IV. Factors Affecting Nitrogen Fixation in Grasses

NITROGEN FIXATION IN GRASSES



27



1976), maize (von Bulow and Db'bereiner, 1975), and sorghum (van Berkum and

Neyra, 1976), it does appear that a full establishment of the root system is

necessary for maximal rates of N2 fixation to occur. On the other hand, it is

very likely that competition for available photosynthate by the grain could be

the cause for the observed decline of nitrogenase activity during the seed-filling

stages. Similar patterns of N2 fixation with ontogeny of the plant have been

reported for legumes (Harper and Hageman, 1972; Thibodeau and Jaworsky,

1975).

Diurnal fluctuation patterns have also been observed for several grasses. A peak

of nitrogenase activity has been observed around midday and in some species a

second peak was observed during the night. This pattern was observed for

Paspalum notatum and sorghum (Dobereiner and Day, 1975) and for Panicum

maximum and maize (Balandreau, 1975). Rice plants did not show any peak at

night (Balandreau et al., 1974).

The night peak was attributed to hydrolysis of carbon storage products

accumulated during the day and their subsequent translocation and exudation in

the rhizosphere (Balandreau et al., 1974). In general, most of the nitrogenase

activity computed over a 24-hour period occurs during the light period and it

may reflect the dependence of nitrogenase activity in grasses upon available

photosynthate, as is the case in symbiotic systems. Recent results indicate that

malate metabolism may play a major role in the supply of energy for nitrogen

futation (van Berkum and Neyra, 1976). Malate is a primary product of C 4

photosynthesis in some species, e.g., maize, sorghum, and sugar cane (Chollet

and Ogren, 1975). Malate is also one of the best substrates for the growth of

Spinllum lipofemm, whereas glucose is a poor substrate. Addition of malate or

bicarbonate during preincubation doubled the nitrogenase activity of excised

sorghum roots (van Berkum and Neyra, 1976). Addition of similar concentrations of glucose had no effect.



B. PLANT GENOTYPE



Use of the excised root assay has revealed a wide range of nitrogenase activities

for maize genotypes. Mean nitrogenase activities of some S1 maize lines were 10

to 20 times higher than the original cultivar UR-1 (von Biilow and Dobereiner,

1975). Crosses between higher fixing versus less fixing maize cultivars showed

significant heterosis effects (von Biilow et al., 1976). Genotypic differences have

also been shown in Paspatum notatum, Pennisetum purpureum, and wheat

(Dobereiner and Day, 1974, 1975; Day et al., 1975b; Dobereiner, 1976a).

Larson and Neal (1976) presented evidence for a selective colonization of a

genetically defined line of wheat by a facultative N2-fixing Bacillus sp. All these

results reveal the importance of plant genotype for optimal associations and



28



CARLOS A. NEYRA AND J . DOBEREINER



suggest the possibility of improvement of Nz -fixing associations by plant breeding.



C. TEMPERATURE



Because of the relatively high temperature requirements for Spirillum lipoferum development, tropical areas appear more favorable for a higher incidence

of this bacterium (Dobereiner et at., 1976). However, differences among plant

species are expected to occur in relation to tolerance to relatively low temperatures. Optimal temperature (31°C) for nitrogenase activity of pure cultures of S.

Zipofemm and of isolated maize roots were coincident (Dobereiner et al., 1975).

Soil temperatures below 25°C were found to be a major limiting factor to

nitrogen futation in roots of Digitaria decumbens cv. transvala (Abrantes et al.,

1975). Nitrogenase activity of Spirillum lipoferum isolated from Digitaria roots

was also inhibited by temperatures below 25°C. Although Spirillum strains

isolated from maize and Digitaria did not show differences in nitrogenase

activities at optimal temperatures, they seemed to behave quite differently at

lower temperatures. At 22°C the maize strains were five times more active than

the strains isolated from Digitaria (Neves et a l , 1975). Nitrogenase activity on

forage grass roots is also very low once night temperatures fall below 18°C. This

has been attributed to effects upon plant growth (DSbereiner and Day, 1974).



D. OXYGEN

Because of the oxygen sensitivity of the nitrogenase enzyme system (Ljones,

1974; Postgate, 1974) and the known effects of oxygen on nitrogen fixation by

bacterial cultures (Day and Dobereiner, 1976), it is not surprising that optimal

nitrogenase activities on the roots of forage grasses are found at pOz far below

that of air. Studies on the Paspalum notatum-Azotobacter paspali association

revealed that nitrogenase activity on the roots was extremely sensitive to changes

in pOz. Nitrogenase activity was almost completely inhibited in air or in the

absence of Oz and was greatest at p02 0.04 atm (Dobereiner et al., 1972a). In

contrast, the activity of soil cores containing P. notatum plants was little

affected by changes in pOz above the soil (Dobereiner et al., 1972a). Very little

is known about the oxygen protection mechanism in the intact soil grass system.

It is known, however, that Spirillum lipofemm has a very poor oxygen protection mechanism for its nitrogenase (Day and Dobereiner, 1976; Okon et aZ.,

1976a). This would explain the oxygen sensitivity of the nitrogenase activity on

the roots of tropical grasses in association with this bacterium. In Digitaria,

maize, and sorghum roots maximal activities are found at p 0 2 0.01 to 0.02 atm



NITROGEN FIXATION IN GRASSES



29



and pure cultures of Spirillum lipoferum show the same pOz requirements as the

isolated roots (Day and Dobereiner, 1976; Abrantes et al., 1975). Roots preincubated under a p 0 2 of 0.02 atm gave maximal activities without additional

supply of O2 at the time acetylene was injected into the vials.



E. COMBINED NITROGEN



High levels of combined nitrogen in the soil or the application of heavy

nitrogen fertilization reduce the potential for nitrogen fixation in grasses. Exposure of excised roots to NO3-, NOz-, or NH4' during the preincubation period

inhibited nitrogenase activity in sorghum and maize roots (van Berkum and

Neyra, 1976; Neyra et al., 1976). In sorghum roots a 50% inhibition was

observed at 1 0 mM NO3- while a similar effect was obtained by an exposure to

0.1 mM NH4+ or NOz-(van Berkum and Neyra, 1976). These results indicate

that the level of NH4+ in the soil solution may be a limiting factor for the

development of nitrogenase activity in grasses. T h s has been confirmed in

experiments conducted with Digitaria decumbens cv. transvala. Ammonium

concentrations in the soil solution above 200 ppm severely inhibited nitrogenase

activities (Abrantes et al., 1975). From the multiple regression equation for

NH; and soil temperature (r = 0.75) it was calculated that at 27°C (soil

temperature) in the absence of NH4' or at 32.5"C in the presence of 50 ppm

NH4' (soil water basis) a nitrogenase activity of 50 nmoles CzH4 per gram of

dry root per hour, could be obtained.

Nitrogenase activities of Pennisetum purpureum and Digitaria decumbens

roots, determined throughout the season, were not reduced even after eight

applications of 20 kg N per ha as NH4N03 (Dobereiner and Day, 1975). Balandreau et d. (1975) reported that upon the application of different amounts

of (NH4)2S04 at sowing there was a marked effect on the nitrogen fixation in

rice seedlings. For additions of up t o 40 ppm, Nz fixation was stimulated, but

higher applications resulted in a marked decrease. It was assumed that the slight

increase of nitrogenase activity at low nitrogen levels could be attributed to an

increase of root exudate (Balandreau et al., 1975). Watanabe and Kuk-Ki-Lee

(1975) reported that NPK fertilizer appears to promote heterotrophic Nz

fixation in paddy rice, while phototrophic N2 fixation appears to be predominant in nonfertilized soils.

These observations are of great importance because at low levels of combined

nitrogen in the soil the simultaneous utilization of biological Nz fixation and

mineral N fertilizer may be possible. This possibility is further illustrated in Fig.

2. The addition of 40 kg N/ha at planting allowed the development of nitrogenase activity in maize roots while reasonable nitrate reductase activities (in vivo

assay) were determined in the leaves of the same plants (Neyra et al., 1976).



30



CARLOS A. NEYRA AND J. DOBEREINER

1



Days after Germination

FIG. 2. Seasonal variations of nitrogenase and nitrate reductase activities in field-grown

maize plants. Nitrogenase activity was determined in excised preincubated roots and nitrate

reductase activity (in vivo assay) in leaf blades. Nitrogenase activity under low N (40 kg/ha)

(0-0)

and high N (200 kg/ha) (0-0)

fertilizer. Nitrate reductase activity of low N (40

kg/ha) treatment (n---o).

Silk emergence (1) and mid-grain filling (2) stages are indicated

by arrows.



Similar results were obtained with field-grown sorghum (van Berkum et al.,

1976). However, heavy doses of fertilizer (200 kg N/ha) severely inhibited

nitrogenase activities in both maize and sorghum plants which illustrates the fact

that, in areas receiving continuously high doses of nitrogen fertilizer, the potential for N2 fixation may not be realized.

V. General Discussion



Several lines of evidence presented throughout this review have shown the

existence and operation in nature of grass-bacteria associations able to bring

about N2 fixation. Several observations also indicate that these associations are

contributing significantly to the N economy of the plants. However, the actual

contribution of Nz furation to plant N is not known, as "N studies of the

amount and rate of transfer of the fixed nitrogen to the host plant are lacking

(Day et aL, 1975a). Nevertheless, nitrogen fixation in Digitaria decumbens and

Paspalum notatum cores has been confirmed by the incorporation of "Nz

(De-Polli et al., 1976).



NITROGEN FIXATION IN GRASSES



31



Acetylene reduction assays over a 24-hour period in cores indicating fixation

of more than 100 g N/ha per day have been obtained with several tropical forage

grasses, rice (algal fixation substracted), and occasionally with wheat and rye

grown under tropical conditions (Dobereiner, 1976a,b; Watanabe, 1976). In

most instances, a significant correlation of core assays with preincubated

excised root assays has been obtained, with the latter usually underestimating

(Dobereiner, 1976b). On the other hand, N2 furation estimated by the excised

root method in maize and sorghum has been shown to be several-fold higher

than the amount of N2 fixation obtained from core assays (Barber et al., 1976;

Tjepkema and van Berkum, personal communication). These results suggest that

the excised root assay does not provide a reliable measure of N2 fixation in

maize and sorghum (Barber et al., 1976). However, the possibility of underestimation by the core assays has not been ruled out as yet. The major argument

against the excised root assay is that all grasses assayed by this method show a

lag period (8 to 18 hours) before acetylene reduction starts (Dobereiner et al.,

1972a; Abrantes et aZ., 1975; Dobereiner and Day, 1975). All attempts to

eliminate this lag have been unsuccessful (Dobereiner and Day, 1975). Thus, in

order t o obtain linear rates of acetylene reduction, a preincubation overnight at

low O2 tensions (2% 0,) appears necessary. Nevertheless, recent reports (Sloger,

1976; Sloger and Owens, 1976) indicate that high rates can be obtained in

freshly harvested surface-sterilized maize roots (up to 1000 nmolds C2 H4/hour

per gram of dry root). Still in other assays a 20-fold increase in activity was

observed after an overnight preincubation at 2% 0 2 .Although the lag has been

observed by several workers, no satisfactory explanation has been forthcoming.

Barber et al. (1976), Okon et al. (1977a), and van Berkum (unpublished data)

working with excised roots of maize and sorghum, have. observed a substantial

increase, during the preincubation period, in numbers of Nz -fixing bacteria

capable of growing in malate, which could explain the higher nitrogenase

activities observed after preincubation of these plants. On the other hand very

high numbers of S. lipoferum have been found on young maize roots and on

roots from nitrogen-fertilized plots which exhibited no nitrogenase activity

(Podesta et aZ., 1976). Therefore, higher numbers of Nz-furing bacteria do not

necessarily mean higher fixation. There might also be differences according to

the localization of the bacteria outside or inside the roots.

A great deal of information and insight into the field of Nz fixation in grasses

has been obtained using the excised root assays. Because this method allows the

handling of large numbers of samples from the field and the good reproducibility

of the results, it has proven to be a valuable tool for investigating many aspects

of N, fixation in grass-bacterial associations, such as the distribution of Nz-fixing associations among different plant species, as well as plant genotype and

physiological studies. The seasonal pattern of nitrogenase activity associated

with plant ontogeny and the genotypic differences observed with several plants

show that the physiology of the host can control the level of nitrogenase activity



32



CARLOS A. NEYRA AND J. DOBEREINER



of the bacteria associated with their roots. Furthermore, the high degree of

specificity of certain associations as in Paspalum notarum-Azotobacter paspali

(Dobereiner and Campelo, 1971) or the highly specific association of an Nz-fixing Bacillus sp. with a genetically defined line of wheat (Larson and Neal, 1976)

clearly indicate the potential of plant breeding as a promissory tool for achieving

better plant-bacteria associations with a greater Nz-fming ability.

The participation of Spirillum lipofemm in all but one (nitrification) of the

steps in the nitrogen cycle and its widespread distribution in soils and roots

makes this organism very useful for studying the various aspects of nitrogen

transformation in nature. While some strains of S. Zipofemm fur Nz and denitrify

(groups I and II), others do not denitrify (group 111). Studies of methods to

optimize N2 fixation and minimize denitrification are of obvious importance. In

view of the existence of three different groups of S. Zipofemm, the exact role of

this organism in the overall nitrogen metabolism in the soil-plant system needs

to be studied in more detail.

While biological Nz fixation could be sufficient for the maintenance of forage

grasses growing in their natural habitat, it is unlikely that biological N2 fixation

alone could satisfy all the nitrogen requirements of high yielding agricultural

crops and, therefore, studies on the interaction between combined N and

biological N2 assimilation should be ranked as a high research priority.

We know that Spirillum lipofemm grows well in the presence of NO; or NH4+

(Okon et aZ., 1976a; Neyra and van Berkum, 1977) but nitrogenase is inhibited.

On the other hand, NO3- is usually the main form of nitrogen in well-aerated

soils, where conditions are favorable for nitrification. Therefore, it is likely that

under field conditions NO3- will limit Nz fixation. The search for strains which

fix Nz in the presence of NO3- has good possibility of success in the near future.

Development of nitrate-reductase deficient mutants of S. Zipofemm as it has

been shown for Rhizohium (Sik et QZ., 1976; Gibson, personal communication)

should be possible.

Although good progress has been made in understanding the importance of

environmental and plant factors, the exact nature of the grass-bacteria associations is stdl unclear. Recent observations on other N2-furing plant-bacteria

associations might help to advance a working hypothesis for studies of this kind.

Rhizobium can now be grown in culture media and produce active nitrogenase

(Pagan et al., 1975; Kurz and La Rue, 1975) but combined nitrogen is still

necessary for growth (Bergersen, 1976; Keister, 1976). The fixed nitrogen is

excreted as NH4+into the medium and partially reassimilated (Bergersen, 1976).

In the nodules, bacteroids excrete the fixed Nz as NH4' which is assimilated by

plant enzymes (Scott et al., 1976). Ammonia does not repress nitrogenase

synthesis in free-living rhizobia grown either on agar (Kurz and La Rue, 1975) or

in shake cultures under microaerophilic conditions (Keister, 1976).

More similar still to the grassSpiriZlum association are the symbioses of

blue-green algae where the algae are able both to fur Nz in vitro and to associate



NITROGEN FIXATION IN GRASSES



33



with plants. Examples are several lichens and the liverwort symbioses. Stewart

(1976) reported profound changes in the physiology of these algae when they

are living in symbiosis with the procaryotic macrophyte: The percentage of

heterocysts increased, photosynthesis ceased, and NH4’-assimilating enzymes

were inhibited and therefore the algae excreted the fured Nz as NH4+ and the

efficiency (nmol C2H4/mg protein) increased by a factor of 3 to 7. Algae

separated from the macrosymbiont needed more than 24 hours to reassume

growth. On the basis of these observations Stewart (1976) suggested that the

restriction of Nz-fixing symbioses to a few species is linked to the necessity of a

plant factor which switches the bacterial NH4+ assimilation pathway off. How

far these findings apply to the grass associations remains to be seen.



ACKNOWLEDGMENTS

We acknowledge the support of the Program for International Cooperation in Training and

Basic Research on Nitrogen Fixation in the Tropics, sponsored by the Brazilian National

Research Council (CNPq), the Empresa Brasileira de Pesquisa Agropecuaria (EMBRAPA),

and the Universidade Federal Rural d o Rio de Janeiro.

We wish to thank Dr. D. B. Scott for helpful discussions and revision of this manuscript

and Mrs. C. Scott for helpful assistance.



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