Pergmon

Soit Bidt. Biochem. Vol. 29, No. 516, pp. 911-922, 1997
c 1997 Elsevier Science Ltd. Al1 rights reserved
Printed in Great Britain
PII: S0038-0717(%)0021&0 0038-0717/97 $17.00 + 0.00
RECENT ADVANCES IN BNF WITH NON-LEGUME
PLANTS
JOS 1. BALDANI,* LEONARDO CARUSO, VERA L. D. BALDANI,
SILVIA R. GOE and JOHANNA DBEREINER
EMBRAPA-Centro Nacional de Pesquisa de Agrobiologia (CNPAB), 23851-970, Seropdica, RJ,
Brazil. and UFRRJ, IF, DCA, 23850-970, Seropdica, RJ, Brazil.
( Accepted 5 July 1996 )
Summary-It is now wel1 accepted that nitrogen-fxing bacteria colonising graminaceous plants can be
grouped into three categories: 1, rhizosphere organisms; 2, facultative endophytes and 3, obligate endo-
phytes. In the first category are included al1 species that colonise the root surface such as Arotobacter
pasputi, Beijerinckia spp. Facultative endophytes are those nitrogen-fixing bacteria that can colonise the
surface and interior of the roots principally the four species of Azospirihum, except Azospirillum hato-
praeferuns. The third category is constituted mainly by diazotrophs isolated more recently such as Acet-
obacter diazotrophicus, Herbaspirillum spp and Azoarcus spp which are able to colonise the root interior
and aerial tissues of the plan& Although most of the studies related to nitrogen fixing bacteria have
been concentrated on Azospirillum spp, it is the obligate endophytes, isolated more recently, that have
attracted the attention of scientists working in this field. The ability to colonise the entire plant interior
and locate themselves within niches protected from oxygen or other factors make them the most prom-
ising group of diazotrophs associated with graminaceous and other non-leguminous plants. In this
review we compare these three groups of nitrogen-fixing bacteria, their interaction with the host plants
and discuss the potential of their use in agricu1ture.O 1997 Elsevier Science Ltd
INTRODIJCTION
It has now been almost 40 years since Dbereiner
and Ruschel (1958) isolated the nitrogen-fixing bac-
terium named Beijerinckiu flurninensis from the rhi-
zosphere of sugarcane plants grown in tropical soils
and demonstrated the potential of diazotrophs to
associate with graminaceous plants. However, it was
only after the rediscovery of the genus Azospirillum
by Dbereiner and Day (1975) that scientists in the
world became interested in diazotrophic bacteria as-
sociated with graminaceous plants. During the last
two decades many new nitrogen-fixing bacteria have
been isolated and identified, including species of the
genera Azospirillum, Herbaspirillum, Acetobacter
and Azonrcus. The greater part of these diazotrophs
have been isolated from tropical regions, especially
in Brazil, and they have been the main source for
groups in the world working with associative diazo-
trophs. Other plant-associated nitrogen-fixing bac-
teria have been identified, but probably because of
their low number, or restricted occurrence, they are
not wel1 explored. The interest in the association of
diazotrophs with graminaceous plants reinforces the
importante of the biological nitrogen fixation pro-
cess to sustainable agriculture systems in Brazil
where low amounts of nitrogen fertilisers have
always been applied (Dbereiner, 1995).
Author for correspondence: Fax: 55 021 682-1230.
An analysis of the studies carried out on the
interactions of diazotrophic bacteria with graminac-
eous plants over the last 40 years has shown a clear
change in the way scientists have focused on these
interactions. Initially, most of the studies were con-
centrated on bacteria isolated from soil or rhizo-
sphere soil. The best example is Azotobacter puspali,
a bacterium that colonises specifically the rhizo-
sphere of Puspalum notatum (Dbereiner, 1966).
Later, came the era of Azospirillum, nitrogen-fixing
bacteria able to colonise either the rhizosphere or
the root interior (here called facultative endophytes)
of several forage grasses and cereals (Dbereiner
and Baldani, 1982). Five species have been identified
so far: A. Iipoferum, A. brasilense, A. amazonense,
A. halopraeferans and A. irakense (Tarrand et
Magalhaes et al., 1983; Reinhold et al., 1987;
Khammas et al., 1989). In the last decade, new
nitrogen-fixing genera were identified and because
of their occurrence mainly within plant tissues, they
have been called endophytes (obligate) instead of
endorhizosphere-associated bacteria, a term used
until recently for the root interior (Dbereiner,
1992a). Among the obligate endophytic diazotrophs
are Acetobacter diuzotrophicus (Cavalcante and
Dbereiner, 1988), Azoarcus spp (Reinhold-Hurek
et al., 1993), Herbaspirillum seropedicae (Baldani et
al., 1986a), the recently described species
Herbaspirillum rubrisubalbicans (Gillis et al., 1991;
911
912 Jos 1. Baldani ef al.
Baldani et al., 1996) and the partially characterised
Burkholderia sp. (Hartmann et al., 1995b; Baldani et
al., unpublished). Very recently many of these dia-
zotrophs (facultative and obligate) were isolated
from palm oil trees (Ferreira et al., 1995), fruit
(Weber et al., 1995) and toffee plants (Jimnez-
Salgado et al., 1995) and therefore extending the
endophytic colonisation of diazotrophic bacteria to
plants other than graminaceous.
RHIZOSPHERE DIAZOTROPHS
Rhizosphere diazotrophs were mainly isolated in
the early sixties and their biological nitrogen fix-
ation (BNF) contribution to the sustainable agricul-
ture system is stil1 unknown. There is a debate
about the actual role of these bacteria in the associ-
ation with graminaceous plants. Much evidente has
pointed towards the role of phytohormones
(Zimmer et al., 1988), and because of their ability to
interfere with the root morphology and growth of
the plant they have also been considered plant-
growth promoting rhizobacteria (PGPR) (Bashan et
al., 1993).
At least in one case, however, there is strong evi-
dence of a rhizosphere nitrogen-fixing bacterium
contributing to the nitrogen accumulated in the
plant (Boddey et al., 1983). Using the N isotope
dilution technique the authors demonstrated that
about 20 kg ha-y- of the N accumulated in
Paspalum notatum CV. batatais was derived from
BNF. An important aspect of this association is the
strict specificity of this plant with the rhizosphere
diazotroph named Azotobacter paspali (Dbereiner,
1966). A survey on the occurrence of this diazo-
troph among several ecotypes or varieties of
Paspalum and other grasses showed the presence of
A. puspali mainly in the rhizosphere of the ecotype
P. notatum CV. batatais (93% of 247 samples tested),
rarely associated with other Paspalum spp and none
in other plants. The specificity makes this associ-
ation unique and could be compared with the endo-
phytic association of Acetobacter diazotrophicus
with sugar cane (Dbereiner, 1992b). The com-
pounds involved in the plant-bacteria interaction
are stil1 unknown, although one could speculate
about the role of molecular signals similar to those
observed in the rhizobia-legume interaction. A
complete understanding of this association could
open new avenues to manipulate other associations,
such as the facultative endophytic association, so
that their maximum potential of nitrogen fixation
could be explored.
Another rhizosphere diazotroph that seems to
behave quite differently from the common rhizo-
sphere diazotrophs is Beijerinckia, principally B.
indica and B. fluminensis found mainly in associ-
ation with sugarcane plants grown in tropical cli-
mates (Dbereiner, 1959). Although no biological
nitrogen fixation contribution by these diazotrophs
has been demonstrated, the release of compounds
by the sugar cane plants, regulating the colonisation
of the root as we11 as establishing the bacteria limit-
ing area around the plant, led US to speculate about
the importante of Beijerinckia spp in association
with sugar cane. It has been shown that these bac-
teria preferentially colonise the rhizoplane and rhi-
zosphere of sugar cane plants as do other
rhizosphere diazotrophs, and that the exudates,
mainly sugar compounds, are involved in the associ-
ation because the bacteria are found principally in
areas where these compounds are released or run
off from the leaves (Dbereiner and Alvahydo,
1959). Nothing is known about the interaction of
rhizosphere diazotrophs like Beijerinckia spp with
obligate endophytes like Acetobacter diazotrophicus
that appear to colonise sugar cane plants exclu-
sively. It is possible that competition for these com-
pounds, or the amount released by the plant, may
account for some of the variability in biological
nitrogen fixation observed among sugar cane var-
ieties.
More recently, a very specific rhizosphere associ-
ation was also demonstrated between Azospirillum
halopraeferans and kallar grass plants grown in
saline-sodic soil in Pakistan (Reinhold et al., 1987).
Attempts to isolate this bacterium from grasses
grown in saline regions of the Brazilian toast were
unsuccessful (Reinhold et al., 1989). However, no
survey on the occurrence of this rhizosphere diazo-
troph has yet been carried out in other countries.
ENDOPHYTIC DIAZOTROPHS
The term endophyte was coined some years ago
to refer to root interior colonisation of plants by
micro-organisms (bacteria and fungi) that usually
do not cause damage to the host and live most of
their life inside of the plant tissue without eliciting
any pathogenic symptoms (Pereira, 1995). Later, the
definition was extended to bacteria and fungi that
colonise the root interior and promote benefits to
the plant (Kloepper and Beauchamp, 1992). Due to
the ability of some diazotrophs to colonise primar-
ily the root interior of graminaceous plants, to sur-
vive very poorly in soil and to fix nitrogen in
association with these plants, the term endophyte
was also introduced into this area by Dbereiner
(1992a, 1992b) and became widespread among
scientists interested in nitrogen-fixing bacteria.
Splitting the term endophyte into facultative and
obligate was suggested to distinguish, respectively,
strains that are able to colonise both the surface
and root interior and to survive we11 in soil from
those that do not survive we11 in soil but colonise
the root interior and aerial parts. The ecological
aspects used to distinguish these two groups as we11
BNF with non-legume plants
913
as the rhizosphere diazotrophs
Table 1.
are presented in Table 2. Occurrence of faculatative and obligate endophytic diazo-
trophs in non-legurne vlants
Facultative endophytic diazotrophs
This group of diazotrophs, composed mostly of
Azospirillum species, can be considered the starting
point of most ongoing biological nitrogen fixation
programs with non-legume plants worldwide.
Although Azospirillum hpoferum (Tarrand et al.,
1978) was the first species of the genus to be iso-
lated, it is A. brasilense among al1 of the five known
species that is the best characterised diazotroph at
the physiological and molecular level. Azospirillum
spp have been found associated with several cereals
and forage grasses grown in temperate and tropical
climates (Dbereiner and Baldani, 1982; Baldani,
1984; Germida, 1986). However, not al1 species have
been demonstrated to colonise graminaceous plants
grown al1 over the world (Table 2). The species A.
amazonense (Magalhes et al., 1983), for example,
has been isolated only from cereals, forage grasses
and palm trees collected in Brazil (Baldani, 1984;
Magalhes and Dbereiner, 1984). The species A.
irakense, described more recently, has been isolated
from roots of rite plants grown in the subtropical
region in Iraq (Khammas et al., 1989). NO other
report on the occurrence of this diazotroph has
been published as yet.
Among the several physiological characteristics
used to distinguish the Azospirillum species, there
are a few that may play a role in the association
with the plants. Both A. lipoferum and A. brasilense
utilise malate as a carbon source, but only the for-
mer is able to use glucose. Besides the ability to fix
nitrogen both species are able to produce phytohor-
mones like IAA as demonstrated by the presence of
the IAA biosynthesis genes (vande Broek and
Vanderleyden, 1995). The authors also showed the
presence of these genes in A. amazonense but not in
A. irakense. The ability to utilise sucrose is the main
characteristic of A. amazonense and A. irakense
species, although only the first is able to grow in a
Table 1. Ecological aspezts to differentiate rhizosphere and endo-
phytic diazotrophs associated with non-legume plants
Aspects Rhizosphere Endophytes
Facultative Obligate
Survival in soil
Natura1 Good Good Vely poor
Sterile Good Good POOI
Colonization
Root surface Yes Yes NO
Root interior NO Yes Yes
Aerial parts NO Yesjno Yes
More
Host plant Various/restricted Various restrictedb
Arotobacter paspoli/Paspolum notatum CV. batatais; A. haloprae-
ferans/Kallar grass.
b Acetobacter diazotrophicus/sugarane, weet potato and
Pennisetum; Azoarcus/Kallar grass.
Diazotroph Plant Plant parts
A. brasilense
A. lipoferum
A. amazoneme
A. irakense
Forage grasses
Sugar cane
Tuber plants
Palm trees
Cereals
Sugar cane
Palm trees
Rite
Obligates
H. seropedicae Cereals
H. rubrisubalbicans
A. diazotrophicus
Aroarcus spp
Burkholderia spp
Faculatatives
Cereals
Forage grasses
Sugar cane
Palm trees
Cereals
Sugar cane
Forage grasses
Palm trees
Sugar cane
Rite
Sugar cane
Pennisetum
Sweet potato
Kallar grass
Cereals
Sugar cane
Tuber plants
Palm trees
Roots, sterns, seeds
Roots, sterns
Roots, sterns, leaves
Roots, sterns, fruits
Roots, sterns, seeds,
xylem sap
Roots, leaves
Roots, sterns, leaves
Tubers, roots
Roots, sterns, fruits
Roots, sterns, seeds
Roots, sterns
Roots, sterns, fruits
Roots
Roots, sterns, leaves,
seeds
Roots, sterns
Roots, sterns, leaves
Roots, sterns
Roots, sterns, leaves
Roots, sterns, leaves
Roots, sterns, leaves
Roots, sterns
Roots, sterns, tubers
Roots, base of sterns
Roots, sterns, leaves
Roots, sterns, leaves
Roots, tubers
Roots, sterns
After Dbereiner et al. (1994).
wide range of pH. In addition, A. irakense hydroly-
ses pectin and tolerates high concentrations of salts
(3%). The knowledge that many Azospirillum
strains dissimilate nitrite to nitrous oxide and dini-
trogen based on physiological tests, was recently
confrmed by the detection of the N20 reductase
gene involved in the denitrification with a probe
constructed from the enzymes present in
Pseudomonas stutzeri (Bothe et al., 1994). A signal
of about 20 kb was observed in A. lipoferum and A.
brasilense but not in A. amazonense and A. irakense.
Due to their ability to colonise the root surface
and interior of many cereals and forage grasses, the
first report showing the presence of Azospirillum in
cells of the cortex, in intercellular spaces between
the cortex and endodermis and in the xylem cells of
maize roots by applying the tetrazolium reduction
staining technique (Patriquin and Dbereiner, 1978)
was accepted with some skepticism, because the for-
masan crystals formed could be produced by any
type of reducing agent. Later on, using a very
specific method of root sterilisation, Baldani (1984)
and Baldani et al. (1986b) showed that certain
strains of Azospirillum spp in fact colonise the root
interior and that there are sites of colonisation by
the bacteria along the root. Afterwards, the same
group working on the colonisation process of wheat
plants by Azospirillum spp demonstrated that a
homologous strain A. brasilense Sp245, isolated
914 Jos 1. Baldani et al.
from surface sterilised roots of wheat, colonised and
established itself inside the roots, whereas strains
considered heterologous like A. brasilense Sp7 were
unable to colonise the root interior as evaluated by
the Most Probable Number counting of these
strains in N-free semisolid NFb medium after sur-
face sterilisation of the roots (Baldani et al., 1987).
In addition, it was shown that homologous strains
contributed much more to the nitrogen incorporated
in the plant. Recently, studies with strain-specific
monoclonal antibodies confirmed the ability of
strain Sp245 but not Sp7 to colonise the root in-
terior of wheat plants (Schloter et al., 1994).
Identical results were obtained using a more sophis-
ticated approach, TRITC-labelled species-specific
oligonucleotide probe (BRA 18bl6b) for A. brasi-
lense coupled with confocal laser scanning mi-
croscopy (Hartmann et al., 1995a). The
methodology clearly visualised an in-situ interior
colonisation of wheat roots by strain Sp 245
whereas strains Sp7 or strain Wa3, an isolate from
wheat rhizosphere, only colonised the root surface.
Also quite recently, vande Broek et al. (1993) study-
ing the colonisation of wheat roots under axenic
conditions by strain Sp245 expressing the gusA gene
(GUS reporter system) showed that the bacteria in-
itially concentrate in the root hair zones and at sites
of lateral root emergence and that the proliferation
to other parts of the root is dependent on the status
of the nitrogen and carbon source present in the
solution.
Although many ecological and physiological
properties of Azospirillum are already known, there
is stil1 a lack of information on the mechanisms
involved in the plant-bacteria interaction. Studies
of this interaction at the molecular leve1 have not
advanced rapidly, despite the availability of tech-
niques, probably because of the difficulties in iso-
lation of the genes directly involved in the
processes, and because of the absente of an easily
detectable plant phenotype after inoculation with
the mutants strains (see review of vande Broek and
Vanderleyden, 1995). Nevertheless, many genes
homologous to rhizobia genes already known to be
involved in the legume symbiosis have been detected
in Azospirillum using indirect approaches.
Homologies to the Rhizobium meliloti genes essen-
tial for nodulation in alfalfa such as nod DABCIJ
and nodQPGEFG with A. brasilense and A. lipo-
ferum (Fogher et al., 1985) as wel1 as to the chvA
and chvB genes involved in the root attachment of
A. tumefaciens (Waelkens et al., 1987) have been
demonstrated. More recently, Raina et al. (1995)
confirmed the presence of the chvB homologous
gene in A. brasilense Sp7 and demonstrated that this
lotus has a good homology to the ndvB gene of
Rhizobium meliloti. Loei exoB and exoC that comp-
lement R. meliloti exoB and exoC mutants have also
been identified in A. brasilense (Michiels et al.,
1988), but a mutation in the exoC lotus in strain
Sp7, affecting the EPS biosynthesis, suggests that, at
least in this case, this lotus does not play a role in
the root attachment capacity of this strain (Michiels
et al., 1991). In contrast, recent results with a
mutant of strain Sp7 impaired in exopolysaccharide
production and hence the flocculation process,
suggest that exopolysaccharide is involved in the
root colonisation of wheat (Katupitiya et al., 1995).
The mutant showed a different pattern of colonisa-
tion (concentrated on crevices surrounding the site
of lateral root emergence) and a superior ability to
fix nitrogen in association with the plant. Similar to
that observed for rhizobia species, some of these
homologous genes observed in A. brasilense are
located in the plasmid p90 (Croes et al., 1991). The
authors also demonstrated the presence of three loei
involved in motility of A. brasilense in solid (Mot1
and Mot2) and liquid (Mot3) medium in the plas-
mid ~90. In addition, the same group working with
a mutant affected in the lotus Mot3, which lost
both polar and lateral flagella, demonstrated that
the polar flagellum could be involved in the first
step of the root attachment (Croes et al., 1993;
more detail in vande Broek and Vanderleyden,
1995). Recently, signal molecules were isolated from
the gramineous plants and partially identified that
might provide new insights on the plant-bacteria
associations (van Bastelaere et al., 1995).
Nevertheless, we need to keep in mind that other
species of Azospirillum and other facultative diazo-
trophs also colonise graminaceous plants and there-
fore a much more complex interaction should be
expected when seeds are sown in the field.
Although many inoculation experiments with
Azospirillum spp have been carried out al1 over the
world showing a positive effect of the bacteria on
yield (Boddey and Dbereiner, 1995) there is stil1 a
debate about the main mode of action by which
facultative endophytic diazotrophs contribute to the
nitrogen accumulated in the plants. Effects of plant-
growth promoting substances (Zimmer et al., 1988)
nitrogen fixation per se (I.E. Garcia de Salamone,
unpubl. M.Sc. thesis, University of Buenos Aires,
1993) or the ability of the bacterial nitrate reductase
to help in the incorporation of the nitrogen assimi-
lated from soil by the plant (Boddey et al., 1986;
Ferreira et al., 1987) have been demonstrated.
Despite these different mechanisms exerted by facul-
tative endophytic diazotrophs in association with
graminaceous plants, increases in the range of 5 to
30% in yield have been observed in several inocu-
lation experiments with Azospirillum (Baldani et al.,
1983; Okon and Labandera-Gonzalez, 1994).
Since Azospirillum spp do not excrete ammonium
during the process of nitrogen fixation in culture,
several approaches have been developed to provide
this characteristic to Azospirillum envisaging
improvement of the association. Mutants able to
BNF with non-legume plants 915
excrete ammonium have been obtained from A. bra-
silense Sp7 using EDA mutagenesis (Machado et
al., 1991) but no effect on the plant was determined.
Almost at the same time, Christiansen-Weniger and
van Veen (1991) showed that an ammonium excret-
ing mutant of A. brasilense (Wa3) promoted better
growth of the wheat plant as compared with the
wild-type. The same authors, using another am-
monium excreting mutant (strain C3) tested with
the 15Nz gas i o p s to e dilution technique, concluded
that the strain was able to transfer the nitrogen
fixed directly to the maize plant and that this
amount transferred was increased in para-nodulat-
ing plants obtained by treatment with 2,4D
(Christiansen-Weniger, 1994). Responses of grami-
naceous plants to inoculation with facultative endo-
phytic diazotrophs like Azospirillum should always
include the interaction with other diazotrophs as
wel1 as additional characteristics such as excretion
of ammonium or IAA production.
Obligate endophytic diazotrophs
This group includes Acetobacter diazotrophicus, a
nitrogen-fixing bacterium clustered in the alpha sub-
class of the Proteobacteria and Azoarcus spp,
Herbaspirillum seropedicae, H. rubrisubalbicans and
a partially identified Burkholderia sp.; these are clus-
tered in the beta subclass of the Proteobacteria.
Acetobacter diazotrophicus
Studies on the occurrence of this diazotroph have
shown that it has restricted host range (Dbereiner,
1992b). A. diazotrophicus, has been found mainly
associated with sugar-rich plants such as sugarcane,
sweet potato and Cameroon grass, al1 of which pro-
pagate vegetatively (Table 2). It colonises roots,
sterns and leaves of sugar cane in numbers up to
106 cells g- fresh weight or more if an improved
testing medium is used (Reis et al., 1994); in sweet
potato it has been found in numbers up to 105
(Paula et al., 1993). Similar values als0 were
observed in sugar cane grown in Australia by Li
and MacRae (1992) using the ELISA method. In
our laboratory with the ELISA technique it was
possible to detect differences in the colonisation of
various nitrogen-fixing varieties of sugar cane by A.
diazotrophicus. However, no correlation was
observed in the number of the bacteria and the ca-
pacity of the sugarcane varieties to obtain N from
BNF (Silva et al., 1995). The restricted occurrence
of A. diazotrophicus was confirmed by its complete
absente in soil and in tissue of weed plants grown
between rows in a sugar cane field (Dbereiner et
al., 1988). These results also were confirmed using a
species-specific primer designed to discriminate A.
diazotrophicus from Acetobacter species and other
diazotrophs (Reis et al., 1995). The best confir-
mation that this bacterium is an obligate endophyte
comes from the results obtained with 10 month old
plants grown in a sugarcane field which had been
planted by the micropropagation process normally
used to produce disease-free plants. This bacterium
could not be detected in plants at ages varying from
3 to 10 months (Reis Jr, unpubl. results). Other sup-
port for this endophytic characteristic comes from
recent results from our laboratory showing that A.
diazotrophicus does not survive in the soil (Fig. 1).
Using a lacZ-fusion it was observed that this bacter-
ium cannot be detected 2 days after inoculation in
natura1 soil, although it can survive longer (up to
10 days) in sterile soil (Caruso and Baldani, 1995).
The infection and colonisation processes of sugar
cane by Acetobacter diazotrophicus have been exam-
ined by James et al. (1994) and Reis Jr et al. (1995).
On the root surface the bacteria were concentrated
at the root tips and around lateral root junctions,
and the bacteria infected the plants at these sites.
Inside the roots, the bacteria were observed in
apparently intact, enlarged epidermal cells, and also
at the base of the stem within xylem vessels through
which the bacteria appeared to migrate to the shoot
tissues. The confirmation that A. diazotrophicus
colonises the intercellular spaces of sugar cane stem
parenchyma comes from the work of Dong et al.
(1994) who showed the presence of the bacteria in
numbers of 104 cells ml- of fluid inside the apo-
plast. Despite the restricted host plant occurrence, a
few isolates with many characteristics similar to
Acetobacter were isolated recently from toffee plants
in Mexico (Jimnez-Salgado et al., 1995).
The most common physiological characteristics of
A. diazotrophicus are the high sucrose tolerante
(10%) growth and nitrogen fixation at low pH (5.0
or less), chocolate colonies on potato agar medium
with 10% sucrose, absente of nitrate reductase,
nitrogen fixation not affected by high concen-
trations of NO; (25 mM) and partial inhibition by
NH,+ especially at high sucrose concentrations
(Teixeira et al., 1987; Boddey et al., 1991; Stephan
et al., 1991). Another unique characteristic of this
bacterium is the ability to excrete part of the fixed
nitrogen into the medium as demonstrated by
Cojho et al. (1993) using an amylolytic yeast to
mimic the plant. More recently, Cruz et al. (1995)
determined that ammonium was the product
excreted by A. diazotrophicus under nitrogen fix-
ation conditions.
Not much is known about the genetics of A. diu-
zotrophicus, as the bacterium was isolated quite
recently. Nevertheless, it is known that some strains
carry plasmids of sizes varying from 50 to 110 Mda
and that the nif genes are located in the chromo-
somes (Teixeira et al., 1994). It was also shown that
this obligate diazotroph has a narrow genetic diver-
sity (Caballero-Mellado and Martinez-Romero,
1994); n$HDK and nifA have already been
sequenced (Sevilla et al., 1995; Teixeira, unpubl.
results). Other genes like ntrBC, n$B, (Meletzus,
916 Jos 1. Baldam et ul
10
Q Sterile soil
8 0 Natura1 soil
z
%
OO 6
2
0 4
!!
2
0
0 2 7 15
Days after inoculation
Fig. 1. Survival of Acetobucter diuzotrophicus strain PAL5
in natura1 and sterile soil with moisture tension at
-0.03 MPa and temperature of 32C. Asterisks indicate
that the population had declined to less than the leve1
detected by the Most Probable Number (MPN) method
unpubl. results) and nifV and n!fE have also been
detected in A. diuzotrophicus by complementation
(Sevilla et ~1.. 1995).
Herbuspirillum seropedicue
The second obligate endophyte is Herbaspirillum
seropedicae that has a wider host range specificity
than A. diazotrophicus since it has been isolated
from many graminaceous plants such as maize, sor-
ghum, rite, sugar cane and forage grasses grown in
Brazil (Table 2). Quite recently, it was isolated from
sugar cane plants grown in South Africa (J.
Thomson. pers commun.). It has also been isolated
from other non-legume plants including oil palm
trees (Ferreira et al., 1995). It has been found inside
roots, sterns and leaves of these graminaceous plants
(Dbereiner ef al., 1994) but not within leaves of
sugar cane plants harvested from the field (Olivares
et al.. 1996). Artificial inoculation of sugar cane
leaves with H. seropedicue showed that the bacteria
do not spread into the xylem vessels of the leaves
but remain localised at the point of inoculation.
The pattern of sugar cane root colonisation by H.
seropedicae is quite similar to that observed for A.
diazotrophicus, except that the attachment of the
cells occurs al1 along the roots and that invasion of
the roots occurs mostly at junctions of secondary
roots where through the intercellular space it colo-
nises the xylem. (F.L. Olivares, pers commun.). On
the other hand, the colonisation of rite plants by a
selected strain of H. seropedicue showed that the
bacteria first colonise the epidermal cells ofthe root
surface and then enter through the intercellular
spaces derived from cells or secondary root emer-
gence (Baldani et al., 1995).
The natura1 dissemination of H. seropedicue is
not yet clear, although it seems to be transferred
mainly through the seeds, as it can occasionally be
isolated from seeds of cereals (Baldani et al., 1992).
In plants which are micropropagated vegetatively
like sugarcane, it can be transmitted in the same
way as observed for A. diazotrophicus. This was
confirmed by the presence of the bacteria in sugar
cane plants originating from the micropropagation
process where the innermost apical meristems have
not been extracted carefully (Olivares et UI ., 1996).
Like other endophytes, H. seropedicae does not sur-
vive wel1 in natura1 soil; it is less affected in sterile
soil indicating that biotic factors interfere with the
survival of this bacterium in natura1 soil.
Nevertheless, the survival of H. seropedicue in both
sterile and natura1 soils was better than that
observed for A. diazotrophicus (Caruso and Baldani,
1995).
Herhaspirillum rubrisubulbicans
This obligate endophytic diazotroph was found
recently among several strains of Pseudomonus
rubrisubulbicans. a species considered a mild phyto-
pathogenic agent causing mottle stripe disease in
some susceptible varieties of sugarcane grown in
countries other than Brazil. (Pimentel et al.. 1991).
Based on its DNA:rRNA homology, and some
physiological characteristics as wel1 as 15Nz gas in-
corporation, these strains were included in the
genus Herbuspirillum (Gillis ef al., 1991; Baldani et
al., 1996). Inoculation of H. rubrisubulbicuns by
injection into leaves of the sugarcane variety B-
4362, susceptible to mottled stripe disease, produced
the typical symptoms of the disease (Olivares et al.,
1993). NO symptoms, however, were observed in the
commercial Brazilian sugar cane varieties CB45-3,
NA 56-79 or SP 70-1143 inoculated with this bac-
terium. Similarly, inoculation of these commercial
varieties as wel1 as the susceptible variety B-4362,
with a strain of H. seropedicae did not produce any
red and white stripes symptomatic of the mottled
stripe disease (Olivares et al., 1993). Studies on the
colonisation of sugar cane by this endophyte, have
concentrated mainly on the aerial tissue parts and
have shown the presence of the bacterium within
the meta- and protoxylem (Olivares et al., 1993). In
the case of the variety B-4362, H. rubrisubulbicuns
was found to completely black some of the xylem
vessels and to colonise the intercellular spaces of the
mesophyll cells. On the other hand, in the variety
SP 70-1143, resistant to mottled stripe disease, the
bacteria also colonise the xylem but form clusters of
10 to 20 cells encapsulated by membranes, probably
of plant origin (Boddey, 1995). More recently, ac-
tivity of nitrogenase was detected in the xylem
vessels of sugar cane leaves infected with this obli-
gate endophyte; nitrogenase was demonstrated with
an antiserum raised against the FeMoCo subunit of
nitrogenase (F.L. Olivares et al., unpubl. data). The
pattern of colonisation of roots of sugarcane var-
ieties showed an infection and colonisation process
similar to that of the H. seropedicue. Preliminary
BNF with non-legume plants 917
results on the colonisation of rite roots by H. rubri-
subalbicans showed that the bacteria concentrate at
the point of secondary root emergence and first
colonise cells from the epidermis and hypodermis
where they begin to multiply and colonise the upper
part of the plant, probably through the xylem
(Fig. 2).
Although it was initially thought that H. rubrisu-
balbicans would be restricted to sugarcane (Table 2),
it was recently isolated from rite plants (V.L.D.
Baldani, unpubl. results) and palm tree plants
(Ferreira et al, 1995). Occurrence of H. rubrisubal-
bicans in rite plants may not be a surprise because
inoculation of rite seedlings grown axenically with
strain M4 increased nitrogen accumulated in the
plant about 30% (Baldani et al., 1995).
Azoarcus spp
This obligate endophytic diazotroph, including
the species A. indigens, A. communis and a few iso-
lates that do not show high homology to these two
species but are called Azoarcus sp. (Reinhold-Hurek
et al., 1993), has been isolated from Kallar grass
grown in saline-sodic soils in Pakistan. In contrast
to Azospirillum halopraeferans, that has been found
only on the surface of the roots of this plant
(Reinhold et al., 1987), Azoarcus was repeatedly
found inside Kallar grass roots or stem bases
(Table 2). More recently, the authors developed
genus-specific primers for Azoarcus which could
differentiate strains of this genus from other nitro-
gen-fixing bacteria and suggested PCR amplification
using the genus-specific primers to determine the
presence of Azoarcus in plant tissues of Kallar grass
(Hurek et al., 1993a). Like other endophytes already
discussed, Azoarcus does not cause pathogenic reac-
tions when inoculated into Kallar grass grown axe-
nically (Hurek et al., 1994). The authors showed
that strain BH72 is capable of invading roots of the
original host as wel1 as rite plants, infecting the cor-
tex region and penetrating the stele of the roots.
Spreading of the bacteria into the plant shoot is
probably by the colonisation of the xylem vessels
where it could be detected by the use of specific
antibodies or by its nitrogenase activity. Enzymes
like cellulases, an exoglucanase and an endogluca-
nase, already detected in Azoarcus, might contribute
to the process of infection of this obligate endo-
phyte into these plants (Hurek and Reinhold-
Hurek, 1994; Reinhold-Hurek et al., 1994). A close
interaction of this bacterium with a rhizosphere fun-
gus was also demonstrated by these authors. They
observed the induction of tubular arrays of internal
membrane stacks in Azoarcus which they demon-
strated to be functional membranes related to nitro-
gen fixation (confirmed by antiserum raised against
nitrogenase), and they named these structures diazo-
somes (Hurek er al., 1993b).
Burkholderiu spp
This genus was recently created to include strains
belonging to the (Pseudomonas) rRNA complex
including (P.) solanacearum and (P.) cepuciu
(Yabuuchi et al., 1992). Quite recently, a new nitro-
gen-fixing bacterium was isolated from the rhizo-
sphere of a rite plant grown in Vietnam, and
because.of its high DNA:DNA, DNA:rDNA hom-
ology and phenotypic characteristics close to the
genus Burkholderia, the name B. vietnamiensis was
proposed (Tran Van et al., 1994). It cannot be con-
cluded yet that this species is an endophyte, because
it has been isolated only from rhizosphere macerates
(roots plus adhering soil).
In a survey to determine the occurrence of nitro-
gen-fxing bacteria in rite plants grown in Brazil,
Oliveira (unpubl. MSc thesis, Federal Rural
University of Rio de Janeiro 1992) isolated a new
group of bacteria on a N-free semi-solid medium
containing glucose, oxalate and citrate. The bacteria
were different from the other nitrogen-fixing bac-
teria known in our Centre and were designated E.
This bacterium was also isolated at the same time
from cassava plants (roots, tubers and sterns) in
numbers quite similar to Azospirillum (Balota et UI.,
1994). Using an improved N-free semi-solid medium
called JMV (mannitol as a carbon source and pH
4.5 to 5.0), several isolates were obtained from
roots, sterns and leaves of rite plants (Table 2)
(V.L.D. Baldani, pers commun.). Morphological
and physiological characteristics as wel1 as a partial
sequencing of the 23s rRNA indicate that these
strains as wel1 as isolates from cassava and sweet
potato belong to the Burkholderia genus (Hartmann
et al., 1995b) but do not share similarities that per-
mit them to be included in the species B. vietnamien-
sis. It is interesting that a specific oligonucleotide
probe generated to identify these rite isolates per-
mitted the identification of another group of strains
that had been isolated from sugar cane. This new
group was also confirmed to belong to the genus
Burkholderia by partial sequencing of the 16s
rRNA of a strain Ppe8; its phenotypical and mor-
phological characteristics suggest it may represent a
new species or subspecies (Hartmann et al., 1995b).
A oligonucleotide probe generated from the hyper-
variable region of the 23s rRNA of the strain PPe8
confirmed these results and distinguished them from
the other group (Hartmann et al., 1995b). More
recently, strains of these two groups were also iso-
lated from fruit plants (Weber et al., 1995). The role
of these new endophytic nitrogen-fixing bacteria to
an association of a plant and bacteria is not yet
known, although they may exercise the same func-
tions as the other endophytes.
Preliminary results from the infection and coloni-
sation process of rite by Burkholderia sp. strains
showed that the bacteria first colonise the root sur-
faces and them enter the cells through the intercellu-
Fig. 2. Rite root Lissues inoculated with ffe~hu.cfJ i,-i//rlr,l ~~h~rsd~o/l~i~UI.\ straln M4. (21) Entrance of the
bacteria through a wound on the root surface. Bar = 10 pm. (b) Detail of hacteria growing under the
loose epidermis (B). Bar ~ 10 pm.
BNF with non-legume plants 919
Fig. 3. Detail of the root surface colonisation of rite by the Burkhoideriu sp. strain M209. Bar = 10 Pm
lar spaces caused by broken membranes (Fig. 3). needed to identify the limiting factors of the associ-
The bacteria also can penetrate through crdcks of ation. Nif minus or other regulatory nif mutants as
the epidermal cells caused by loss of surface cells we11 as mutants affected by molecular signal inter-
and also at the points of secondary root emergence actions wil1 be very helpful in providing a better
(Baldani et al., 1995). A massive number of bacteria understanding of associative endophytic bacteria.
have been found inside rite roots without causing
cel1 damage (V.L.D. Baldani and S.R. Goi, unpubl.
results).
REFERENCES
CONCLUSIONS
Baldani J.I. (1984) Ocorrncia e caracterizao de
Azospirillum amazonense em comparao com as outras
Obligate endophytic diazotrophs have an enor-
espcies deste gnero. em raizes de milho. sorgo e arroz.
mous potential for use because of their ability to
Itaguai, U.F.R.R.J., 1lOpp. Tese Mestrado.
Baldani V.L.D., Baldani J.l. and Dbereiner J. (1983)
colonise the entire plant interior and establish them- Effects of Azospirillum inoculation on root infection and
selves within niches nrotected from oxvgen or other
nitrogen incorporation in wheat. Canadian Journal of
1 <.,
inhibitory factors; thus their potential to fix nitro-
Microbiology 29, 869988 1.
gen can be expressed at the maxima1 level. These
Baldani J.l., Baldani V.L.D., Seldin L. and Dbereiner J.
properties may be the reason for the high nitrogen
(1986a) Characterization of Herbaspirillum seropedicae
pen, nov.. SD. nov.. a root-associated nitroeen-fixine bac-
fixation observed in sugarcane plants (Urquiaga et Serium. Int&wational Journal of Systemat& B&ter~olog?
al.. 1992). Nevertheless, it should be kept in mind
36, 86693.
that other nitrogen-fixing bacteria such as rhizo-
Baldani V.L.D., Alvarez M.A., De B.. Baldani J.I. and
spheric diazotrophs, especially those showing
Dbereiner. J. (1986b) Establishment of inoculated
narrow host plant range, colonise the root system
Azospirillum spp in the rhizosphere and in roots of field
grown wheat and sorghum. Plant and Soil90, 35-46.
and therefore may also play an important role in
Baldani V.L.D., Baldani J.I. and Dbereiner J. (1987)
the association.
Inoculation of field-grown wheat (Triticum aestivum)
The potential of the endophytes to be used on a
with Azospirillum spp in Brazil. Biologl, and Fwtility of
large scale in the field is very promising. However,
Soils 4, 57-60.
Baldani V.L.D., James E.K., Baldani J.I. and Dbereiner
more studies on the plant-bacterial interactions are J. (1992) Localization of the Nz-fixing bacterium
920 Joac I. Balduni (/ rrl.
Hwhuxpirillwr .wr~~/wrli~~r~~~ within root cells of rite. Chrlstlansen-Wcniger c. and van Veen J.A. (IYY 1) :L/f ;
.417(1;.~ Aoxlentirr Bur.~il~~i,o de Cimcitr.s. Vol. 64. p. 43 1 cxcreting A:o.spiri/hm! hrr/.\i/m.w mutants enhancc the
Rio de Janeiro. nitrogen %lpply of a wheat host. .App/i~~t/ trrrtl
Baldani V.L.D.. Goi SR.. Baldani J.I. and Diibereiner J. ~~r~,;~~>rln?rr~/ol M/crohio/o,qj~ 57, 3006 30 11.
( 1995) Localization of Hwhuspirilhrm spp and Co.iho E.H.. Reis V.M.. Schenberg A.C.G. and DObereinei
Burhholdwirr sp. in rite root system. In I~rle~ri(riiotrtrl J. ( l9Y3) Interactions of Aw/ohtrc/rr </ic/-orr<)/~/li(,ll with
S,wyx~siruit 011 Miuohirrl f?o/ocq~. Vol. 7. p 133. an amylolytic yeast in nitrogcn-free batch culture.
Brazilian Society for Microbiology. Santos. Sao Paulo. FEMS Microhio/o,q>, Lc//erv 106, 341 346.
Baldani J. 1.. Pot B.. Kirchlaof G.. Falsen E.. Baldani Croes C., Van Bastelaere E.. Declercq E.. Eyers M..
V.L.D., Olivares F.L.. Horte B.. Kersters K., Hartmann Vanderleyden J. and Michtels K. (1991) Identilication
A.. Gillis M. and Dbereiner J. (1996) Emended descrip- and mapping of loei involved in motility, adsorption to
tion of Therbaspirillium: inclusion of [pseudononas]
wheat roots. colony morphology. and growth in minimal
rubruisu haltican. a mild plant pathogen as
medium on the Azoxpirilhm hrmi/uw Sp7 90.Mda plas-
Herhaspirillium subuisubdlacaus comb- nov. and classi-
mid. Plrrstnid 26, X3 93.
tication of a group of clinical isolates (EF group 1) as
Croes C.L.. Moens S., Van Bastelaere E.. Vanderleyden
Herbaspirillium species 3. f.J.S.B 46, 802 810.
J. and Michiels K.W. (1993) The polar Aagellum med-
Balota E.L.. Hungria M. and Dbereiner J. (1994)
Occurrence of diazotroph bacteria and vesicular-arhus-
tates A:.~ppiri//un~ hru.si/mw adsorption to wheat root\.
cular mycorrhizal fungi associated with
Jurw/ Getwnl Microhiolog~~ 139, 226 1 -2269.
cassava Cruz L.M.. Teixeira K.R.S. and Baldani J.l. (1995)
(Mtrnihot csc~ulr~~t~r Crantz). In Nitro,qw Fi.x-rrtim wilh
Non-L~~unrm. (N.A. Hegazi. M. Fayez and M. Monib.
Expressao fenotipica de estirpes de Ac~tohucrer di~otro-
Eds), pp. 53-58. American University in Cairo Press.
plri<,r~.r contendo o gene nifA de ,4:tohac~r1~~ ~~inclwulii.
In Ancris 20. Reuniao Anual de Gentica dc
Cairo. Egypt.
Bashan Y.. Holguin G. and Lifshitr R. (1993) Isolation
Microorganismos. Vol. 20. p. 116. Sociedade Brasileirn
and characterization of plant growth-promoting rhizo-
de Gentica. Piracicaba, So Paulo.
bacteria. In Mrthods NI Plru/ Molwultrr Bioloxj, crnd
Dbereiner J. (1959) Influncia da cana-de-accar na
Biotechnologie (B.R. Click and J.E. Thompson. Eds). pp.
populacao de Br&vinckirr do solo. Revistu Brcr.si/eirrr de
331-343. CRC Press. Boca Raton.
Biolqiu 19, 251 25X.
Boddey R.M. (1995) Biological nitrogen fixation in sugar
Dobereiner J. ( 1966) A:o/ohoc~tcr pu.spu/i sp., uma bactria
cane: A key to energetically viable biofuel production.
lxadord de nitrognio na rizosfera de Pa~pcrlun~.
Critical Revim in Pkrnt Scimws 14, 263 219.
Pm/uistr Agropecbici Brasilriru 1, 357-365.
Boddey R.M.. Baldani V.L.D.. Baldani J.l. and
Dbereiner J. (1992a) Recent changes in concepts of plant
Dbereiner J. (1986) Effect of inoculation ol
bacteria interactions: Endophytic Nz fixing bacteria.
Axspiri//um spp on thc mtrogen assimilation of tield
C&ci0 c ~rr//mr 44, 3 1 Om 3 13.
grown wheat. Pkrnt crut/ Soil 95, 109 12 1
Dhereiner J. (1992h) History and new perspectives of dia-
Boddey R.M.. Chalk P.M., Victoria K.L.. Matsui E. and
zotrophs in association wsith non-leguminous plunta.
Dbereiner J. (1983) The use of the lSN isotope dilution
.Sjwhio.ti.c 13, I 13.
techniaue to estimate the contribution of associated hio-
Dobereiner J. (1995) Biological nitrogen tixation in the tro-
logical nitrogen fixation to the nitrogen nutrition of
Pnspultun notutm CV. h0tcrttri.c. Ctrnodion .Jmrrr~rrl of
Microhiolqg 29, 1036~-1045.
Boddey R.M. and Dbereiner J. (1995) Nitrogen tixation
associated with grasses and cereals: Recent progress and
perspectives for the future. Ferti/ix,r Rcwcwch 4, I 10.
Boddey R.M.. Urquiaga S.. Reis V. and Dbereiner J.
(1991) Biological nitrogen tixation associated with sugar
cane. P/mt cm/ .Soi/ 137, I 1I 117.
Bothe H.. Zimmer W.. Kloss K. and Kaldorf M. (lYY4)
Azospirillum and related organisms: ecological. physio-
logical. biochemical and genetics aspects. In Nitrogcw
Fi.\-ntion with Non-Lquvm. (N.A. Hegazi. M. Fayez
and M. Monib, Eds), pp. 43-52. American University in
Cairo Press, Cairo. Egypt.
pits: Social and conomic- contributions. In
It~trrntrtionrrl S~~mpo.vium ou Suc tcrinuhle Agriculturc for
the Tropic,.sv T/w R/c of BioloRicrrl Nitrogen Fiuation, p.
3 4. Angra dos Reis.
t jbereiner J. and Alvahydo R. (lY59) Sobre a influncia
da cana-de-accar na ocorrncia de Beijerinckia no solo.
Il-Influncia das diversas partes do vegetal. Rrl,isttr
Brmikeirrr de Biologia 19, 25 1-25 8.
jhereiner J. and Baldani J.I. (1982) Bases cientiticas para
uma agricultum hiolgica. Ci&ciu c Cu/turu. So Puulo
34, X69 8X1.
Caballero-Mellado J. and Martinez-Romero E. (1994)
Limited genetic diversity in the endophytic sugarcune
bacterium Awtohocter clicrsotrophi<,u,c. Applied trud
Emironmwtcd Microhiology 60, 153221537.
Caruso L.V. and Baldani J.I. (1995) Monitormg the survi-
val of endophytic diazotrophic bacteria in soil using
Lac-Z fusion. 1 n Intrrnationul S~wpovium m Susttrirwhle
ARriculturr fiw the Tropicx: The Roio of Biologiurl
Nitrogrn Fi.ytrtion. p. 108. hgrd dos Reis. Rio de
Janeiro.
Cavalcante V.A. and Dbereiner J. (198X) A new acid-tol-
erant nitrogen-tixing bacterium associated with sugar-
cane. Plant and Soil 108, 23 -3 I
Christiansen-Weniger C. (1994) Pard-nodule induction m
maize with indole-acetic-acid (IAA) and its infection
with ammonia excreting Ax~pirillum hrusi/mw. In
Nitrogew Fi\-rrtion wirh Non-Lqqmes (N.A. Hegazi. M
Fayez and M. Monib, Eds). pp. 5266536. American
University in Cairo Press, Cairo. Egypt.
jiihereiner J.. Baldani V.L.D.. Olivares F.L. and Reis
V.M. (1994) Endophytic diazotrophs: The key to grami-
naceous plants. In Nitrogcn Fiction with NmLqymr.s
(N.A. Hegazi. M. Fayez and M. Monib, Eds). pp. 395
408. American Umversity in Cairo Press, Cairo. Egypt.
Dbereiner J. and Day J.M. (1975) Associative symbioses
in tropical grasses: Characterization of microorganisms
and nitrogen-fixing sites. In Intwnotionol _mposium 0>1
hitrogen Fisution. Vol. 1. (W.E. Newton and C.J.
Nyman. Eds). pp. 518~538. Pullman Proceedings
Washington. Washington State University.
Dbereiner J.. Reis V.M. and Lazarini A.C. (1988) New
N2-hxing bacteria in association with cereals and sugar-
cane. In Nitrogcn Fi.wtim: Hundred Ymm After (H.
Bothe. F.J. De Brmjn and W.E. Newton. Eds). pp. 717
722. Gustav Fischer, Stuttgart.
Dhereiner J. and Ruschel A.P. (1958) Uma nova espcte
de Beijerinkicr. Rcvista de Biologie, Rio dc Jrrnciro 1,
26l- 272.
Dong Z.. Canny M.J.. McCully M.E., Roboredo M.R.,
Cabadilla C.F.. Ortega E. and Rods R. (1994) A nitro-
gen-fixing endophyte of sugarcane sterns. A new role for
the apoplast. Plant Ph~.~io/o,qy 105, 1 13991 147.
BNF with non-legume plants 921
Ferreira M.C.B., Fernandes M.S. and Dbereiner J. (1987) Katupitiya S., Millet J., Vesk M., Viccars L., Zeman A.,
Role of Azospirillum brasilense nitrate reductase in Lidong Z., Elmerich C. and Kennedy I.R. (1995) A
nitrate assimilation by wheat plants. Biology and mutant of Azospirillum brasilense Sp7 impaired in floccu-
Fertility of Soils 4, 47-53. lation with a modified colonization pattern and superior
Ferreira A.C., Cozzolino K., Carvalho A.R.V. and nitrogen fixation in association with wheat. Applied and
Dbereiner J. (1995) Isolation and characterisation of Environmental Microbiology 61, 1987-1995.
diazotrophic bacteria in oil palm trees. In I nternational Khammas K.M., Ageron E., Grimont P.A.D. and Kaiser
Symposium on Sustainable Agriculture for the Tropics- P. (1989) Azospirillum irakense sp. nov., a nitrogen-8xing
The Role of Biological Nitrogen Fixation, p. 210. Angra bacterium associated with rite roots and rhizosphere
dos Reis, Rio de Janeiro. soil. Research in Microbiology 140, 679-693.
Fogher C., Dusha I., Barbot P. and Elmerich C. (1985) Kloepper J.W. and Beauchamp C.J. (1992) A review of
Heterologous hybridization of Azospirillum DNA to issues related to measuring colonization of plant roots
Rhizobium nod and 3.x genes. FEMS Microbiology by bacteria. Canadian J ournal of Microbiology 38, 1219-
Letters 30, 245-249. 1232.
Germida J.J. (1986) Population dynamics of Azospirillum Li R. and MacRae I.C. (1992) Specific identification and
brasilense and its bacteriophage in soil. Plant and Soil6, enumeration of Acetobacter diazotrophicus in sugarcane.
189-203. Soil Biology & Biochemistry 24, 4 13-419.
Gillis M., Dbereiner J., Pot B., Goor M., Falsen E., Machado H.B., Funayama S., Rigo L.U. and Pedrosa
Hoste B., Reinhold B. and Kersters K. (1991) F.O. (1991) Excretion of ammonium by Azospirillum
Taxonomie relationships between (Pseudomonas) rubrisu- brasilense mutants resistent to ethylenediamine.
balbicans, some clinical isolates (EF group l), Canadian J ournal of Microbiology 37, 549-553.
Herbaspirillum seropedicae and (Aquaspirillum) autotro- Magalhes F.M., Baldani J.I., Souto S.M., Kuykendall
phicum. In Nitrogen Fixation (M. Polsinelh, R. J.R. and Dbereiner J. (1983) A new acid-tolerant
Materassi and M. Vincenzi, Eds), pp. 292-294. Kluwer Azospirillum species. Anais Academia Brasileira de
Academie Pubhshers, Dordrecht. Cincia 55, 417430.
Hartmann A., Assmus B., Chatzimotas A., Schloter M., Magalhes F.M. and Dbereiner J. (1984) Ocorrncia de
Ludwig W. and Kirchhof G. (1995a) Use of molecular Azospirillum amazonense em alguns ecossistemas da
probes to study Azospirillum-plant interactions. In Amaznia. Revista de Microbiologia 15, 246-252.
I nternational Workshop on Associative I nteractions of Michiels K., Croes C. and Vanderleyden J. (1991) Two
Nitrogen-Fixing Bacteria with Plants, pp. 59-63. Russia different modes of attachment of Azospirillum brasilense
Academy of Science Saratov. Sp7 to wheat roots. J ournal of General Microbiology
Hartmann A., Baldani J.I., Kirchhof G., Assmus B., 137, 2241-2246.
Hutzler P., Springer N., Ludwig W., Baldani V.L.D. Michiels K., Vanderleyden J., Van Go01 A. and Signer E.
and Dbereiner J. (1995b) Taxonomie and Ecologie stu- (1988) Isolation and characterization of Azospirillum
dies of diazotrophic rhizosphere bacteria using phyloge- brasilense loei that correct Rhizobium meliloti exol and
netic probes. In Azospirillum VI and Related exoC mutants. J ournal of Bacteriology 170, 540-5404.
Microorganisms (1. Fendrik, M. del Gallo, J. Okon Y. and Labandera-Gonzalez C. (1994) Agronomie
Vanderleyden and M. de Zamaroczy Ed.) Springer- application of Azospirillwn: An evaluation of 20 years
Verlag, Berlin. worldwide field inoculation. Soil Biology and
Hurek T., Burggraf S., Woese C.R. and Reinhold-Hurek Biochemistry 26, 1591-1601.
B. (1993) 16s rRNA-Target polymerase reaction and oli- Ohvares F.L., Baldani V.L.D., Reis V.M., Baldani J.I. and
gonucleotide hybridization to screen for Azoarcus spp Dbereiner J.I. (1996) Occurrence of endophytic diazo-
grass-associated diazotrophs. Applied and Environmental trophs Herbaspirillum spp in roots, sterns and leaves pre-
Microbiology 59, 3816-3824. dominantly of gramineae. Biology and Fertility of Soils
Hurek T., Kellenberger E., van Montagu M. and 21, 197-200.
Reinhold-Hurek B. (1993b) Formation of internal mem- Olivares F.L., James E.K., Reis V.M., Baldani V.L.D.,
brane stacks, diazomes, in Azoarcus sp. In New Horizons Baldani J.I. and Dbereiner J. (1993) Colonizao do
in Nitrogen Fixation (R. Palacios, J. Mora and W.E. tecido vascular por Herbaspirillum spp em sorgo e cana-
Newton, Eds), p. 619. Kluwer Academie Publishers, de-aucar. In Fitopatologia Brasileira. Supplemento no.
Dordrecht. 18, p. 313. Sociedade Brasileira de Fitopatologia,
Hurek T. and Reinhold-Hurek B. (1994) Identification of Aracaju, Sergipe.
Azoarcus spp grass-associated diazotrophs, by analysis Oliveira E. (1992) Estudo da associaco entre bactrias
of partial 16s rRNA sequences. In Nitrogen Fixation diazotrficas e arroz. MSc. Thesis. Universidade Federal
with Non-Legumes (N.A. Hegazi, M. Fayez and M. Rural do Rio de Janeiro, Itaguai, RJ.
Monib, Eds), pp. 59-68. American University in Cairo Patriquin D.G. and Dbereiner J. (1978) Light microscopy
Press, Cairo, Egypt. observations of tetrazohum-reducing bacteria in the
Hurek T., Reinhold-Hurek B., van Montagu M. and endorhizosphere of maize and other grasses in Brazil.
Kellenberaer E. (1994) Root colonization and svstemic
~ I
Canadian J ournal of Microbiology 24, 734-742.
spreading of Azoarcus sp. strain BH72 in grasses. Paula M.A., de, Siqueira, J.O. and Dbereiner, J. (1993)
J ournal of Bacteriology 176, 1913-1923. Ocorrncia de fungos micorrzicos vesculo-arbusculares
James E.K., Reis V.M., Baldani J.I. and Dbereiner J. e de bactrias diazotrficas na cuhura de batata doce.
(1994) Infection of sugarcane by the nitrogen-fixing bac- Revista Brasileira de Ciencia do Solo 17, 349-356.
terium Acetobacter diazotrophicus. J ournal of Pereira J.O. (1995) Microrganismos endofiticos em espcies
Experimental Botany 45, 757-766. tropicais. In Anais 20. Reunio Anual de Gentica de
Jimnez-Salgado T., Fuentes-Ramires L.E., Tapia- Microrganismos, Vol. 20, p. 3. Sociedade Brasileira de
Hemndes A., Mascara-Esparza M.A., Martinez- Gentica, Piracicaba, So Paulo.
Romero E. and Caballero-Melado J. (1995) Acetobacter Pimentel J.P., Olivares F.L., Pitard R.M., Urquiaga S.C.,
endophytic from Coffea arabica, In I nternational Akiba F. and Dbereiner J. (1991) Dinitrogen fixation
Symposium on Sustainable Agriculture for the Tropics- and infection of grass leaves by Pseudomonas rubrisubal-
The Role of Biological Nitrogen Fixation, p. 212. Angra bicans and Herbaspirillum seropedicae. Plant and Soil
dos Reis, Rio de Janeiro. 137,61-65.
SBB 29/5-6F
922 Jos 1. Baldani et al
Raina S., Raina S., Venkatesh T.V. and Das H.K. (1995)
Isolation and chardcterization of a lotus from
Azospirillum hrasiiense Sp7 that complements the
tumorigenic defect of Agrohacterium tumefaciens CAVE
mutant. Moiecular Plant-Microbe Interactions 8, 322.~
326.
Reinhold B., Hurek T., Baldani J.I. and Dbereiner J.
(1989) Temperdture and salt tolerante of A-_ospiri//um
spp from salt-affected soils in Brazil. In Ax~spirillum II.
Genetics. Physiology, and Ecology, (W. Klingmeller. Ed. 1.
pp, 234-241, Springer-Verlag, Berlin.
Reinhold B., Hurek T., Fendrik 1.. Pot B., Gillis M..
Kersters K.. Thielemans S. and De Ley J. (1987)
Azospirillum halopraef?wns sp. nov.. a nitrogen-fixing
organism associated with roots of kallar grass
(Leptochloa fusca (L.) Kunth). Internutional Journul o/
Systemutic Bucteriology 31, 4335 1.
Reinhold-Hurek B., Hurek T.. Gillis M.. Hoste B..
Vancanneyt M.. Kersters K. and De Ley J. (1993)
Azourcus gen. nov.. nitrogen-Ixing proteobacteria as-
sociated with roots of kallar grass (Leptorhloa fusca (L.)
Kunth). and description of two species, Azourcus indi-
gens sp. nov. and Azourcus communi.\ sp. nov.
International Journul c!f Sy:vtcmutic Bucteriology. 43, 574
584.
Reinhold-Hurek B.. Hurek T.. Vlaeyssens M. and Van
Montagu M. (1994) Cloning. expression in E.schrrhhia
coh, and characterization of cellulolytic enzymes 01
Azourcus sp.. a root-invading diazotroph. Journol of
Bacterioiogy 175, 70% 7065.
Reis Jr F.B., Olivares F.L.. Oliveira A.L.M . . Reis V.L.M ._
Baldani J.I. and Dbereiner J. (1995) Infection and colo-
nisation of ilcetohacler diaxnrophicus in sugar cane
plantlets. In International Symposium on Sustuinahle
Agriculture for the Tropics: The Rok of Biological
Nitrogen Fisation. p. 225. hgrd dos Reis. Rio de
Janeiro.
Reis V.M., Baldani J.I.. Kirchhof G. and Hartmann A.
(1995) Development of a molecular assay to identify
Acetohacter diazotrophic~us species and detect their
occurrence in sugar cane and other plants grown in the
field. In hternational Symposium on Suslainahie
Agriculture fiw the Tropics: The Roie of Biological
Nilrogen Fixation. p. 130. Angra dos Reis. Rio de
Janeiro.
Reis V.M.. Ohvares F.L. and Dbereiner J. (1994)
Improved methodology for isolatton of Acetohacter dia-
zotrophs and confirmation of its endophytic habitat,
World Journal of .Microhiology~ and Biotechnology 10,
401-405.
Schloter M.. Kirchhof G.. Heinzmann U.. Dbereiner J.
and Hartmann A. (1994) Immunological studies of the
wheat-root-colonization by the A:o.spirii/um hrasilense
strains Sp7 and Sp245 using strain-specific monoclonal
antibodies. In Nitrogen Fiuation with Non-Legumes
(N.A. Hegdzi , M. Fayez and M. Monib, Eds). pp. 291l
297. American University in Cairo Press, Cairo, Egypt.
Sevilla M.Q., Meletzus D.. Teixeira K.R.S., Lee S., Pero
R.. Nutakki A., Baldani J.I. and Kennedy C. (1995)
Analysis of nif and regulatory genes in Acetohacter diu-
zotrophicus. In International Sy~mposium on Sustainahle
Agriculture ,for the Tropicx The Role of Biological
Nilrogen Fixation. pp. 27 28. Angra dos Reis, Rio de
Janeiro.
Silva L.G. da, Reis V.M., Reis Jr F.B. and Boddey R.M.
(1995) Ontogenie variation of diazotrophic bacteria in
sugar cane (Sacchurum spp) tissues. In International
Symposium on Sustainahle Agriculture ,for the Tropics
The Role of Biologieut Nilrogen Fixation. p. 234235.
Angrd dos Reis, Rio de Janeiro.
Stephan M.P., Oliveira M., Teixeira K.R.S., Martinez-
Dretz G. and Dbereiner J. (1991) Physiology and dini-
trogen fixation of Acetohacter diazotrophicus. FEMS
Microhioiogy Letters 77, 67-72.
Tarrand J.J., Krieg N.R. and Dbereiner J. (1978) A taxo-
nomic study of the Spirilium lipoferum group. with
descriptions of a new genus, Azospirillum gen. nov. and
two species, A:ospirillum lipoferum (Beijerinck) comb.
nov. and Azospirillum brasilense sp. nov. Canadian
Journal of Microbiology 8, 9677980.
Teixeira K.R.S., Stephan M.P. and Dbereiner J. (1987)
Physiological studies of Saccharobacter nitrocaptans a
new acid tolerant Nz fixing bacterium. In International
.Sysmposium On Nitrogen Fixation with Non-Legumes, p.
149. Rio de Janeiro.
Teixeira K.R.S.. Galler R., Kennedy C. and Baldani J.I.
(1994) Plasmid contents and nif gene detection in
Acetobucter diazotrophiucs strains. In Nitrogen Fixation
nYth Non-Legumes (N.A. Hegazi, M. Fayez and M.
Monib. Eds), pp. 2744281. American University in
Cairo Press, Cairo. Egypt.
Tran Van V.. Gillis M.. Hebber K.P., Fernandez M.,
Segers P.. Martel M.H.. Berge 0.. Meyer J.M. and
Heulin T. (1994) Isolation from the rite rhizosuhere of a
new species of nitrogen-fixing Proteobacteria, belonging
to the genus Burkholderia. In Nitrogen Fixation with
Non-Legumes (N.A. Hegazi, M. Fayez and M. Monib.
Eds). pp. 2999309. American University in Cairo Press.
Cairo, Egypt.
Urquiaga S.. Cruz K.H.S. and Boddey R.M. (1992)
Contribution of nitrogen fixation to sugarcane:nitrogen-
15 and nitrogen balance estimates. Soil Science Society
of America Journal 56, 1055114.
Van Bastelaere E.. Keijers V., Vermeiren. H. and
Vanderleyden J. (1995) Isolation and characterization of
plant-inducible genes in Azospirillum hrasilense. In IOth
International Congress on Nitrogen Fi.uation, p. 100. St.
Petersburg, Russia.
vjande Broek A., Michiels J., Van GOOI A. and
Vanderleyden J. (1993) Spatialltemporal colonization
patterns of Azospirillum brasilense on the wheat root
surface and expression of the bacterial nlfH gene during
association. Molecular Plant-Microbe Interactions 6,
592m 600.
vande Broek A. and Vanderleyden J. (1995) The genetics
of the Arospirillum-plant root association. Critical
Reviews in Plant Sciences 14, 445-466.
Waelkens F.. Maris M., Verreth C., Vanderleyden J. and
Van GOOI A. (1987) Arospiriihon DNA homology with
.4grohacteriuni tumefuciens chromosomal virulente
genes. FEMS Microhiology Letters 43, 241-246.
Weber O.B.. Teixeira K.R.S., Kirchhof G., Baldani J.I.
and Dbereiner J. (1995) Occurrence of endophytic dia-
zotrophic bacteria in pineapple (Ananas sp.) and banana
(Musrr sp.). In International Symposium on Sustainable
Agriculture ,for the Tropics. The Role of Bioiogical
Nitrogen Fixution, pp. 23X-239. Angra dos Reis, Rio de
Janeiro.
Yabuuchi E., Kosako Y., Oyaizu H., Yano I., Hotta H.,
Hashimoto Y., Ezaki T. and Arakawa M. (1992)
Proposal of Burkhoideria gen. nov. and transfer of seven
species of the genus, with the genus Pseudomonas hom-
ology group 11 to the new genus. with the type species
Burkholderia cepacia (Palleroni and Holmes, 1981)
comb. nov. Microbiology Immunoiogy 36, 1251-1275.
Zimmer W.. Roeben K. and Bothe H. (1988) An alterna-
tive explanation for plant growth promotion by bacteria
of the genus A:ospirillum. Planta 176, 3333342.