lack of adequate education was the major problem. An increasing number of queries have been received by the Cedara Research Station in 1984, concerning nitrate toxicity problems on farms, and a thorough investigation into the problems associated is necessary.

A number of factors have been isolated as being causative agents of nitrate accumulation. Since nitrate levels often approach or exceed levels of known toxicity or sub-clinical toxicity, this paper has been written to present some observations and parameters on these factors.


CHAPTER 1

THE GENERAL METABOLIC ROLE OF NITRATE

The nitrate ion is important to protein synthesis in plants, as most of the nitrogen absorbed by plants is in the form of nitrate (NO3) (Wright & Davison 1964; Huffaker & Rains 1978). This is true as long as the soil pH is not limiting, as mineralisation or, in particular, nitrification in soils is restricted by a low pH (Nyborg & Hoyt 1978). Mclean, Halstead and Finn (1972) showed that, at higher pH levels the nitrate contents in the soil increased markedly.

Soil microflora are principally responsible for the nitrification process and these microbes are more inhibited at low pH levels. These micro-organisms derive their energy by oxidising ammonia with the formation of nitrate. The abundance of these micro-organisms under reasonable pH levels results in most of the soil nitrogen being taken up by the plant in the form of nitrate (Mclean, Halstead & Finn 1972; Nyborg & Hoyt 1978).

The micro-organisms involved in the process of nitrification are, firstly, the aerobic chemolithotroph Nitrosomonas, which oxidises ammonia to nitrite, thus gaining an electron in its respiration cycle and secondly, Nitrobacter, which obtains its energy from the oxidation of nitrite to nitrate (Lehninger 1979).

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Nitrate is the principle form of nitrogen available to higher plants from the soil (Huffaker & Rains 1978) and it's metabolic assimilation into the form of protein nitrogen proceeds in two major steps:

a) Reduction of nitrate to nitrite and;

b) Reduction of nitrite to ammonia (Lehninger 1979).

The accumulation of nitrate implies that the rate of assimilation has not kept pace with the rate of uptake of nitrate from the soil. Often this accumulation is only temporary, diminishing over a short period (Wright & Davison 1964). The accumulation of nitrate is not, as it is known, injurious to the plant (Wright & Davison 1964; Barker, Peck & McDonald 1971; Denium & Sibma 1980). It must be noted that herbage production is not retarded by a lack of nitrogen as long as nitrate content of 0.3% to 0.6% of the dry matter is maintained (Van Burg 1962, cited by Denium & Sibma 1980). This indicates that nitrate is the major source of nitrogen for the plant.

Sites of reduction of nitrate have been found in the roots and in the leaves of the plant, but the nitrate reductase enzyme is most active in plant leaves, especially younger leaves at higher irradiance (Darwinkel 1975; Viets & Hageman 1971, cited by Denium & Sibma 1980). This suggests that most nitrate is reduced in the leaves. Dijkshoorn (1971, cited by Denium & Sibma 1980) found that almost all nitrogen in maize xylem sap was in the form of nitrate. Wheat however, has been shown to reduce 88% of nitrogen in the roots before uptake (Miflin 1979, cited by Denium & Sibma 1980). Perennial ryegrass (L. perenne) was found to follow the same trend whereas Italian ryegrass (L. multiflorum) was found to follow the trend of maize by reducing most of its nitrogen to organic form in the leaves (Alberda 1965; Darwinkel 1975; Denium & Sibma 1980).

CHAPTER 2

INTERNAL FACTORS

2.1 Interspecific Differences

Taxonomic units of plants are known to differ in their tendency to accumulate nitrate. Both surveys of vegetation and planned comparisons in experiments have provided examples of varietal, specific, generic and familial rankings. The most basic of these rankings is the division of plant species into, either:

a) Accumulators of nitrate and;

b) Non-accumulators of nitrate.

The following plant families are reckoned to belong to the nitrate accumulating group: Amaranthaceae, Chenopodiaceae, Compositeae, Convulvulaceae, Polygonaceae, Solanaceae, Gramineae and Cruciferae (Kingsburg 1958; Wright & Davison 1964). In the family of Gramineae the following are distinguished as being:

a) Nitrate accumulators: oats, maize, rye, wheat, barley and Italian ryegrass and meadow fescue in early cuts.

b) Nitrate Non-accumulators: Timothy, brome-grass, alfalfa, cocksfoot, white clover and perennial ryegrass (Crawford, Kennedy & Johnson 1961; Wright & Davison 1965; Darwinkel 1975).

In Table 1. differences between species as to nitrate content are shown with Lolium multiflorum being rich in nitrate, whereas differences in yield relative to the other species were small (Denium & Sibma 1980). In Table 2. the nitrate content of Italian ryegrass is shown to be significantly higher than that of perennial ryegrass. Investigation into a number of species (Behaeghe & Carlier 1974, cited by Denium & Sibma 1980) revealed Festuca pratensis and Lolium multiflorum as being richest in nitrate, followed in decreasing order by Phleum pratense, Dactylis glomerata and finally Lolium perenne.

Table 1. Interspecific differences as to nitrate content on three species at three irradiance levels (after Denium & Sibma 1980).

Irradiance

(J/cm2 /d)

Nitrate Content in Dry Matter

(g/kg)

  Lolium multiflorum Brachiaria ruziziesis Setaria sphacelata
715

1515

3000

65.5

35.4

19.9

39.9

13.3

8.4

38.5

9.3

4.4

There is ample evidence that hierarchies, as to nitrate content, do occur between plant species and families, but, plant to plant variation within a species can be enormous due to a number of factors (Wright & Davison 1964). Some plant species, normally low in nitrate can, under specific conditions, accumulate nitrate to dangerous levels. As an example of this, the perennial forage crops have been found to be relatively low in nitrate and discounted as a source of high nitrate. Murphy and Smith (1967) have, however, observed perennial forage crops to accumulate nitrate to dangerous levels under favourable conditions as did Carey, Mitchell and Anderson (1952); Kretzchmer (1958); Smith and Sund (1965) and Darwinkel (1976).

Table 2. A comparison of the nitrate content of Italian ryegrass (L. multiflorum) to that of perennial ryegrass (L. perenne) in a pot experiment (after Darwinkel 1976).

Species Dry Matter Yield (g) Nitrate (%)
multiflorum 1st cut

5th cut

Perenne New Sown Plants

Old Plants

10.74

18.73

22.12

16.75

1.55

0.67

0.62

0.38

The data presented in Table 2. (after Darwinkel 1976) indicates that, at the high nitrogen fertilisation rate, toxic or potentially sublethal levels of nitrate can accumulate even in perennial ryegrass. Levels of 1,55% as obtained in the first cut of Italian ryegrass, would be lethal and the lowest level of 0,38% in the old plants of perennial ryegrass are above the 0,21% to 0,35% lower limit for toxicity to ruminants (McCreery, Hojjati & Beaty 1966; Lawrence, Warder & Ashford 1967; Lovelace, Holt & Anderson 1968; Gomm 1979; White & Halvarson 1980).

Such varied differences between forage species, may well be due to the genetic make-up of the plants, where one variety can assimilate the nitrates into proteins more rapidly than others (Gul & Kolp 1960) or, such differences in NO3 content can he explained by differences in the distribution of dry matter. If a considerable portion of the dry matter produced accumulates in plant parts poor in organic nitrogen, such as roots and stems, nitrate conversion is restricted. On the contrary, a large accumulation of dry matter in leaves stands for a good conversion. It is possible that the lower nitrate levels in perennials and the higher levels in annuals are connected with this difference in dry matter distribution (Darwinkel 1976). On the basis of the above argument it may be possible to select high-yielding varieties which will contain a smaller amount of nitrate, since forage yield and plant height are correlated with the amount of nitrate accumulated (Gul & Kolp 1960).

2.2 Localization

It is well established that nitrate is not uniformly distributed throughout the plant to all the tissues. In terms of grass structure stems usually contain more nitrate than do the roots, roots usually more than the leaves and leaves usually more than floral parts (Crawford et al 1961; Wright & Davison 1964; Alberda 1965; Darwinkel 1976; Denium and Sibma 1980). The upper regions of the stems also tend to have a lower nitrate content than the lower regions (Wright & Davison 1964).

The vast difference in nitrate content, over the profile of the profile of the plant, emphasises the importance of controlling cutting or grazing height of forages, as a lower cut or heavier grazing regime will lead to the inclusion of more stems in the forage , thus a higher nitrate percentage.

The younger leaves on a plant are low in nitrate and the older leaves are generally higher in nitrate as nitrate reductase activity decreases with age and thus less nitrate is assimilated (Darwinkel 1975). Nitrate content of petioles, leaf sheaths and stems can be very high as in inferred in Fig. 1. in which herbage was cut in layers of 5 cm. The trend was always increasing towards the bottom. Slopes were steeper when average nitrate content was lower (Denium & Sibma 1980). This phenomenon is expected since flow of nitrate into the leaves continues during the life span of the plant whereas nitrate reductase declines rapidly with age (Darwinkel 1976).

Figure 1. Distribution of nitrate content over the profile (steps of 5 cm height) of some grass crops. (Darwinkel 1975).

Table 3. presents the distribution of nitrate between herbage stubble and roots. With ageing of the sward an even smaller proportion of the nitrate is found in the herbage (Darwinkel 1975). The high nitrate content of the stubble indicates that nitrate accumulates there.

Table 3. Nitrate content of herbage as compared to stubble and roots in a pot experiment with high nitrogen supply (after Darwinkel 1976).

SPECIES
HERBAGE
HERBAGE
STUBBLE
+ ROOTS
DISTRIBUTION OF
TOTAL NITRATE
 
Yield (g)
NO3-N (%)
Yield (g)
NO3 -N (%)
Herbage(%)
Stubble + Roots(%)
L.multiflorum
1st cut
5th cut
6.43
15.96
1.66
0.84
4.31
2.77
1.37
3.72
65
57
35
43
L. perenne
New sown plants
Old sown plants
14.53
16.13
0.65
0.32
7.59
0.62
0.73
6.94
63
54
37
46

Sites of reduction play an important role in localisation of nitrates. Woody plants have been shown to reduce nitrate in the root system (Bollard 1960, cited by Wright & Davison 1964). Herbaceous species, however, tend to reduce nitrate in the photosynthetic part of the leaves and nitrate is transported via the xylem from the roots (Wright & Davison 1964). The accumulation of nitrate at higher levels, in the lower regions of the plants, indicate that this could not he as a result of storage in the xylem, as concentrations of nitrate found in lower stems exceed the capacity of the xylem. A possible answer proposed by Wright and Davison (1964) is that of active accumulation by tissues adjacent to the xylem. Vascular parenchyma and other highly protoplasmic cells might thus be the main sites of heavy nitrate accumulation.

To conclude on the data and information presented on localisation of nitrate, is to point out that an animal grazing from top to stubble on a rotational grazing system, could be in danger of toxicity if return to grazing is too rapid, or if camps are over grazed.

2.3 Age

The title of age falls into two distinct groups: a) The age of the plant and; b) The age of the regrowth of herbage.

The age of the plant is from planting to sampling date and the age of the herbage is from the last cut or grazing to the sampling date.

Experiments involving periodic sampling of plants through a cycle of growth, have shown that, nitrate content first rises and then, after reaching a peak at the prebloom stage, declines as the plant matures (Wright & Davison 1964). This would mean that nitrate content decreases over the life of the plant, but is high in recent growth.

Figure 2. Effect of age of regrowth on nitrate content of herbage, with cutting and nitrogen application on 2 June 1972 (after Prins & Von Burg 1974, cited by Denium & Sibma 1980).

Figure 2. shows a rapid increase in nitrate, soon after the fertiliser application, to a peak at approximately seven days (fourteen days for highest level), thereafter a gradual decline of accumulated nitrate as the herbage matures. This is explained by Darwinkel (1975) as, new regrowth demanding a higher level of nitrate to assimilate to plant protein for growth. Newly sown swards and annual grasses have only a limited capacity to store nitrate in the stubble and roots and consequently a major part of the nitrate is translocated to the herbage.

A study on oats by Gul and Kolp (1960) revealed highly significant differences between stages of growth of the plant in nitrate accumulation. Each stage of growth studied was observed to be significantly lower in percentage nitrate than the preceding stage. The samples containing the largest percentage of nitrate were at the prebloom stage and nitrate content decreased as the plant matured thereafter. This indicates that young plants that are toxic to livestock may become less toxic as the plants mature.

Table 4. Nitrate decrease with age of plant and regrowth at high nitrogen supply in a pot experiment (after Darwinkel 1976).

SPECIES
DRY MATTER YIELD
(g)
HERBAGE NO3-N
(%)
L. multiflorum
1st cut
5th cut
10.47
18.73
1.55
0.67
L. perenne
Newly sown plants
0ld sown plants
22.12
16.75
0.62
0.38

The results of Darwinkel’s (1976) pot experiments presented in Table 4., show clearly how, with ageing of the plant sward, a smaller proportion of nitrate is found in the herbage of Lolium multiflorum. The data shows that, regardless of age of regrowth, there is still a decrease in the nitrate percentage accumulated in the herbage with age. In the same experiment on a perennial species (Lolium perenne) Darwinkel (1976) saw a similar decrease in accumulated nitrate with age.

2.4 Nitrate Reducing Enzymes

In recent years much attention has been given to the enzyme, nitrate reductase (Hageman, Flesher & Gitter 1961; Hageman, Creswill & Hewitt 1962; Miflin 1968; Beeves & Hageman 1969; Darwinkel 1975; Huffaker & Rains 1973; Denium & Sibma 1980). Measuring the activity of the enzyme may permit a more direct determination of the reduction of nitrate in the plant (Darwinkel 1976).

Darwinkel (1975) found, for most forage crops, that the nitrate reductase activity (N.R.A.) is considerably higher in leaf blades than in other regions of the plant. This evidence is substantiated by Beevers and Hageman (1969), who found that nitrate reductase activity in the roots was limited and made very little contribution to the nitrogenous components of the mature plant, except under conditions of severe nitrogen deficiency, where the reduction of nitrate in the roots is important. Evidence is strong (Beevers & Hageman 1964; Huffaker & Rains 1978) that after nitrate is inside the plant, nitrate reductase is a rate-limiting enzyme in the biochemical pathway for reduction of nitrate to plant protein, along with nitrite reductase.

The enzymatic pathway of reduction or assimilation proceeds to two major stages:-

a) Reduction of nitrate to nitrite and;

b) Reduction of nitrite to ammonia (Lehninger 1979).

The first reduction is catalysed by nitrate reductase. Nitrate reductase is a molybdenum based metalloflavoprotein, which employs NADPH as an electron donor. The overall process of electron flow to nitrate may be summarised in the following scheme:

wpe1F.jpg (2054 bytes)

The molybdenum appears to undergo cyclic valance changes between Mo(IV) and Mo (VI) during the reduction of nitrate (Beevers & Hageman 1969; Lehninger 1979).

The reduction of nitrite to ammonia, by nitrite reductase in plants requires a highly electronegative reductant. In green plants, ferredoxin reduced during the light reactions, may serve as the ultimate reductant of nitrite in the following chain of reactions:-

wpe20.jpg (2370 bytes)

(after Lehninger 1979; O1sen & Kurtz 1982).

Summarising the above reactions into the overall assimilation process:-

wpe22.jpg (3790 bytes)

(after Olsen & Kurtz 1982).

Nitrate reductase activity is influenced by the age of the leaf. Darwinkel (1975) found a drop in nitrate reductase activity with increased age. This decreasing effect is also found between leaves and tillers of Italian ryegrass. This means that, with age, less nitrate is converted per unit dry matter produced and thus herbage protein levels decline (Fig. 3.).

The potential accumulation of nitrate in the herbage, due to the decreased nitrate reductase activity, is marked by the increased accumulation of nitrate in the stubble. This leads to less nitrate being trans-located to the herbage with increase in age of the plant. Another factor marking the accumulation of nitrate would be the dilution effect that the large increase in dry matter of herbage has on the nitrate in ageing herbage (Darwinkel 1975).

Figure 3. Organic nitrogen content in herbage over the growing period (after Darwinkel 1975).

Any limit imposed on the enzyme system could however, lead to an increase in the potential for nitrate to accumulate in the herbage. A deficiency of molybdenum is known (McKee 1962) to inactivate nitrate reductase and on addition of molybdenum only, the enzyme is reactivated.

Likewise nitrite reductase is a metalloflavoprotein and uses ferredoxin, which is iron containing (McKee 1962; Lehninger 1979), as a co-factor. A deficiency of iron would inactivate nitrite reductase and cause nitrate to accumulate.

A number of other factors have been known to affect nitrate reductase activity. Light intensity and a diurnal variation in nitrate content was observed by Hageman, Flesher and Gitter (1961). Thus the accumulation of nitrates under shaded conditions may be a direct result of a decrease in nitrate reductase activity. The reason for such accumulation could be that, in green tissue, both Ferredoxin and nitrite reductase are located in the chloroplasts and as a result nitrite reduction could be closely linked to solar energy (Huffaker & Rains 1982). Temperature and moisture have also been shown to be positively correlated to nitrate reductase activity (Huffaker & Rains 1982). It would appear that nitrate reductase does affect nitrate accumulation but, from the literature, it is not clear as to the significance of these effects.


CHAPTER 3

EXTERNAL FACTORS

3.1 Moisture

The incidences of nitrate poisoning in livestock have, in many cases, been connected with marked reductions in the normal rainfall levels, as well as being more frequent in semi-arid and sub-humid areas (Wright & Davison 1964). Denium and Sibma (1980) explained this as nitrogen in moist conditions being depleted and also diluted over a greater biomass.

Table 5. Nitrate concentration (% NO3-N) in four species of meadow plants, grown for 45 days at 30C with 440 kg N/ha with three moisture level treatments (low, medium & high) (after Gomm 1979).

SPECIES
LOW
MEDIUM
HIGH
A
1.32
1.07
0.01
B
0.83
0.61
0.08
C
0.77
0.96
0.04
D
0.94
0.99
0.04

In the experiments of Gomm (1979) the dominating effect of soil moisture on the concentration of nitrate is well demonstrated (Fig. 4,). The plants grown in saturated soils were low in nitrate and, even at high levels of nitrogen, the nitrate contents were well below known toxicity levels.

Several authors have reported studies that explain the effect of moisture on nitrate concentration. Tisdale and Nelson (1975) and Gomm (1979) reported that waterlogged soils tend to be more anaerobic than most soils, thus inhibiting microbial conversion of ammonia to nitrate. These conditions also enhance the conversion of nitrate to ammonia, a process known as Denitrification (Olsen & Kurtz 1982). On the other hand, nitrate is released from the more complex organic forms by microbial activity that requires moisture. The nitrogen thus released, as well as any nitrogen added as fertiliser, must then move, through water, to the absorbing roots (Wright & Davison 1964; Gomm 1979; Olsen & Kurtz 1982). Thus a certain amount of moisture is needed in the soil, and either extreme in soil moisture would lead to abnormal conditions.

Figure 4. Average nitrate NO3-N concentration in meadow plants as affected by soil moisture and fertiliser levels (Gomm 1979).

3.2 Irradiance

A number of factors associated with irradiance are known to affect nitrate content in herbage. These factors include, soil temperature and water supply as these two factors stimulate nitrogen mineralisation, nitrification and root functions (Denium & Sibma 1980). Increased irradiance can increase the temperature of the top layers of the soil by a far greater extent in the short tern, than air temperature does. Higher soil temperatures will be caused by increased solar energy and thus increase available nitrogen (Alberda 1965; Denium & Sibma 1980).

Darwinkel (1975) found an increase in the activity of nitrate reductase with increased solar. energy. In most experiments with varying irradiance, biomass is stimulated by higher irradiance and thus nitrogen supply becomes limiting, leading to low nitrate and protein levels. On trials where nitrogen was not limiting, nitrate levels were lower and protein content higher with increased irradiance (Denium 1971, cited by Denium & Sibma 1980).

The relation between nitrate reductase in the leaves and irradiance has often suggested that, nitrate content of herbage would be low after a bright day and high after a cloudy day. This suggestion has rarely been substantiated by the facts (Denium & Sibma 1980) as all associated factors tend to increase available nitrate to the herbage with increased irradiance. A positive correlation can even be found between irradiance and nitrate content if the high irradiance induces a water shortage. As a result, the net effect of increased irradiance on the herbage plant nitrate levels is not profound.

Table 6. presents data indicating an inverse relation of nitrate to irradiance in the short term. In this trial, Denium (1965; cited by Denium & Sibma 1980) covered the soil, to control soil temperature. Significant effects of irradiance changes were only found after four to five days. This time. lag of four to five days would appear to be general, since it also takes four to five days after nitrogen dressing before nitrate content in the herbage increases (Alberda 1965; Denium & Sibma 1980). So it is extremely difficult to detect re-liable and significant short tern trends in nitrate content of herbage.

3.3 Temperature

It is difficult to detect the true effects of temperature on herbage nit rate levels, as the effect of temperature is often marked by the release of nitrogen from the soil, due to nitrification as well as the dilution of the available nitrogen over the herbage produced as a result of temperature (Olsen & Kurtz 1982).

Alberda (1965) found a small positive effect of temperature on nitrate content in Italian ryegrass. The possibility still exists however, that the effect of temperature on the nitrate content in the plant is indirect, as transpiration is higher at a higher temperature and thus resulting in a difference in the amount of nitrate carried into the leaves by the transpiration stream (Alberda 1965).

Table 6. Effect of change of irradiance on content of nitrate in L. perenne at high Nitrogen levels on successive days (after Denium 1965; cited by Denium & Sibma 1980).

TIME AFTER SWITCH
IRRAD IANCE
400 to 2,000
(J/'cm2 /d)
IRRAD IANCE
2,000 to 400
(J/'cm2 /d)
(Days)
NO3 - N(%)
NO3 - N(%)
0
1
2
5
2.36
2.98
2.46
1.56
0.98
0.96
1.27
1.72

Figure 5. Effect of temperature on nitrate content with age (after Crijns 1979; cited by Denium & Sibma 19S0).

The nitrate content of the leaves in Fig. 5. show nitrate as being higher at 200C than at 120C. The converse was true for the roots however, with higher nitrate in the roots at the lower temperature. This indicates that uptake of nitrate was not affected by the change in temperature as much as nitrate reduction was. Reduction of nitrate was less as organic nitrogen was less at the higher temperatures, as was water soluble carbohydrate. The accumulation of nitrate could be explained as being a lack of carbohydrate as substrate in the roots. At a higher level of carbohydrate Alberda (1965) found that the positive effect of temperature on nitrate uptake tends to dominate over the negative effect of reduction. Gomm (1979) found a positive effect of temperature on nitrate accumulation, but indicated that, many environmental interactions linked to temperature were combined to cause the overall increase in nitrate with temperature rise (Table 7).

3.4 Herbicides

The effect of a herbicide (2,4D) on nitrate levels was Looked at as early as 1950 Stahler and Whitehead (1950, cited by Wright & Davison 1964 reported nitrate levels in herbage, sprayed inadvertently with 2,4D, to be as much as twenty times that of unsprayed plots. Wright and Davison (1'~64) indicate that herbicides do have a slight effect on increasing nitrate levels but that satisfactory generalisations regarding the influence of herbicides on nitrate levels are not available. Recent work on the effect of herbicides on nitrate levels in herbage would appear to be lacking.

Table 7. Nitrate content in meadow plants as effected by air temperature at two levels (15C and 30C). Plants were grown for 45 days at nitrogen fertilisation rates of 440 kg N/ha and at two light intensities (after Gomm 1979).

 
15C TEMP
15C TEMP
30C TEMP
30C TEMP
SPECIES
HIGH
IRRADIANCE
LOW
IRRADIANCE
HIGH
IRRADIANCE
LOW
IRRADIANCE
1
2
3
4
0.27
0.39
0.32
0.55
0.47
0.56
0.70
1.24
1.07
0.61
0.96
0.99
0.82
0.84
1.80
2.43

CHAPTER 4

MANAGEMENT FACTORS

4.1 Nutrient Supply

Nitrogen, in plant nutrition, requires more attention than any other nutrient. Nitrogen is subject to losses via ammonia volatilisation, denitrification and leaching. Most of the soil derived, taken up by plants, is in the form of nitrate (Olsen & Kurtz 1982) in well drained soils. With nitrogen application, nitrate content of the plant rises rapidly to a peak within two to three weeks and declines less rapidly over a period of months (Fig. 6.) (Denium & Sibma 1980).

Figure 6. Effect of nitrogen application on nitrate content ~ kg) of herbage. Cutting and nitrogen applications on 2 June (after Prins & Van Burg, cited by Denium & Sibma 1980).

This decline with age (Fig. 6 & 74) is rather rapid in spring and summer, but may be slow or even non-existent in late autumn (Denium & Sibma 1980; White &Halvarson 1980). Consequently it not always helpful to take precautions against nitrate toxicity by delaying harvesting of the crop in autumn.

Figure 7. Relationship between nitrate nitrogen and days after nitrogen fertilisation with three levels of nitrogen application on Italian ryegrass (after Bredon 1979, unpublished data; Eckard 1984, unpublished data).

In a number of experiments on Cedara (Fig.7., applied nitrogen to Italian ryegrass, was shown to markedly affect nitrate content in the herbage. At higher levels of application nitrate took approximately ten days to reach toxic levels and only after sixteen to twenty days was the pasture regarded as safe with regard to know toxic levels of nitrate. These results correlate closely with those of ap Griffith (1960) who found a linear response of nitrate to applied nitrogen fertiliser.

In Figure. 8., a marked increase in nitrate is shown to occur from 20 -22% protein upwards (after ap Griffith 1960), and Fig. 9., Bredon ~ 979, unpublished data) gives an advisable range of crude protein for grazing animals after nitrogen fertilisation. Both Fig. 8 and Fig 9. indicate that, to avoid nitrate toxicity, as well as possible ammonia toxicity, animals should only be returned to grazing a pasture in which crude protein levels are 22% or below (Bredon 1979, unpublished data).

Figure 8. Relationship between percentage crude protein and nitrate nitrogen in herbage (After ap Griffith 1960).

Figure 9. Effect of level of application of nitrogen and days since application on percent of crude protein in the dry matter of Midmar Italian ryegrass (Bredon 1979, unpublished data).

It should also be emphasised that organic fertilisers and slurry lead to nitrate accumulation, if heavily applied (Wright & Davison 1964). The form of nitrogen applied to the soil is affected by soil conditions. In moist, warm and well-aerated soils, most of the nitrogenous compounds will be converted to the nitrate form. If the soil is badly aerated or under anaerobic conditions, the applied nitrogen may be converted more to the ammonia form and plant uptake will be restricted (Tisdale & Nelson 1975; Olsen & Kurtz 1982). Soil pH has an effect on nitrate uptake by the plant as liming of soils increases available nitrate (Maclean, Halstead & Finn 1972).

Applied nitrogen fertiliser is thus an important factor influencing nitrate accumulation in herbage, as long as normal soil conditions are maintained. Return to grazing or cutting would therefore be important in management as, returning animals to grazing or cutting herbage too soon after nitrogen fertilisation, could lead to toxic levels of nitrate being present in the ingested forage.

Phosphorous has an indirect effect on nitrate levels in plants due to it's multiple functions in plant metabolism. A plant deficient in phosphorus will lack NADPH, on which the nitrogen reducing enzymes depend (Lehninger 1979) and any nitrate taken up will thus accumulate.

Potassium in soils is taken up as an ion by the plant in the K+ form, and is thus important to the uptake of the nitrate ion (NO3) in maintaining ionic balance. The influence of potassium on nitrate levels is therefore largely catalytic (Tisdale & Nelson 1975). Potassium is however, essential in protein synthesis and photosynthesis is decreased with insufficient potassium. Studies on potassium deficient plants (Tisdale & Nelson 1975) have shown an accumulation of nitrate in the herbage as, conversion to protein is restricted. With the addition of potassium, the levels of nitrate decreased with a corresponding increase in protein levels. The accumulation of nitrate under potassium deficient conditions is possible as, nitrate may use the sodium ion (Na+) or the calcium ion (Ca2+) to maintain ionic balance on uptake by the plant (Tisdale & Nelson 1975).

Sulphur is an important constituent of many proteins and a deficiency could lead to a nitrate accumulation (Adams & Sheard 1966). Studies in the Netherlands (Tisdale & Nelson 1975) showed that the danger of high nitrate levels in plants could be reduced by adequate sulphur fertilisation. High nitrate levels were shown to be associated with wide N : S ratios in plants. Ratios of 10% N 1% S to 20% N : 1% S are usually considered suitable for plants (Olsen & Kurtz 1982). Ferredoxin is the physiological co-factor of nitrite reductase (Lehninger 1979) and any deficiency of iron could lead to a build up of nitrate in the herbage.

Molybdenum, as in iron, is essential to the enzyme pathway of nitrate assimilation, as nitrate reductase is a molybdenum containing flavoprotein (Beevers & Hageman 1969; Lehninger 1979). Although molybdenum is only required in trace amounts in the soil (Kubota & Allaway 1972), a deficiency would inhibit the activity of nitrate reductase leading to an accumulation of nitrate in the herbage.

Manganese functions in the plant in the activation of numerous enzymes and acts with other metals like molybdenum (Darwinkel 1975), in the activation of enzymes (Tisdale & Nelson 1975). A deficiency of manganese has been reported (Darwinkel 1975) to cause nitrate accumulation.

Calcium is important in the uptake of nitrate from the soil and is associated in the activation of certain enzyme systems (Tisdale & Nelson 1975). Calcium is also important in the form of lime as the liming of acid soils has been shown (Maclean, Halstead & Finn 1972, Nyborg & Hoyt 1978; Sorensen 1982) to markedly increase nitri-fication and thus make nitrate available in the soil.

Boron deficiency has also been cited (Darwinkel 1975) to lead to nitrate accumulation due to it's activity in the regulation of plant functions.

In general, higher nitrate levels are found in plants under any mineral deficiency stress (Hewitt 1970, cited by Darwinkel 1975).

4.2 Re-Seeding

Herbage of newly sown pastures often have a high nitrate content, especially in Italian ryegrass (Denium & Sibma 1980). Darwinkel (1976) showed that as plants age and develop greater root and stubble, these high nitrate levels decline.

Table 8. Nitrate content of herbage in relation to stubble and root weight on five consecutive cuts at intervals of three weeks for newly sown Lolium multiflorum with ample supply of nitrogen (after Darwinkel 1976).

CUT
STUBBLE AND ROOT
PER POT (g)
RATIO OF HERBAGE TO
STUBBLE AND ROOTS
NITRATE CONTENT
(NO3-N %)
1
2
3
4
5
4.31
7.63
9.99
15.04
17.81
1.49
1.32
1.26
0.92
0.91
1.66
1.27
1.24
0.94
0.84

During the five consecutive growth periods in Table 8., nitrate content decreased by 80% on the lower nitrogen application experiment and by 50% on the higher nitrogen application experiment (Darwinkel 1976).

Young seedlings have a small root system that absorbs nitrate well (Denium & Sibma 1980) but reduces little and translocates much to the herbage. In later cuts, the expanding root and stubble system may accommodate more nitrate and also reduce more into organic form. tiller density would therefore appear to be important for nitrate ass~i1ation as w(l1 (Denium & Sibma 1980).

If newly sown pastures are to be heavily grazed nitrogen supply should be carefully calculated and excessive use of organic manures as well as applied nitrogen on the reseeding pasture should be carefully controlled to avoid a build up of nitrate.

4.3 Cutting Interval and Height of Cut

Sibma (1979 cited by Denium & Sibma 1980) shows that cutting interval has a distinct effect on nitrogen assimilation. A cutting interval of five weeks showed the highest nitrogen yield and the lowest nitrate accumulation. Similar results were obtained from a trial by Denium and Sibma (1980) (Table 9.), where cutting intervals were varied between one to seven weeks with several nitrogen applications.

Table 9. Effect of cutting interval on yield of dry matter (DM) and of nitrogen (N) and on nitrate-nitrogen (NO3-N) content in herbage with 600 kg nitrogen/ha/a (after Denium & Sibma 1980).

CUTTING INTERVAL
(WEEKS)
TOTAL YIELD
(t DM/ha)
TOTAL YIELD
(kg N/ha)
NO3-N
1st cut (%)
NO3-N
Last cut (%)
1
3
5
7
13.2
14.8
17.0
19.0
617
580
554
530
0.04
0.04
0.05
0.09
0.13
0.30
0.52
0.58

With shorter cutting interval the dry matter yield is lower but nitrogen uptake is high and nitrate content is lower. In the first cuts, nitrate content is already somewhat higher after longer intervals than shorter intervals, due to the greater stem volume of the plant. (Denium & Sibma 1980).

The marked effect of cutting height or grazing intensity on nitrate in forage can be seen in Fig. 1. and is explained in Section 2.2., where stems have a higher nitrate content that leaves. Thus it follows that the lower the cutting height or grazing height, the greater the amount of stems that will be included in the ingested forage and therefore the greater the amount of nitrate ingested by the animal.

To avoid the excessive inclusion of stems on potentially toxic pastures, management practises would need to avoid the over grazing or over-stocking of such pastures as well as controlling the cutting height of the herbage.


DISCUSSION AND CONCLUSION

Much research has been done in the last forty years into the occurrence of nitrate in plants and the circumstances under which accumulation takes place. The presence of nitrate in plants is important for two main reasons. On the one hand, a large quantity of nitrate in plants is undesirable, because it is poisonous to humans and animals but, on the other hand, the presence of nitrate in the plant is desirable as an indication of a good supply of nitrogen. From the review of literature, Italian ryegrass (of all the species reviewed), would appear to be capable of accumulating the most nitrate. As this is a common pasture species in Natal, it is surprising that, as yet, no significant research has been carried out in South Africa to test for nitrate accumulation. Of the plant species that have the potential to accumulate high levels of nitrate Italian ryegrass has, in the Netherlands, received the most attention. The limited evidence available at the present time, would seem to suggest that potentially harmful levels of nitrate may accumulate in kikuyu (Pennisetum clandestinum) at high rates of nitrogen fertilisation.

From the literature review, it would appear that, most of the determinants reviewed do not, on their own, cause a significant accumulation of nitrate. In most cases, the positive interaction of two or more factors is necessary before a significant accumulation of nitrate is noted. As an example, the effect of a change in solar radiation from day to day is not, on its own, significant in its influence on nitrate accumulation. If the change in solar radiation, due to a cloudy day happens to coincide with a significant drop in soil moisture, then the accumulation of nitrate could be significant. In the majority of cases, however, these factors tend to interact negatively so as to produce an insignificant end result. By way of example, the increased solar radiation is often coupled with an increase in evaporation of soil moisture. This means that the effect of increased nitrate reductase activity, on assimilating accumulated nitrate in the herbage, is reduced by the increase in accumulation of nitrate due to the lack of adequate moisture.

From the review, some influences on nitrate in herbage did appear as being significant on their own without the positive interaction of other factors. These major influences include the following:

The differences between plant species, as to their potential to accumulate nitrate, is vast. As mentioned earlier, Italian rye-grass emerged as one of the highest potential accumulators. In such a pasture species, the cutting or grazing height could present a problem. This is as a result of the stem to leaf ratio, the stems containing a higher proportion of nitrate than the leaves. Thus the lower the cutting or grazing height, the greater the proportion of nitrate in the ingesta.

The age of the herbage was noted to have a significant effect on nitrate levels. Older herbage tends to have less accumulated nitrate than younger herbage. The young herbage would only be significant as to nitrate levels, if nitrogen supply was high or recently applied. Nitrogen supply on its own is obviously the most important single factor influencing nitrate levels in the herbage. Nitrogen applied to a pasture either as organic manure or fertiliser will increase plant nitrate levels rapidly and to significant levels. Of particular significance is evidence of a linear increase in nitrate content at fertiliser nitrogen rates in excess of those required for maximum dry matter production (Fig. 8.).

In conclusion the problem of nitrate toxicity in herbage may be controlled, to a large extent, by management practices. If a nitrate accumulator species of grass is to be grazed, suggested management practices to minimise the possibilities of nitrate toxicity in live-stock could be the following:

SUMMARY

  1. Nitrate is the principle form of nitrogen available to plants from the soil, under normal circumstances.
  2. The accumulation of nitrate means that the rate of assimilation of nitrate to protein has not kept pace with the rate of nitrate uptake from the soil.
  3. Herbage nitrate levels of approximately 0,6% to 1% NO3-N are cited as being lethal to grazing cattle and levels of 0,3% to 0,5% NO3-N have sub-clinical or sub-lethal effects.
  4. Of all the grass species in review, the annual grasses accumu-late more nitrate than perennial grasses and, of the annual species in review, Lolium multiflorum potentially accumulates the most nitrate.
  5. Nitrate tends to accumulate to higher levels in grass stubble than in the roots, which in turn usually accumulate more nitrate than the leaves or herbage, which tend to contain more nitrate than the floral parts of the plant.
  6. Nitrate in herbage tends to increase rapidly to a peak at the prebloom stage and then declines as the plant matures. After cutting, nitrate levels rise rapidly in the regrowth to a peak in seven to fourteen days and then declines as the regrowth matures.
  7. The enzyme. nitrate reductase has been shown to decrease in activity with age. For most grasses nitrate reductase is more active in the leaf blades than in other regions of the plant. Any limits placed on the activity of nitrate or nitrite reductase could lead to an accumulation of nitrate.
  8. Under dry soil conditions nitrate tends to accumulate in herbage and under saturated soil conditions nitrate, in the soil, is converted to ammonia causing a deficiency of nitrate in the herbage.
  9. Increased solar energy has an effect on increasing the activity of nitrate reductase along with an effect on increasing soil temperature. The effect of varied solar energy on nitrate levels is only significant after a period of five days after which a decrease in solar energy can be shown to increase nitrate content in herbage.
  10. Increased temperature has a slight positive effect on nitrate accumulation. This effect is difficult to determine, as many environmental interactions linked to temperature mask the true effect.
  11. Liberal applications of nitrogen fertilisers lead to an accumulation of nitrate in the herbage rapidly and decreases slowly with time. A number of other elements, if deficient, lead to a potential nitrate accumulation. These elements include:-Phosphorus, potassium, calcium, sulphur, iron, molybdenum, manganese and boron.
  12. A newly reseeded pasture has a higher nitrate content than an older pasture.
  13. Nitrate content of cut herbage tends to increase with a longer cutting interval and the lower the cutting height, the greater the proportion of stems are included in the cut, the higher the nitrate content of the cut forage.

LITERATURE CITED

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