Plant Physiology
VARIETAL DIFFERENCES AFFECTING NITROGENASE ACTIVITY
IN THE RHIZOSPHERE OF SUGARCANE
and Renato Ruschel.**
Alaides Puppin Ru~chel:~
*Centro de Energia Nuclear na Agricultura (CENA/CNEN/USP) ,
Piracicaba, SP, Brazil and **IAA-PLANALSUCAR, Piracicaba, SP, Brazil
ABSTRACT
Nitrogenase (ethylene) activity was estimated in roots, germinated
cuttings and whole plants of sugarcane (2 months old) in intact and
disturbed systems, under low and normal (air) oxygen atmosphere.
Strong evidence of a genetic effect on nitrogenase activity of the
sugarcane rhizosphere was observed; this activity was high for
NA56-79 and CB46-47 varieties and almost nil in CB41-76 variety.
Correlation of rhizosphere nitrogenase activity and N-content of leaves
was postulated. The rate of nitrogenase activity increased with time
on NA56-79 and CB46-47. Nitrogenase activity was observed on roots
and on germinated cuttings withouth roots and shoots.
INTRODUCTION
The effect of some plants on N-fixing nlicroorganisms is known. Ruschel and Dobereinerg studing the effect of rice (Oryza sativa L.), beans
(Phaseolus sp) and maize (Zea mays L.) on Azotobacter and Beijerinckia,
noticed high populations of those bacteria in the rhizosphere of gramineae.
DobereineF and Dobereiner and Alvahydos showed higher numbers of
Beijerinckia on the sugarcane rhizosphere than in soil alone. Ruschel and
Brittolo, studying N-fixing bacteria in relationship with nitrogen content of
three forage gramineae, observed a higher population for Paspalum notatum
than for Hiparrehenia rufa and Particum maximum, with or without inoculation and that, when inoculated with free living bacteria, Paspalum
showed increased nitrogen in the top part of the plant.
Evaluation of nitrogenase activity in the rhizosphere of field grown
forage grasses showed differences among crops (754 nmoles of ethylene
g-1, root h-1 for Pennisetum purpuraceaum, 341 for Digitaria decumbens,
and 29 for Hyparrehenia rufa - Day et al.3). These authors also observed
significant differences among Paspalum notatum ecotypes and cultivars, in
Azotobacter paspali ocurrence and nitrogenase activity, indicating the possibility of plant breeding to enhance N-fixation.
Nitrogenase activity in the sugarcane rhizosphere has been established
(Dobereiner et a16, Ruschelll), and has been proved by 15-dinitrogen incor-
1942
PLANT PHYSXOLOGY
poration through biological fixation to the plant in periods of 24 and 30
hours (Ruschel et ~ 1 1 2 1 1 3 ) .
,
Sugarcane is a plant propagated vegetatively. Every bud of a variety
has the same genetic constitution which makes it easier to separate a genetic
effect from the phenotype. The objective of this work, as a first step in
this field of study, is to detect any varietal effects on N-fixation by microorganisms using the acetylene reduction technique with cane plants of same
and different genetic constitutions.
MATERIAL AND METHODS
Six of the most commonly planted sugarcane varieties in the State of Sio
Paulo were used in this study: CB41-76, CB46-47, CB47-355, CP51-22,
IAC51/205 and NA56-79. Plants were started from single node cuttings in
200 g of soil plus vermiculite (4 : 1 v/v) in plastic bags. Two and three
months old plants were tested for nitrogenase activity using the acetylene
reduction method.
Nitgogenase is an enzyme common to all N-fixers, that reduces several
substrates. Knowledge of its activity indicates the potential of microbial
population to N-fixation. Acetylene reduction to ethylene is a quick reaction
used to measure nitrogenase activity (Hardy et a17). In this i,nvestigation,
the ethylene was measured by a GC-65 Beckman gas chromatograph, with
Porapak N in a 2 m glass column at the temperature of 1lOC.
The following parts were incubated under 10% acetylene air atmosphere, under normal and/or low (2%) oxygen atmosphere: whole plant
plus soil (intact system); whole plant with roots free of soil; roots plus
shoots without cuttings; cuttings use'd for germination (roots and shoots
removed); external and internal parts of cuttings; detached roots. Except
for the first of these all others were considered to be disturbed systems.
Samples were placed into containers (100 ml, 500 or 70 ml bottles)
in accordance with sample size. After being sealed with a rubber stopper,
10% of the atmosphere of each container was withdrawn, and an equal
volume of acetylene injected. Samples were analysed for ethylene content
at one, two and four hours after acetylene injection.
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The data on amounts of ethylene evolved per container with plants
or plant parts were analyzed according to completely random expgkiments
on a model were the effects are fixed.
RESULTS, DISCUSSION AND CONCLUSIONS
The results presented in Table I, show evidence of a varietal effect on
N-fixing (ethylene) activity of microorganisms in sugarcane. Varieties
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A.P. RUSCHEL AND R. RUSCHEL
NA56-79 and CB46-47 had, on the average, the greatest nitrogenase activity,
which in the intact system (normal oxygen atmosphere), differed statistically
from the others. In a low oxygen atmosphere CB47-355 as well a's CB46-47
and NA56-79 showed high nitrogenase activity. Variety CB41-76, had the
lowest nitrogenase activity in all experiments.
I
TABLE I Nitrogenase activity (nmoles ethylene evolved/plant two months old) of
different parts of sugarcane. Averaged measurements taken after one, two and four
hours of exposure to acetylene (mean of five plants).
Varieties
CB41-76
CB46-47
CB47-355
CP51-22
IAC5 1/205
NA56-79
Mean
*
B
A
84,9
551,l
159,l
214,7
299,4
683,6
332,l
b*
a
b
b
b
a
D
E
b
248,2
76,3
313,4
471,5
ab
-
-
C
66,4 b
1485,8 a
1033,2 a
482,2 b
449,4 b
1754,9 a
878,5
32,l
375,6
29,5
226,3
75,4
415,9
192,5
-
b
a
b
a
152,5
159,O b
613,l
466,O a
= In the same column, values followed by different letters are statistically different at 5%
leved of significance.
A = Whole plant plus soil (intact system) - air 0, atmosphere.
B = Whole plant plus soil in low 0, atmosphere.
C = 'Whole plant with roots free of soil (disturbed system).
D = Roots plus top part without cuttings (disturbed system).
E = Cuttings used for germination (roots and shoots removed).
Strong evidence of a genetic effect in controlling nitrogen fixation might
open a new field in sugarcane breeding. Experiments will soon be started
to detect the degree of heritability of this trait. It is already known that
the nitrogen concentration in sugarcane leaves varies according to the genotype (Orlando Fo. and Haag8). These authors, from a nutritional survey
by foliar analysis, found a varietal effect on leaf nitrogen content independent of soil type. A high correlation was obtained between concentrations
of nitrogen found by these authors in five of the six varieties studied, in
the sape soil, and the average volume of ethylene evolved from these
varieties in the intact system under normal oxygen from air (r ,= 0,821,
on the verge of significance at the 5% level). This positive correlation might
be an indication that the differential ability of two month old plants to fix
nitrogen might influence N-balance of these varieties.
It is interesting to mention that mature stalks of varieties CB46-47 and
NA56-79 are high in sucrose conient, while CB41-76 has a low sucrose
content, although it has gooa productivity (Bassinelld). CB41-76 is the variety most frequently planted in the State of SBo Paulo, occupying 45%
of the total area of sugarcane i(Anonymous1).
*
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PLANT PHYSIOLOGY
The volume of ethylene produced after four hours of exposure to
acetylene was higher than that measured at earlier intervals, indicating that
the nitrogenase activity continued with time (Fig. 1). However, the rate
of ethylene evolution decreased in the fourth hour in all varieties, with the
exception of CB46-47 under normal and low oxygen contents and NA56-79
under low oxygen (Fig. 2). For both of these varieties, the microorganisms
still appeared to be very active after four h o u ~ sof incubation in a sealed
atmosphere.
FIGURE I . nmoles of ethylene evolved per plant two months of age in intact system,
after one, two and four hours of exposure to acetylene.
I
I
I
1 hr
I
I
2 hr
Hours
4 hr
FIGURE 2. nmoles of ethylene evolved per plant two months of age in an intact system in normal air and in low oxygen, during
of exposure to acetylene.
the second hour ( A ) and fourth hour (£3)
1 1 '
1-1
i n normal a i r
i n low oxygen a i r
A = During second hour
= During f o u r t h hour
A.P. RUSCHEL A N D R. RUSCHEL
PLANT PHYSIOLOGY
Nitrogenase activity measured in intact systems seems to be greater
than in disturbed (Table I) as shown by Ruschelll. This indicates that sugarcane influences soil activity of N-fixing populations, which is in accord with
Dobereiner3 who observed an increase of N-fixing bacteria in the cane soil
rhizosphere.
Under a low oxygen atmosphere in an intact system, nitrogenase activity
appears to be higher than for a system where oxygen is normal; this was
most evident for the variety CB47-355 (Table I). This is a very interesting
fact since the system in both cases was intact. Although it is well known
that, in the soil, the oxygen level is lower than in air, this increase observed
under low oxygen atmosphere, suggests that the microaerophylic population
of bacteria in the system is inhibited by oxygen, and indicates that when
soil microflora respiration is high, causing depletion in oxigen tension, the
activity of N-fixers can be enhanced.
Ruschel et a1.12 working with three months old cane seedlings under
15 N atmosphere noticed that the sites 01 N-fixation were In the roots.
However, results of the present paper indicate that germinated cuttings of
some varieties from which the roots and shoots were excised, had a higher
nitrogenase activity than the other parts of the plant (Table I). There appear
to be differences (not statistically significant) in nitrogenase activity among
the internal part of the cuttings, the external part (rind), and the roots
(Table 11), the rind showing the highest activity. Investigations have
to be made to study the ca.rbohydrate contents of germinated cuttings,
nature of exudates around them, and their effects on N-fixing microorganism.
Varieties, such as CB46-47 and NA56-79, that maintain a high level of nitrogenase, even without the germinated cuttings, are desirable since it is not
yet known how the cuttings are able to cause proliferation of the N-fixers.
TABLE 11. Nitrogenase activity (nmoles ethylene evolved/parts of a single plant three
months old) of germinated cuttings and roots of sugarcane after four hours of exposition to acetylene (mean of three plants).
Varieties
A
3
CB46-47
CB47-355
IACS 1/205
NA56-79
Mean
A = Internal part of germinated single node cuttiligs.
B = External part (rind) of germinated single node cuttings.
C = Detached roots.
C
1947
A . P . R U S C H E L A N D R . RUSCHEL
REFERENCES
1. Anonymous. (1976). PLANALSUCAR Annual Report. (1975). G.M. Azzi, L.R.
G r a ~ aand G.A. Gilvert. Piracicaba, p. 80.
2 . Bassinello, A.I. (1976). Apreciaglo sobre experimentos de competi~lode variedades da shrie 71. Brasil A~ucareiro87 (5), 42-57.
3 . Day, J.M., M.C.P. Neves and J. Dobereiner. (1975). Nitrogenase activity on the
roots of tropical forage grasses. Soil Biol. and Biochem. 7, 107-112.
4 . Dobereiner, J. (1959). Influcncia d a cana-de-agficar n a popula~5ode Beijerimkia
do solo. Rev. Brasil. Biol. 19, 251-258.
5 . Dobereiner, J., R. Alvahydo. (1959). Sobre a influ6ncia da cana-de-apicar na
ocorri2ncia de Beijerinckia no solo: I1 Influencia das diversas partes do Vegetal.
Rev., Brasil. Biol. 19, 401-412.
6 . Dobereiner, J., J.M. Day and J. Dart. (1972). Nitrogenase activity in the rhizosphere of sugarcane and others tropical grasses. PI. and Soil 37, 191-196.
7 . Hardy, R.W.F., R.D. Holstein, E.K. Jakson and R.C. Bruns. (1968). The acetylene
ethylene assays for NI fixation: laboratory and field evaluation. Plant Physiol.
43, 1185-1207.
8 . Orlando F . O J., H.P. Haag. (1976). Influencia varietal e do solo no estado nutricional na cana-de-a~ficar(Saccharum spp) pela anhlise foliar. Boletim dcnico
n.O 2 da Coordenadoria Regional Sul. PLANALSUCAR, Araras. p. 52.
9 . Ruschel, A.P. and J. Dobereiner. (1961). Equilibria microbiano na rizosfera de
arroz, feijlo e milho. Comunicado Tbcnico 14. Instituto de Ecologia e Experim e n t a ~ l oAgricola M.A. Rio de Janeiro. p. 22.
10. Ruschel, A.P. and D.P.P.S. Britto. (1966). F i x a ~ l oassimbibtica de nitrogtnio atmosfkrico em algumas gramineas e na tiririca pelas bacthrias do genero Beijerinckia
Derx. Pesq. agrop. brasil. 1, 65-69.
11. Ruschel, A.P. (1975). F i x a ~ l oBiol6gica de Nitrogenio em cana-de-ac6car. Doctor
Brazil.
Sci. Thesis ESALQ -,USP
12. Ruschel, A.P., Y Henis and E. Salati. (1975). Nitrogen - 15 tracing of N-fixation with soil grown sugarcane seedlings. Soil Biol. Biochem. 7, 181-182.
13. Ruschel, A.P., R.B. Victtbria, E. Salati, Y. Henis. (1976). Biological Nitrogen
fixation in Sugarcane (Saccharum officinarum, L.). Intern Symp Environ. Role
N-fixing Blue - green Algae and Assymb Bact. The Swedish University of Agriculture, Forestry and Veterinary Medicine Uppsala, Sweden.
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DIFERENCIAS VARIETALES QUE AFECTAN LA ACTIVIDAD
NITROGENASA EN LA RIZOSFERA DE ZA CARA DE AZUCAR
A.P. Ruschel y R. Ruschel
RESUMEN
I
Fue estirnada la actividad de nitrogenasa (etileno) en raices,
tallos gerrninados y plantas cornpletas de caiia de azlicar de dos
rneses de edad, tanto en sus sisternas intactos como alterados, bajo
condiciones de contenido normal y pobres de oxigeno atrnosferico.
Fue observada en la rizosfera de la caAa de azlicar una gran evidencia de efectos geneticos sobre la actividad de nitrogenasa. Esta
actividad fue elevada para las variedades NA 56-79 y CB 46-47 y casi
nula en la variedad CB 41-76. Fueron postuladas correlaciones entre
la actividad nitrogenasa de la rizosfera y el contenido de nitr6geno
en las hojas. El ritmo de la actividad de nitrogenasa se incrernenta
con el tiernpo en la variedad NA 56-79 y CB 46-47. Fue observada
actividad de nitrogenasa en raices y en tallos gerrninados sin raices.
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1