Plant and Soil 252: 139–149, 2003.

© 2003 FAO. Published by Kluwer Academic Publishers. Printed in the Netherlands.

139

Endophytic nitrogen fixation in sugarcane: present knowledge and future

applications

Robert M. Boddey1 , Segundo Urquiaga, Bruno J.R. Alves & Veronica Reis
Embrapa Agrobiologia, km 47, Estrada Antiga Rio-São Paulo, Seropédica, 23890-000, Rio de Janeiro, Brazil.
1 Corresponding author∗

Received 7 January 2002. Accepted in revised form 20 August 2002

Key words: endophytic bacteria, Gluconacetobacter diazotrophicus, Herbaspirillum spp., molybdenum, N2 -

fixation, sugarcane

Abstract
In Brazil the long-term continuous cultivation of sugarcane with low N fertiliser inputs, without apparent depletion
of soil-N reserves, led to the suggestion that N2 -fixing bacteria associated with the plants may be the source of ag-
ronomically significant N inputs to this crop. From the 1950s to 1970s, considerable numbers of N2 -fixing bacteria
were found to be associated with the crop, but it was not until the late 1980s that evidence from N balance and 15 N
dilution experiments showed that some Brazilian varieties of sugarcane were able to obtain significant contributions
from this source. The results of these studies renewed the efforts to search for N2 -fixing bacteria, but this time the
emphasis was on those diazotrophs that infected the interior of the plants. Within a few years several species
of such ‘endophytic diazotrophs’ were discovered including Gluconacetobacter diazotrophicus, Herbaspirillum
seropedicae, H. rubrisubalbicans and Burkholderia sp. Work has continued on these endophytes within sugarcane
plants, but to date little success has been attained in elucidating which endophyte is responsible for the observed
BNF and in what site, or sites, within the cane plants the N2 fixation mainly occurs. Until such important questions
are answered further developments or extension of this novel N2 -fixing system to other economically important
non-legumes (e.g. cereals) will be seriously hindered. As far as application of present knowledge to maximise BNF
with sugarcane is concerned, molybdenum is an essential micronutrient. An abundant water supply favours high
BNF inputs, and the best medium term strategy to increase BNF would appear to be based on cultivar selection on
irrigated N deficient soils fertilised with Mo.

Introduction production rose, passing the 100 000 Mg sugar year−1

The Portuguese commander Pedro Alvarez Cabral first
sighted the coast of the land that became called Brazil
on Easter Sunday 1500. The first colonising exped-
ition brought sugarcane from Madeira in 1532 and
the first sugar mill was built at São Vincente on the
coast of what is now São Paulo State, in the follow-
ing year (Machado et al., 1987). Sugar production was
estimated to be approximately 10 000 Mg in 1600 and
did not exceed 20 000 Mg until the start of the 19th
century (Galloway, 1989). During the 19th century
∗ FAX No: +21-2682-1230.
E-mail: bob@cnpab.embrapa.br
mark in about 1850. By the late 1960s, sugar produc-
tion was almost 5 000 000 Mg a year and this rose
gradually until the 10 million Mg mark was passed
in 1994 (Figure 1). By the mid-1970s, the Brazilian
government found itself with a rapidly growing eco-
nomy which brought with it a large oil import bill
at the time of the first Middle-East oil crisis. With
support from the sugarcane producers the ‘ProÁlcool’
Program was launched to produce ethanol from cane
juice as a biofuel. This program grew rapidly, so that
today almost 5 million ha are planted to cane, over half
of which is processed to produce approximately 13 bil-
lion litres of alcohol annually (Figure 1). All gasoline
in Brazil, which fuels 10–12 million cars and other
140
Figure 1. Total area (A) in m ha planted to sugar cane in Brazil, and (B) annual production of sugar (millions of tons) and ethanol (billions of
litres) produced from sugar cane from 1972 to 2000. (Sources: FAO, IBGE and Copersucar).
light vehicles, contains 20–22% ethanol, that totally
replaces other octane-enhancing additives such as tet-
raethyl lead or MTBE (methyl tert-butyl ether) used in
other parts of the world. Furthermore, approximately
3 million vehicles are powered by hydrated (95%) eth-
anol (for details of the very significant environmental
advantages of this biofuel program see Boddey, 1993
and Macedo, 1998).
In the 19th century, the sugarcane was generally
harvested ‘green’, that is to say without burning off
the trash before harvesting. Yet the immense volume
of trash makes manual harvesting extremely difficult,
and for the same labour input three times more burned
cane can be harvested than ‘green’ cane. As labour
became more expensive, preharvest burning became
more widely practised. Since the 1940s and until re-
cently, pre-harvest burning was almost universally ad-
opted. Present yields of sugarcane in Brazil are about
70 Mg ha−1 and mean fertiliser N additions, 60 kg
N ha−1 . With trash (senescent leaves) burned off and
cane being taken to the mill/distillery between 80 and
100 kg N ha−1 is exported from the field. This means
that soil N reserves should become depleted. That this
did not appear to occur led some Brazilian scientists
to suggest that sugarcane could benefit from N2 -fixing
bacteria associated with the plants (Döbereiner, 1961;
Ruschel et al., 1975).
N2 -fixing bacteria isolated from sugarcane
Many N2 -fixing bacteria have been found to be asso-
ciated with sugarcane. Döbereiner and Ruschel (1958)
isolated a new species of Beijerinckia (B. fluminense)
from the surface of sugarcane roots, and members
of this genus were found to be particularly prevalent
in the sugarcane rhizosphere/rhizoplane (Döbereiner,
1961). Subsequently, N2 -fixing Azospirillum, Azoto-
bacter, Bacillus, Derxia, Enterobacter and Erwinia
species were isolated (Arias et al., 1978; Graciolli et
al., 1983; Hegazi et al., 1979; Purchase, 1980; Rennie
et al., 1982; Seldin et al., 1984). Unfortunately, few of
these reports list the numbers of diazotrophs present,
or how this was related to plant-associated N2 -fixing
activity.
To show that there could be agronomically signi-
ficant inputs of BNF to sugarcane plant proved more
difficult. Ruschel et al. (1975) working in Piracicaba
showed the incorporation of 15 N-labelled N2 gas into
just one sugarcane plant, and had negative results with
others. An ambitious attempt to expose a sugarcane

plant in the field to 15 N2 by Matsui et al. (1981) was
did not demonstrate any 15 N incorporation into the
plant principally because of problems of leakage of the
labelled gas.
At Embrapa Agrobiologia in Seropédica (Rio de
Janeiro) a 15 N-aided N balance study on 4 sugarcane
varieties was performed in large pots containing 64
kg of soil (Lima et al., 1987). The N content of this
soil was approximately 0.8 g kg−1 and each pot con-
tained a total of approximately 52 g N pot−1 (Table
1). In the first year, one of the cane varieties, CB
47–89, accumulated 16.5 g of N and in the second
year a further 13.8 g N pot−1 . Including roots, this
variety accumulated 34.8 g of N while the soil N con-
tent only decreased by 6.8 g N pot−1 . Even including
the 2.65 g of N pot−1 added as 15 N-labelled urea fer-
tiliser, the large positive balance of 25.3 g N pot−1
clearly showed that there was a very large input of N
apparently coming from the atmosphere via BNF. Un-
planted pots lost a mean of almost 10 g N pot−1 . The
fact that two other cane varieties in the same study,
IAC 52–150 and NA 56–79, showed only small and
statistically insignificant positive N balances demon-
strated that this BNF input was variety-associated.
The 15 N enrichment of the CB 47–89 variety was al-
most half that of the other three cane varieties, which
confirmed the large BNF input to this variety.
The success of this pot experiment encouraged the
group to plant a larger experiment in a concrete tank
(20 × 6 × 0.8m) filled with 15 N-labelled soil with 10
varieties of sugarcane using the grass Brachiaria ar-
recta (syn. B. radicans) as a non-N2-fixing reference
plant (Urquiaga et al., 1992). Although there was a
large temporal change in 15 N enrichment in soil N,
especially in the first year, which made interpretation
of the results difficult, by the end of the 3-year study
(plant crop followed by two ratoons) it was clear that
there were very large BNF inputs to several of the cane
varieties, especially the Saccharum spontaneum vari-
ety, Krakatau, as well as the commercial interspecific
Saccharum hybrids SP 70–1143 and CB 45–3. These
latter two varieties were those that were most planted
in Brazil at that time (1990–1992), which suggested
that their ability to benefit from BNF, and hence to
yield well on soils low in N, had brought them into
favour with the cane farmers.
141
Endophytic N2 -fixing bacteria
While this study was being conducted, efforts were
redoubled to investigate the populations of N2 -fixing
bacteria associated with cane plants, particularly those
which had shown to benefit most from BNF in the N
balance and 15 N dilution studies. Work in Brazil on
the inoculation of Azospirillum spp. on cereal crops
had suggested that bacterial strains most likely to give
yield responses were those which managed to infect
the interior of the roots (Baldani et al., 1983, 1986a,
1987). For this reason attempts were made to isolate
N2 -fixing bacteria from sugarcane juice and almost
immediately a novel diazotroph, Acetobacter diazo-
trophicus was discovered. This bacteria (now renamed
as Gluconacetobacter diazotrophicus) has many in-
teresting properties that separate it from other diazo-
trophs. In common with several other N2 -fixing bac-
teria, the organism appears as small, Gram-negative,
rods, and is aerobic showing pellicle formation in N-
free semi-solid medium. While it will grow on modest
concentrations of sucrose or glucose (e.g. 5 g L−1 ), its
maximum growth rate occurs at approximately 100 g
sucrose or glucose L−1 , which shows its adaptation to
sugar-rich cane stems. At high sucrose concentrations,
the bacteria produces gluconic acid causing the pH to
fall to less than 3.0 but active N2 fixation continues
(Stephan et al., 1991). G. diazotrophicus lacks nitrate
reductase and can fix N2 in the presence of 20 mM
NO3 − (for further genotypic and phenotypic details
see Baldani et al., 1997; Gillis et al., 1989; James et
al., 1994; Reis et al., 1994). What is remarkable about
this bacterium is that while it is ideally suited to sur-
vival and growth within cane tissues, it does not easily
infect intact sugarcane plants and barely survives in
soil (Baldani et al., 1997). So far it has only been
isolated from sugarcane, elephant grass (Pennisetum
purpureum) (Döbereiner, 1988), sweet potato (Paula
et al., 1989), coffee (Jimenez-Salgado et al., 1997) and
pineapple (Tapia-Hernandez, 2000) as well as other
genera of the sugarcane family (Gonzalez and Bar-
raquio, 2000). The strong preference for the internal
tissues of the plants led Döbereiner (1992) to coin the
adjective ‘endophytic’ for these diazotrophs and more
recently several other species have been discovered.
Recently two other N2 -fixing species of Gluconaceto-
bacter (G. johannae and G. azotocaptans) have been
described associated with coffee plants (Coffea arab-
ica) in Mexico (Fuentes-Ramirez et al., 2001), but so
far these have not been found to be associated with
sugarcane. A further species of Gluconacetobacter
142
Table 1. Total nitrogen balance (g N pot−1 ) on four varieties of sugarcane grown in pots of 64 kg of soil. Plants grown for 10 months then
harvested and allowed to ratoon for a further 12 months. Means of 5 replicates. After Lima et al. (1987)

Cultivar N in Plant Material N in Soil

Harvest 1 Harvest 2 Roots Total Initial Final LossA BalanceB

CB 47–89 16.5 13.8 4.4 34.8 53.6 46.8 6.8 +25.3

CB 47–355 6.7 6.8 3.1 16.5 49.7 44.5 4.9 +9.0
IAC 52–150 6.6 5.0 1.9 13.5 52.7 45.1 7.6 +3.2
NA 56–79 5.7 5.4 2.0 13.0 54.2 45.8 8.4 +2.0
Unplanted - - - - 51.1 44.1 7.0 −9.7
HSD
(Tukey) 4.7 8.6 2.1 12.9 8.5 6.5 7.9 13.7
CV (%) 30.8 59.0 41.8 36.8 7.7 6.6 49.7 (6.4)C

A Loss of N from soil = (initial N in soil) – (final N in soil).

B N balance = (final N in soil + N in plant material) – (initial N in soil – 2.65 g N as 15 N-labelled urea fertiliser).
C Standard error of the mean.

(G. sacchari) has been isolated from pink sugarcane
mealybugs (Saccharicoccus sacchari) and cane leaf
sheaths in Queensland, Australia, but no evidence was
presented to suggest that this species was capable of
BNF (Franke et al., 1999).
Baldani et al. (1986) isolated the N2 -fixing bac-
terium Herbaspirillum seropedicae from the roots of
maize, rice and sorghum, but did not attempt to isolate
it from sugarcane. Subsequently, the bacteria known
as Pseudomonas rubrisubalbicans (Christopher and
Edgerton, 1932), a sugarcane endophyte that causes
mottled-stripe disease in some varieties of sugarcane,
was shown to be closely related genetically to H. sero-
pedicae. Most of the isolates of P. rubrisubalbicans
were able to fix N2 and had phenotypic properties
similar to those of H. seropedicae. Subsequently,
genotyping led P. rubrisubalbicans to be reclassified
as Herbaspirillum rubrisubalbicans (Baldani et al.,
1996; Gillis et al., 1991). There are only a few phen-
otypic properties to differentiate between the species,
such as their ability to grow on meso-erythritol and N-
acetylglucosamine, but they do behave differently in
their infection of plants. Both species survive poorly
in soil, but only H. seropedicae is found in a wide
range of grasses and cereals (Baldani et al., 1997). To
date Herbaspirillum rubrisubalbicans has only been
isolated from sugarcane and Miscanthus spp. (Reis et
al., 2000).
One aspect that differentiates these two Herbaspir-
illum species is in their infection of sugarcane leaves.
H. seropedicae has never been isolated from natur-
ally occurring cane leaves although roots and stems
are often highly infested. James et al. (1997) and
Olivares et al. (1997) compared the inoculation of
sorghum and sugarcane leaves with H. rubrisubalbic-
ans and H. seropedicae. When a suspension of H.
rubrisubalbicans was injected into the leaves of a cane
variety (B-4362) susceptible to mottled stripe, these
bacteria completely blocked some of the xylem vessels
and colonised the intercellular space of the mesophyll
cells. Nitrogenase protein was observed in the centre
of the micro-colonies using an antiserum against the
FeMoCo subunit of the enzyme (James et al., 1997).
In sorghum, the metaxylem was also colonised by H.
rubrisubalbicans and the nitrogenase antigen was also
observed to associate with this bacterium (Olivares et
al., 1997). In the genotype resistant to mottled stripe
disease, SP 70–1143, the bacterium also colonised the
xylem, but formed clusters of 10–20 cells encapsu-
lated by membranes, probably of plant origin (for a
more complete review of the infection and colonisa-
tion of endophytic diazotrophs in graminaceous plants
see James and Olivares, 1998).
While these species of Herbaspirillum show some
capacity to survive in the soil, they nevertheless in-
fect the inner tissues of sugarcane and other grasses,
and hence are also classified as endophytes. Recently,
there have been reports of the isolation of N2 -fixing
members of the genus Burkholderia from cane stems
and roots in numbers as high as 107 viable cells g
fresh tissue−1 (Boddey et al., 1998; Robertson et al.,
2000). Baldani et al. (1997) also reported that species
of Azospirillum (A. lipoferum, A. brasilense and A.
amazonense) as well as other unidentified N2 -fixing
bacteria have been isolated from interior tissues of
sugarcane plants.

Responses of sugar cane plants to inoculation with
endophytic diazotrophs
Sugarcane is propagated vegetatively, and planting
material can either be setts (stem pieces with at least
one node) or from micro-propagated plantlets derived
from meristem culture (Hendre et al., 1983). Setts
are usually observed to be colonised by high numbers
of a diverse range of endophytic bacteria including
N2 fixers (Reis Junior et al., 2000a). On the other
hand, micro-propagated plantlets can be produced free
of microbial contaminants and hence are ideal for
inoculation prior to transplanting in the field. How-
ever, when Reis et al. (1999) inoculated live cell
suspensions of G. diazotrophicus on to intact micro-
propagated sugar cane plantlets the bacterium failed to
establish within the plant tissues. These authors then
developed a protocol to successfully establish high
populations of this diazotroph in micro-propagated
plantlets. This procedure has now been utilised to test
the effects of inoculation of G. diazotrophicus and
other diazotrophs on sugarcane growth.
Sevilla et al. (2001) inoculated micro-propagated
sugar cane plantlets with the G. diazotrophicus type
strain PAL-5 and a nifD non-N2-fixing mutant. In the
absence of a mineral N supply, plants showed signific-
ant increases in dry matter (DM) and N accumulation
after 60 days of growth in the greenhouse. When these
plants were transplanted into the field (under irriga-
tion) for a further 4 months, plants inoculated with the
wild-type strain yielded 40% more fresh cane stems
and 42% more leaf DM than those plants inoculated
with the nif- mutant. Some evidence suggested that
at least part of this yield increase was due to BNF
contributions. Plants 60 days after inoculation were
exposed to 15 N-enriched N2 gas and those inoculated
with PAL-5 (or another wild-type strain PPE-4) incor-
porated significantly more than uninoculated plants or
those inoculated with the nif- mutant of PAL-5. The
low but significant enrichment of these latter plants
was almost certainly due to contamination of the 15 N-
enriched N2 with traces of 15 N-enriched ammonium
used to generate the N2 . The data confirmed that
BNF was taking place in the plants which is a very
encouraging step forward. However, the authors con-
ceded that the observed 15 N enrichment of the tissue
could possibly be explained by the enrichment N in
the bacteria biomass and they did not prove that N
was incorporated into plant tissue. Longer term expos-
ure to labelled N2 gas, or the use of the 15 N dilution
technique, would be required to prove this.
143
A further study on the inoculation of micro-
propagated sugar cane plantlets with endophytic
diazotrophs was reported by Oliveira et al. (2002).
There were 8 different inoculation treatments consist-
ing of strains of 5 different N2 -fixing bacteria (G.
diazotrophicus, Herbaspirillum seropedicae, H. rub-
risubalbicans, Azospirillum amazonense, and Burk-
holderia ‘tropicalis’ [Baldani et al., 2002]), in either
mixtures or alone. After inoculation and acclimatisa-
tion of the plantlets, they were transplanted into 60 L
pots containing 50 kg of 15 N-labelled soil. After 400
days of growth in the open field, the plants inoculated
with all 5 different bacterial species and those inocu-
lated with a mixture of the two Herbaspirillum spp,
increased dry matter yields by 35 and 26%, respect-
ively, over the non-inoculated control (DM yields,
respectively 645 and 599 g plant−1 ). The 15 N enrich-
ment of the 400 day-old plants inoculated with all 5
bacterial species were significantly lower in that of
the control plants, indicating a BNF contribution of
approximately 29% of plant N to the inoculated plants.
Importance of N2 fixation in the field
All the studies on quantification of BNF mentioned
above used 15 N-labelled N2 gas or 15 N-labelled N
balance techniques were performed under controlled
conditions. There appear to be almost no long-term N
balance experiments on sugarcane such as those repor-
ted on wetland rice by Walcott et al. (1977), App et al.
(1984) and Ventura et al. (1986). In Brazil, Oliveira et
al. (1994) reported a study to examine the long-term
effects of pre-harvest burning on cane yield and soil
organic matter levels. This experiment was planted in
1983 on a non-irrigated commercial plantation in Per-
nambuco in NE Brazil. There was a significant N gain
in the soil/plant system equivalent to 38 kg N ha−1
yr−1 (31.5 g N m−2 during 9 years) in the unburned
if the 0–20 cm layer of the soil was taken into con-
sideration (Table 2). When the soil was sampled to 60
cm the overall N balance was higher (54.4 g N m−2 ),
but sampling the soil to a greater depth increased the
variability between replicates and the difference was
not statistically significant at P = 0.05.
Another technique that might be possible to apply
on-farm to quantify contributions of BNF to sugarcane
is the 15 N natural abundance method. This technique
for evaluating N2 fixation inputs to legumes was pi-
oneered by Shearer and Kohl (1986) and has become
widely used for the quantification of BNF contribu-
144
Table 2. Effect of pre-harvest burning on total nitrogen balance (g N m−2 ) of the soil/plant system of field-grown sugar cane over a sequence of
the plant crop followed by 7 ratoon crops. Means of 16 replicates. After Oliveira et al. (1994)
Treatment N Total N in soil/plant system considering soil N content in the layer:
accumulated
by crop
over 8 cuts
1983–1992 Na Initial
Burned 58.3 365.9
Unburned 73.6 369.1
HSDc (P=0.05) 7.0 24.2
CV (%)d 14.2 8.9
a Initial N in soil/plant system = total N is soil at planting + added fertiliser and vinasse.
b Final N in soil/plant system = total N in soil at final harvest + total N accumulated by crop over 8 harvests + roots.
c Honest significant difference (Tukey P = 0.05).
d Coefficient of variation.
e Value in italics = standard error of mean.
tions on-farm with forage, grain and woody legumes
(e.g. Peoples et al., 1996, 1997; Unkovich et al.,
1994). Yoneyama et al. (1997) made a survey in Brasil,
the Philippines and Mijako (Japan), of the 15 N nat-
ural abundance of sugarcane plants in comparison with
neighbouring non-N2-fixing plants (usually weeds).
At many sites, especially in Brazil, the reference plants
showed higher 15 N abundance levels than the cane
leaves, suggesting contributions from BNF. Unfortu-
nately, it was not possible to compare statistically the
15 N abundance of the cane plants and the neighbouring
‘reference plants’, especially as just two, and some-
times, only one, reference plant/s were sampled. Of
the 44 cane plants samples, 32% showed 15 N abund-
ance levels which were the same as, or higher than,
that of the weeds, suggesting that the cane was ob-
taining no contribution from BNF at these sites. There
are many reasons why neighbouring plants growing in
the same soils may have different 15 N abundance, and
BNF inputs are only one cause (Boddey et al., 2000;
Handley and Scrimegeour, 1997; Högberg, 1997).
In a recent study at Embrapa Agrobiologia, we
outlined some strategies to overcome some of these
problems (Boddey et al., 2001). A total of 11 estab-
lished cane fields in commercial plantations in four
different regions of Brazil were sampled. As is usual
practice in Brazil, plant crops were not fertilised with
nitrogen, but on these farms ratoon crops were sup-
plied with 40–60 kg N at full leaf cover. Samples were
taken towards maturity of the cane crop (usually 6–
7 months after the N fertiliser addition). The fields
were divided in to 4 strips, 50 m long and 10 m wide,
0–20 cm 0–60 cm
Nb Final Balance Na Initial Nb Final Balance
g N m−2
354.1 –11.8 789.0 744.6 –44.4
400.6 +31.5 774.3 828.7 +54.4
29.7 30.6 74.5 64.1 61.9
10.6 (10.3)e 12.8 11.0 (20.9)e
to act as replicate blocks. In each block, samples of
the 3rd emergent leaf were taken from 30 cane plants
and the samples bulked for each block. Non-N2-fixing
weeds were sampled throughout the blocks and where
there were several samples of the same weed species
the whole shoots of the weeds were collected and the
samples again bulked by block. In some fields as many
as 5 different weed species were found (e.g. field of
variety RB 72–454, UFRRJ, Campos, RJ) while in
others there were only two (Table 3).
The data were analysed by comparing the 4 rep-
licate values of the 15 N abundance of each weed with
those of the cane samples using the Student ‘t’ test. In
some cases (Sites 1 and 6), the 15 N abundance of one
or more of the reference plants was not significantly
higher than that of the sugarcane. In this case, we feel
that it is not possible to conclude that BNF made a
net contribution to the cane plants. Site 6 planted to
the variety SP 80–1842 and the two reference weed
species were marginally, but not significantly, lower in
15 N abundance than the cane leaves.
For all other sites the 15 N abundance of the weed
species were significantly higher than of the sugarcane
leaves. One limitation of this data is that the N acquisi-
tion strategies of some of these weeds could lead them
to remove N from sources in the soil which had higher
15 N abundance levels than those available to the cane
plants. This would then be wrongly interpreted as an
input of BNF to the cane. In those cases where 4 or
5 different weeds were chosen (eg sites 4, 5 and 8)
where some of the weeds were dicotyledonous and
other monocotyledonous, the fact that all weed spe-
 145
Table 3. 15 N natural abundance of 3rd emergent cane leaves and whole weed plants taken from 11 different sugar cane plantations
(‘Usinas’) in four different states in Brazil (SP – São Paulo, MG – Minas Gerais, RJ – Rio de Janeiro, and PE – Pernambuco). Using
these data estimates have been made of the proportion of N derived from BNF by the cane plants (%Ndfa). Data from J.C. Polidoro
(2001) PhD thesis, Universidade Federal Rural do Rio de Janeiro, Seropédica, Rio de Janeiro

Usina/Town/ State Cane Variety - Reference plant δ 15 N(‰) % Ndfa

Growth cycle (cane plant)

São José/Macatuba/SP SP 80–1842 - – 6.80±0.23 -

2nd Ratoon Sonchus 9.17±0.30 25.9∗
espontaneum
Amaranthus sp. 12.92±0.07 47.4∗
Erechites 7.2 ±0.22 5.6ns
heracifolia
São José/Macatuba/SP RB 72–454 - 5.24±0.37 -
Plant crop Eragrotis pilosa 7.59±0.69 31.0∗
Sida rhobifolia 7.86±0.97 33.3∗
São José/Macatuba/SP RB 72–454 - 3.59±0.07 -
2nd Ratoon Emilia Sonchifolia 6.10±0.46 41.2∗
Panicum maximum 5.35±0.32 32.9∗
São José/Macatuba/SP SP 80–1842 3.34±0.12 -
Plant crop Partenium 7.22±1.02 53.7∗
histerophorus
Lepdium virginicum 6.13±1.24 45.5∗
Panicum maximum 11.08±0.21 69.9∗
Melinus minutifolia 11.79±0.06 71.7∗
Sítio Pedreira/Oratórios/ RB 86–7515 - 5.20±0.97 -
MG Plant crop
Sida rhobifolia 8.60±0.03 39.5∗
Melinus minutifolia 7.57±0.06 31.3∗
Eleusine indica 7.66±0.05 32.1∗
Emilia Sonchifolia 7.11±0.02 26.9∗
Sítio Pedreira/Oratórios/ SP-801–842 –Planta - 8.87±0.07 -
MG
Lepdium virginium 7.90±0.06 −12.3ns
Bidens pilosa 8.20±0.03 −8.1ns
UFRRJ – Campos/RJ CB 45–3 - 5.33±0.22
1st Ratoon Sidrastum sp. 7.85±0.03 32.0∗
UFRRJ – Campos/RJ RB 72–454 5.34±0.24
1st Ratoon Acamthopurpureum 7.96±1.07 32.9∗
australe
Bidens pilosa 8.06±0.27 33.8∗
Croton lobatus 9.82±0.14 45.6∗
Commelina 6.90±0.33 22.6∗
benghalensis
Sida rhombifolia 8.02±0.07 33.4∗

cies showed 15 N abundance values significantly higher
than the 15 N abundance of the cane leaves, is ex-
tremely strong evidence that BNF inputs into the cane
occurred. Only if sugarcane has some almost unique
N acquisition strategy which allows it to tap N sources
with lower 15 N abundance levels than any of the weed
crops would the above conclusion be false. The res-
ults so far suggest that in these commercial plantations
between 0 and 60% of plant N was being derived from
plant-associated biological nitrogen fixation.
146
Table 3. contd.

Usina Barcelos/Campos/ RB 74–454 - 7.07±0.19

RJ
Eclipta alba 9.85±0.01 28.2∗
Ni 9.63±0.22 26.6∗
Usina Cruangi/ RB 78–4764 - 6.20±0.56
Timbaúba/ PE 1st Ratoon
Panicum maximum 7.88±0.05 21.3∗
Brachiaria mutica 9.31±0.21 24.1∗
Capim achó 12.82±0.05 51.6∗
Usina RB 83–102 - 13.20±1.36
Cruangi/Timbaúba/PE 1st Ratoon
Monordica 26.48±0.12 49.1∗
charantia
∗ Indicates that 15 N natural abundance of the reference plants was significantly different from that of the 3rd emergent leaves (bulked samples
for each block) using the student ‘t’ test at p = 0.05.
Ni = Weed species not identified.

Table 4. Response of sugar cane variety RB 72–454 to Mo and

Several field studies conducted in Brazil have
shown that applications of molybdenum often increase
cane yields. To investigate whether this response to
Mo is related to the Mo requirement of nitrogenase,
a field experiment was performed at Campos on a N
and Mo deficient Cambissol. Using the cane variety
RB 72–454, treatments consisted of 3 levels of N (0,
60 and 120 kg N ha−1 as ammonium sulphate) and
4 levels of Mo (0, 100, 200 and 400 g Mo ha−1 as
sodium molybdate) applied as a foliar spray (400 L
ha−1 ). Regression analysis showed that the response to
Mo and to N were both significant (P < 0.01)(Table
4). The crop responded to both Mo and N but the ap-
plication of 100 g ha−1 of Mo alone gave the same
yield increase as 60 kg N. This cane variety is known
to yield well in soils low in available N so the con-
clusion from this study is that this high N utilisation
efficiency is at least partially due to a BNF input that
is limited in this soil by molybdenum deficiency. This
hypothesis is now being tested by examination of the
15 N abundance levels of the cane plants when fertilised
with different levels of Mo.
Future applications
Large and diverse populations of N2 -fixing bacteria
are associated with sugarcane. Abundant microscopic
evidence shows that several species can infect the
internal tissues of sugarcane. It is clear that the ex-
ploitation and improvement of BNF in sugarcane is
severely hindered by the fact that there is no clear can-
didate for ‘the’ N2 -fixing bacterium responsible for the
N application on a Vertic Cambissol (Campos, Rio de Janeiro).
Plots size 60 m2 . Values are means of 4 replicates. Coefficient
of variation 14.1%. Data from J.C. Polidoro (2001) PhD thesis,
Universidade Federal Rural do Rio de Janeiro, Seropédica, Rio de
Janeiro
N addition (kg ha−1 ) Molybdenum addition (g ha−1 )
0 100 200 400
mg fresh cane ha−1
0 47.6 53.7 56.7 61.9
60 51.2 56.9 72.4 65.7
120 55.3 64.4 64.2 60.0
observed N2 fixation. Added to this is the inconsistent
variation in the capacity of different cane varieties to
obtain contributions form BNF. For example, Lima et
al. (1987) compared four varieties and found that IAC
52–150 yielded only a small positive N balance that
was only one eighth of that shown by CB 47–89. In
contrast in the study of Urquiaga et al. (1992) both
varieties showed similar capacities to obtain of their N
from BNF (between 50 and 60%). It is our experience
that conditions (cane variety, N2 -fixing micro-flora,
etc.), which will guarantee agronomically significant
contributions of BNF cannot as yet be defined. It
may well be that complex interactions between plant
genotype, niches appropriate for N2 fixation, selected
diazotrophs to inhabit these niches and specific en-
vironmental conditions are all necessary to stimulate
BNF in sugarcane. Unfortunately, this is the only way

of explaining why the very considerable contributions
of BNF to sugarcane that are detected in the field or
tanks or large pots of soil cannot be extrapolated to
a single plant/diazotroph association which will re-
liably ‘fix’ nitrogen. Nevertheless, certain measures
help maximise any inputs from BNF to sugarcane.
Molybdenum addition is an obvious example, es-
pecially in acid soils where the availability of this
element is depressed. Also first consistent sugarcane
breeding program conducted in Brazil started in the
1930s in Campos, in the north of Rio de Janeiro State.
The soils where the breeding program was conducted
were not highly fertile and N fertiliser was not used.
Perhaps for these reasons, selection pressure for N2
fixation occurred and this may be the reason today
that this trait is particularly prevalent in Brazilian cane
varieties. The lesson from this is that if attempts are
to be made to select cane varieties for high BNF, then
the soils should not be particularly high in available N,
and molybdenum should be added either to the soil or
as a foliar spray. It should be pointed out that recent
evidence from both Mexico, India and Brazil shows a
negative effect of high N fertilisation on the popula-
tions of endophytic diazotrophic bacteria in sugarcane
(Fuentes-Ramíres et al., 1999; Muthukumarasamy et
al., 1999, Reis Junior et al., 2000b). At the present
time, it seems premature to use inoculants of N2 -fixing
bacteria since the main contributors to the observed
BNF are not known and in all studies so far reported
there already seems to be well established populations
of N2 -fixing bacteria within the plants.
Acknowledgements
All authors gratefully acknowledge research fellow-
ships from the Brazilian National Research Council
(CNPq).
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