Journal of Experimental Botany, Vol. 52, No. 357, pp. 747±760, April 2001
Further observations on the interaction between
sugar cane and Gluconacetobacter diazotrophicus
under laboratory and greenhouse conditions1
Euan K. James2,5, Fabio L. Olivares3, Andre L.M. de Oliveira4, Fabio B. dos Reis Jr4,
Lucia G. da Silva4 and VeroÃnica M. Reis4
2
Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, UK
Setor de Citologia Vegetal, Lab. Biologia Celular e Tecidual, Centro de BiocieÃncias e Biotecnologia,
Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ 28015-620, Brazil
4
EMBRAPA-Agrobiologia, km 47, SeropeÂdica, Rio de Janeiro, 23851-970, Brazil
3
Received 22 June 2000; Accepted 12 October 2000
Abstract
Sugar cane (Saccharum spp.) variety SP 70-1143 was
inoculated with Gluconacetobacter diazotrophicus
strain PAL5 (ATCC 49037) in two experiments. In
experiment 1 the bacteria were inoculated into a
modified, low sucrose MS medium within which
micropropagated plantlets were rooted. After 10 d
there was extensive anatomical evidence of endophytic colonization by G. diazotrophicus, particularly
in lower stems, where high numbers of bacteria were
visible within some of the xylem vessels. The identity
of the bacteria was confirmed by immunogold labelling with an antibody raised against G. diazotrophicus.
On the lower stems there were breaks caused by
the separation of the plantlets into individuals, and
at these `wounds' bacteria were seen colonizing
the xylem and intercellular spaces. Bacteria were
also occasionally seen entering leaves via damaged
stomata, and subsequently colonizing sub-stomatal
cavities and intercellular spaces. A localized host
defence response in the form of fibrillar material
surrounding the bacteria was associated with both
the stem and leaf invasion. In experiment 2, stems of
5-week-old greenhouse-grown plants were inoculated
by injection with a suspension of G. diazotrophicus
containing 108 bacteria ml 1. No hypersensitive
response (HR) was observed, and no symptoms
were visible on the leaves and stems for the duration
of the experiment (7 d). Close to the point of
1
inoculation, G. diazotrophicus cells were observed
within the protoxylem and the xylem parenchyma,
where they were surrounded by fibrillar material
that stained light-green with toluidine blue. In leaf
samples taken up to 4 cm from the inoculation
points, G. diazotrophicus cells were mainly found
within the metaxylem, where they were surrounded
by a light green-staining material. The bacteria were
growing in relatively low numbers adjacent to the
xylem cell walls, and they were separated from
the host-derived material by electron-transparent
`haloes' that contained material that reacted with
the G. diazotrophicus antibody.
Key words: Sugar cane, Gluconacetobacter diazotrophicus,
endophytic bacteria, nitrogen fixation, immunogold labelling.
Introduction
Applications of mineral N fertilizer to Brazilian sugar
cane (interspeci®c hybrids of Saccharum sp.) ®elds are
typically much lower than those used in other countries,
and they do not appear to be suf®cient to compensate
for N losses from annual harvesting (Ruschel, 1981;
Boddey et al., 1995). It has long been hypothesized that
the apparent de®cit in N inputs is made up by biological nitrogen ®xation (BNF) (Patriquin et al., 1980;
Ruschel, 1981). Studies using long-term N-balances and
15
N isotope dilution (Lima et al., 1987; Urquiaga et al.,
This paper is dedicated to the memory of Joanna DoÈbereiner (1924 to 2000), the discoverer of Gluconacetobacter diazotrophicus.
Present address and to whom correspondence should be sent: Centre for High Resolution Imaging and Processing, MSI/WTB Complex, School of
Life Sciences, University of Dundee, Dundee DDI 5EH, UK. Fax: q44 1382 345893. E-mail: [email protected]
5
ß Society for Experimental Biology 2001
748
James et al.
1992) have shown that some Brazilian sugar cane varieties
(e.g. SP 70-1143, SP 79-2312, CB 45-3, Krakatau) may
actually ®x up to 70% of their N requirements. Although
the speci®c micro-organisms responsible for the ®xation
have yet to be determined (see reviews by James and
Olivares, 1998; James, 2000), the roots, stems, leaves, and
trash of Brazilian varieties contain substantial numbers
(up to 107 g 1 fresh weight) of diazotrophic bacteria,
such as Acetobacter diazotrophicus (recently renamed
Gluconacetobacter diazotrophicus; Yamada et al., 1997,
1998; Franke et al., 1999) and Herbaspirillum spp.
(Boddey et al., 1995, 2000; Reis et al., 1994; Olivares
et al., 1996; Baldani et al., 1997; DoÈbereiner et al., 1995).
It has been suggested that as these bacteria are endophytes, and hence live within the plant tissues, they can ®x
N2 more ef®ciently than diazotrophs that remain in the
rhizosphere or on the rhizoplane (Patriquin et al., 1983;
DoÈbereiner et al., 1995). This may be due to the plant
directly providing photosynthate for their nutrition, but
also because the interior of a plant could provide a low
O2 environment which is necessary for the expression of
the O2-sensitive enzyme, nitrogenase. In addition, endophytic bacteria will not have to compete with other soil
microbes for scarce resources (Sprent and James, 1995;
Hallmann et al., 1997; Reinhold-Hurek and Hurek,
1998a, b; James and Olivares, 1998). In return for providing this niche, it may be that the bacteria provide ®xed
N anduor plant growth-promoting compounds to the
host, in this case sugar cane (Sevilla and Kennedy, 2000).
Although endophytic diazotrophs, such as G. diazotrophicus, have been isolated from all parts of the sugar
cane plant, their exact location has still to be established
(James and Olivares, 1998). In an earlier study, sterilegrown plantlets of variety NA 56-79 were inoculated with
G. diazotrophicus, and the plants were subsequently
examined under the optical and electron microscopes
(James et al., 1994). After 4 d the bacteria had colonized
the surface of the roots, with the lateral root junctions
and the root tips being the most preferred sites. After
a further 11 d, G. diazotrophicus was observed within the
plants, speci®cally within the xylem vessels at the base of
the stems. The bacteria in the xylem were con®rmed to be
G. diazotrophicus using immunogold labelling. Since that
study was published, further micrographs showing xylem
vessels colonized by G. diazotrophicus have been presented (DoÈbereiner et al., 1995; James and Olivares, 1998;
Reis et al., 1999; Fuentes-Ramirez et al., 1999). However,
results have been presented that contrast with these
studies (Dong et al., 1994). These authors suggested that
the intercellular spaces in the sucrose storage parenchyma
of the stems of mature sugar cane stalks were the most
likely location of a symbiosis with G. diazotrophicus as
(1) the intercellular spaces contain an acidic solution rich
in sucrose (up to 13%), and G. diazotrophicus grows and
®xes N2 well when cultured in a low pH media (pH 5.5)
containing high sucrose (10±12%; Cavalcante and
DoÈbereiner, 1988; Reis et al., 1994), (2) G. diazotrophicus
(1.1 3 104 cells ml 1) could be isolated from `apoplastic
¯uid' obtained by centrifuging stem pieces; this ¯uid is
rich in sucrose (11%) and consists of ¯uid from the
apoplast (i.e. intercellular spaces, cell walls and xylem
lumina), with up to 20% of its volume comprising xylem
sap, and (3) electron microscopy failed to reveal any
bacteria within the vascular tissue, but a few unidenti®ed
bacteria were observed within intercellular spaces.
In a later paper (Dong et al., 1997), it was further
suggested that the xylem of sugar cane stems was an
unlikely location for `symbiotic' G. diazotrophicus as (a) it
is very low in sucrose (0±9%; Hawker, 1965; Bull et al.,
1972; Welbaum et al., 1992), (b) at the stem nodes, the
xylem (particularly in varieties resistant to the xylemdwelling pathogen Clavibacter xyli subsp. xyli) contains
convolutions and discontinuities (Teakle et al., 1977;
Gillaspie and Teakle, 1989; Harrison and Davis, 1988)
that would not allow dye and paint particles of a size
similar to G. diazotrophicus to pass freely through to
the next internode, and (c) when the ends of cut stalks
were placed within liquid cultures of G. diazotrophicus the
bacteria entered the xylem and elicited a `most violent'
reaction from the plant involving the production of a gum
that stained bright red with toluidine blue; this appeared
to arrest the movement of the bacteria and eventually kill
them. Furthermore, Dong et al. (Dong et al., 1997) were
of the opinion that the micrographs presented by James
et al. (James et al., 1994) contained artefacts and that the
`xylem vessels' that the latter authors purported to show
containing G. diazotrophicus were actually dead ligni®ed
xylem parenchyma cells in which the bacteria had been
moved during preparation for microscopy.
In order to resolve these differences, two sets of experiments are performed. (1) Given the above criticisms of
the study of James et al. (1994), the interaction between
G. diazotrophicus and micropropagated sugar cane plantlets was examined in more detail using the methodology
of Reis et al. (Reis et al., 1999). One of the aims of this
infection study was to con®rm the data presented
previously (James et al., 1994; DoÈbereiner et al., 1995;
James and Olivares, 1998), and show that xylem vessels
are indeed a possible site of colonization by G. diazotrophicus. (2) To examine the infection of stems and leaves
of greenhouse-grown sugar cane plants that had been
directly inoculated with high numbers of G. diazotrophicus. This was done in order to determine the preferred
location of the bacteria in older plants, and also to see if
there was any host defence reaction anduor disease symptoms when they are forcibly introduced in large numbers.
This is a method commonly used by sugar cane pathologists to determine resistance or susceptibility to bacteria, such as Xanthomonas albilineans and X. campestris
pv. vasculorum, the xylem-dwelling agents of leaf scald
Sugar cane and endophytic bacteria
disease (LSD) and `gumming disease', respectively
(Ricaud and Ryan, 1989; Rott et al., 1997; Ricaud and
Autrey, 1989). This inoculation technique has been used
successfully to examine the infection of sugar cane and
sorghum leaves by Herbaspirillum spp (Olivares et al.,
1997; James et al., 1997).
Materials and methods
Organisms, growth conditions, bacterial inoculation,
and counts
Experiment 1: Micropropagated sugar cane plantlets (cv. SP
70-1143) were grown for 60 d and then inoculated with
G. diazotrophicus strain PAL-5 according to the procedure
previously used (Reis et al., 1999). Brie¯y, the plantlets were
separated into individuals and transferred to 50 ml tubes
containing a modi®ed MS medium, without vitamins or
hormones, and with the concentrations of salts and sucrose
reduced 10-fold, for example, from 20 mg sucrose l 1 to
2 mg l 1. The plantlets were then inoculated with 0.1 ml
suspensions of G. diazotrophicus containing 108 cells ml 1
(controls were inoculated with sterile distilled water or
autoclaved bacteria). Plants were harvested at 10 d after
inoculation, and pieces of roots, leaves and stems from ®ve
inoculated and ®ve control plants were taken for microscopical analysis. Whole plantlets were surface-sterilized for
5 min in 1% chloramine T and Most Probable Number
(MPN) counts of bacteria were made according to the
methods of Reis et al. (Reis et al., 1994, 1999). Dry weights
were determined according to Reis et al. (Reis et al., 1999).
Acetylene reduction assays were performed according to the
methodology of James et al. (James et al., 1994) on ®ve plants
from each treatment after they were surface-sterilized.
Experiment 2: Greenhouse-grown sugar cane plants (cv. SP
70-1143) were inoculated at 5 weeks after germination with
1 ml of a liquid culture of G. diazotrophicus strain PAL-5
growing in LGI medium, resuspended in sterilized water and
adjusted to 108 cells ml 1. The inoculation method, and the
point of inoculation (the leaf `pocket' at the base of the stem),
was the same as that used previously (Pimentel et al., 1991;
Olivares et al., 1997) with Herbaspirillum spp. Five replicate
plants were inoculated, and ®ve control plants were inoculated
with sterile water, with the exuded excess ¯uid in both cases
being immediately mopped up with sterile cotton wool. At 7 d
after inoculation, leaves with visible inoculation points were
taken for microscopy and bacterial counts. The leaves were
prepared for the counts according to the method of Olivares
et al. (Olivares et al., 1997), and the MPN of G. diazotrophicus
cells were determined as in Experiment 1.
Microscopy and immunogold labelling
Samples from Experiment 1 were ®xed for 24 h in 5%
glutaraldehyde in 50 mM phosphate buffer (pH 6.8). They were
then dehydrated in an ethanol series before being embedded in
LR White resin (Agar Aids, UK) (James et al., 1994). Pieces of
leaves from Experiment 2 were sampled around the inoculation
point and up to 4 cm above the inoculation point, and these
were ®xed and embedded as above. Optical and transmission
electron microscopy (TEM) were performed on the samples
(James et al., 1994; Olivares et al., 1997). The 1±2 mm sections
used in the optical microscopy were stained in 1% toluidine blue
O in an aqueous solution of 1% sodium tetraborate (borax),
749
except for those sections that were used for immunogold
labelling which were left unstained. Immunogold labelling
for optical microscopy (using silver-enhancement) and TEM
used the methods of James et al. (James et al., 1994). Two
antibodies were used in the immunogold analysis: a polyclonal
antibody raised in a rabbit against G. diazotrophicus strain PRJ2
(Silva, 1999; Boddey et al., 2000), and a polyclonal antibody
raised against the Fe(NifH)-protein of nitrogenase from
Rhodospirillum rubrum (a gift from PW Ludden, Madison,
Wisconsin, USA). In cross-reaction tests using enzyme-linked
immunosorbent assays (ELISA) the antibody against strain
PRJ2 gave a very strong reaction with G. diazotrophicus strain
PAL-5, but gave little or no reaction with other bacteria
commonly associated with sugar cane, including Herbaspirillum
and Azospirillum spp., as well as with other members of the
Acetobacteriaceae (Silva, 1999). Pre-immune sera from the same
rabbits before they were immunized were used as negative controls in the immunogold analyses, and additional controls used
substitution of normal rabbit serum (Sigma) for the primary
antibody, and omission of the primary antibody (Olivares et al.,
1997). Five nm gold particles conjugated to goat anti-rabbit
antibodies were used for silver-enhancement, and 15 nm particles were used for TEM (both from Amersham, UK). The
IntenSE M silver enhancement kit from Amersham (UK) was
used according to the instructions of the manufacturer.
Results
Experiment 1: infection of micropropagated plants
At 10 d after inoculation none of the plantlets from either
treatment exhibited any macroscopically visible disease
symptoms (data not shown). The dry weights of the
G. diazotrophicus-inoculated plants (68.7"21.2 mg), were
greater than the controls (50.0"16.1 mg), but owing to
high variation the differences were not signi®cant (values
given are "se, n ˆ 4). There was no acetylene reduction
activity by surface-sterilized plants from any treatment.
There was a slight red colour on most of the
G. diazotrophicus-inoculated plants at breaksuwounds
in the base of the stem caused by the separation of
the plantlets into individuals; the red coloration was not
seen on the control plants (data not shown). After
surface-sterilization there were 1.8 3 107"0.26 3 107 cells
of G. diazotrophicus g 1 fresh weight in the inoculated
plantlets, but none in the controls. The surfaces of the
roots were colonized by the bacteria in a manner similar
to that reported previously (James et al., 1994; Reis et al.,
1999), with the bacterial cells surrounded by a layer of
mucus (not shown). As in these studies, the bacteria
accumulated at lateral root junctions and colonized
damaged epidermal cells (not shown), but in the present
study there were no indications that they had actually
penetrated beyond the root epidermis. However, in contrast to the roots, using immunogold silver-enhancement
to highlight the presence of G. diazotrophicus, there
was clear evidence for internal colonization of the stems,
particularly within the vascular tissue (Fig. 1a, c). In transverse sections taken from approximately 5 mm above
750
James et al.
Fig. 1. Micrographs from Experiment 1 showing colonization of micropropagated sugar cane plantlets by Gluconacetobacter diazotrophicus at 10 d
after inoculation. (a) Light micrograph of a transverse section (TS) through the cortex of the lower stem. This section was incubated with an antibody
raised against G. diazotrophicus followed by 5 nm gold particles conjugated to goat anti-rabbit antibodies. The gold labelling was visualized for light
microscopy using silver enhancement, and the background was lightly stained with a dilute solution of toluidine blue (0.01%). The strong silverenhanced signal con®rms that the bacteria in the xylem vessel are G. diazotrophicus (arrow). Bar ˆ 10 mm. (b) High magni®cation light micrograph of a
TS stained with 1% toluidine blue. This is taken from a serial section to that in (a) and was not immunogold labelled. Note that the vessel is completely
full of bacteria (*). Bar ˆ 5 mm. (c) Light micrograph of a longitudinal section (LS) through the cortex of the lower stem. This section was treated as
(a) and again shows immunogold labelled G. diazotrophicus densely colonizing xylem vessels (arrows). Bar ˆ 10 mm. (d) Light micrograph of a TS
through the cortex of the stem base of an uninoculated control plantlet showing no bacterial colonization. Note that the xylem vessels are occluded by
neither bacteria nor host defence material (*). Bar ˆ 20 mm. (e) Transmission electron micrograph (TEM) of bacteria that are strongly immunogold
labelled with an antibody raised against G. diazotrophicus. Note that much of the antibody reaction is with extracellular material (arrows).
Bar ˆ 500 nm. (f ) Serial section to (e). This was incubated in `blocking' buffer followed by 15 nm gold particles conjugated to goat anti-rabbit
antibodies. There are no gold particles on the section. Note that the bacteria are embedded in a matrix (*). Bar ˆ 500 nm.
Sugar cane and endophytic bacteria
the brokenuwounded regions on the lower stems of ®ve
inoculated plantlets, the proportion of infected vascular
bundles ranged from 4±36%, with a mean of 16.1%
("6.3 s.e.). This infection generally took the form of one
or two of the metaxylem vessels being almost completely
®lled with bacteria (Fig. 1a, c). No bacteria were apparent within control plants (Fig. 1d). The bacteria in the
infected xylem vessels (Fig. 1a, c), as well as those within
some of the intercellular spaces (not shown), and leaf
sheath epidermal cells (not shown) reacted strongly with
the G. diazotrophicus antibody, with the antibody recognizing the surface of the bacteria, and also `mucus' that
was produced by them (Fig. 1e, f ).
At the sites of breaksuwounds in the lower stem there
was considerable colonization and invasion by bacteria
of the wound surface, particularly within broken cells
(Fig. 2a). Further into the wound sites, intercellular and
intracellular bacteria (Fig. 2b) could be seen, and these
were immunogold labelled with the anti-G. diazotrophicus
antibody (Fig. 2c; not shown). The intracellular bacteria
were observed only in cells that appeared to be dead or
damaged, without intact cytoplasm or organelles (Fig. 2c;
not shown). Some xylem vessels in this region (Fig. 2a,
b, d) were densely occluded with a material that stained
pink with toluidine blue (not shown), and these vessels
did not contain bacteria. However, bacteria were observed within adjacent vessels that were much less densely
occluded with pink-staining material, and the bacteria
within them were usually surrounded by a loosely-®brous
matrix (Fig. 2d). Deeper into the stem cortex, and
away from the wound sites, bacteria were seen within
apparently unoccluded xylem vessels (Fig. 2e). Some of
the non-senescent xylem-dwelling bacteria were recognized, albeit sparsely, by an antibody raised against
the Fe-protein of nitrogenase (Fig. 2f ). No intercellular
bacteria were observed within the stem cortex away from
the wound sites (Fig. 2e), and none of the intercellular or
xylem-dwelling bacteria within the wound sites labelled
with the anti-nitrogenase antibody (not shown).
Although they were not usually seen within leaves, in
some specimens bacteria were observed entering via
stomata, and the latter appeared to be damaged by this
invasion process (not shown). These bacteria subsequently colonized the sub-stomatal cavities and adjoining intercellular spaces, within which a host-derived
matrix surrounded the bacteria (Fig. 3b). Both the
bacteria and the matrix reacted with the G. diazotrophicus
antibody in immunogold assays (Fig. 3c), but none
of them reacted with the antibody against the Fe-protein
of nitrogenase (not shown).
Experiment 2: infection of mature leaves
The leaves were examined at 12 h intervals over the
®rst 2 d after inoculation and there were no signs of a HR,
751
i.e. water-soaked necrotic regions around the inoculation
point. At 7 d after inoculation, G. diazotrophicusinoculated leaves contained 1.1 3 105 G. diazotrophicus
cells g 1 fresh weight, whereas the controls had no
detectable bacteria. No macro symptoms of disease were
visible on leaves from either treatment (not shown), and
no disease symptoms appeared in the leaves over the
subsequent 4 weeks. Although the lack of symptoms
made the bacteria dif®cult to localize under the microscope, close to the points of inoculation of both control
and G. diazotrophicus-treated leaves, there were signs of
degradation at the point of inoculation itself (not shown)
as well as in adjacent vascular bundles (Fig. 4a). In the
latter, there was distortion of the metaxylem elements
that were closest to the inoculation point and also gumming of the vessels by a material (Fig. 4a) that stained
light pink with toluidine blue (not shown). In the
G. diazotrophicus-inoculated (but not the control leaves;
not shown), there was an accumulation of (greenstaining) material in the intercellular spaces adjacent to
the occluded vascular bundles (Fig. 4a) and, under the
TEM, a few bacteria were observed within this material
(not shown). However, most bacteria, even some in the
form of micro-colonies, were seen within the metaxylem
(Fig. 4a), again surrounded by pink-staining material
(not shown).
Further from the point of inoculation (up to 4 cm),
bacteria were observed in only 3 out of 10 leaves that were
sectioned. In these samples, bacteria were visible only
within vascular tissue (Fig. 4b, c, d). The bacteria in these
sections labelled strongly with the anti-G. diazotrophicus
antibody (Figs 4b, 5a, c, d), whereas the serial sections
used as immunogold controls did not label (Fig. 5b).
Although some of the bacteria were in the protoxylem
(Figs 4c, 5a), most of them were in the metaxylem vessels,
often arranged in a monolayer immediately adjacent to
the vessel walls (Figs 4b, c, d, 5b). A blue-green gum had
accumulated in the colonized metaxylem vessels (Fig. 4c,
d; data not shown) and, under the TEM, this was revealed
to be a ®brous matrix, probably of plant origin, that
surrounded the bacteria (Fig. 5b±d). This material was
less distinct in the colonized protoxylem (Fig. 5a).
Interestingly, the bacteria were separated from the ®brous
matrix by a distinct, electron-transparent `halo' around
each bacterium. When the sections were immunogold
labelled with the G. diazotrophicus antibody, gold particles
that were not actually attached to the bacteria themselves,
were prominent within these haloes (Fig. 5a, d). Under
the TEM, the appearance of the bacteria ranged from
senescent to obviously healthy (Fig. 5a±c), and some were
dividing (Fig. 5d). No bacteria were observed in the
control leaves (not shown).
None of the bacteria in sections from Experiment 2
gave a signi®cant immunogold reaction with the antibody
raised against the Fe-protein of nitrogenase (not shown).
752
James et al.
Fig. 2. Micrographs from Experiment 1 showing infection of micropropagated sugar cane plantlets by Gluconacetobacter diazotrophicus at `wound'
sites 10 d after inoculation. (a) TS of the stem base showing part of a woundubreak caused by separation of the plantlets at the start of the inoculation
experiment. This section was stained with 1% toluidine blue. Large numbers of bacteria have accumulated at the surface of the wound, and many have
colonized damaged cells (small arrows). The tissue immediately internal to the wound shows signs of bacterial infection and a consequent host defence
response, with the latter taking the form of gumming of xylem vessels with dark-staining material (large arrows). Further in, and adjacent to the wound
site, the tissue appears to be undamaged and the xylem vessels are unoccluded (*). Bar ˆ 50 mm. (b) Detailed view (stained with 1% toluidine blue) of
the internal tissue of a wound, showing bacterial infection of intercellular spaces (small arrows), as well the gumming of a xylem vessel with a darkstaining material (large arrow). Deadudamaged cells (*) also appear to contain bacteria. Bar ˆ 10 mm. (c) TEM of an intercellular space within a wound
site. The space contains a number of bacteria that are immunogold labelled with an antibody raised against G. diazotrophicus (arrows). Bar ˆ 1 mm.
(d) TEM showing a xylem vessel from deep within a wound site. The bacteria within the vessel (arrows) are surrounded by a ®brillar matrix (*). Note
that a vessel adjacent to the infected vessel is ®lled with electron-dense material (E). Bar ˆ 1 mm. (e) TS of a vascular bundle within the stem cortex
adjacent to (but not within) a wound site. The xylem vessels, which appear to be unoccluded (cf. b, d), contain bacteria (small arrows), with one
showing quite dense colonization (large arrow). P, phloem. Bar ˆ 5 mm. (f ) TEM of bacteria within the xylem vessels from (e). This section was
immunogold labelled with an antibody raised against the Fe (NifH ) ± protein of nitrogenase. One of the bacteria is clearly senescent in appearance,
with disintegrating cytoplasm (S), and is not immunogold labelled. The other bacterium appears to be healthier and it is labelled with gold particles
(arrows), thus indicating that it is expressing nitrogenase Fe-protein. W, xylem cell wall. Bar ˆ 100 nm.
Sugar cane and endophytic bacteria
753
Fig. 3. (a) TEM of bacteria within an intercellular space (*) adjacent to an infected sub-stomatal cavity. Bar ˆ 1 mm. (b) Higher magni®cation TEM
of bacteria within the leaf intercellular space shown in (a). This section was incubated with an antibody raised against G. diazotrophicus followed
by 15 nm gold particles conjugated to goat anti-rabbit antibodies. The bacteria (arrows) and the material surrounding them are gold labelled (*). Note
that some bacteria are dividing (large arrow). Bar ˆ 200 nm.
Controls for the immunogold labelling also gave no
signi®cant signal (Figs 1f, 5b).
Discussion
Infection of micropropagated plantlets
Micropropagated plantlets are being used increasingly by
Brazilian farmers as a means of obtaining disease-free
plants (Reis et al., 1999). However, this also means that
unlike the traditional method of vegetative propagation
from seed pieces (settes) there may not be transmission
from generation to generation of possibly bene®cial
bacteria, such as G. diazotrophicus. Therefore, the plantlets need to be inoculated, and a rooting medium has
been devised (based upon a modi®ed MS medium) (Reis
et al., 1999) by which this can be done in order to
maximize the number of G. diazotrophicus within the
plantlets over a short period (7 d) before transplanting
into the ®eldugreenhouse. This medium, which was also
used in the present study, is suf®ciently low in sucrose
(2 g l 1) that the growth of the G. diazotrophicus within it
is not so great that it becomes detrimental to the plants.
On the other hand, there is still enough sucrose to allow
the bacteria to multiply slowly and, as they consume it,
the bacteria appear to be induced to invade most of
the plantlets within 7±10 d after inoculation (Reis et al.,
1999; Sevilla et al., 1998; Olivares and James, 2000). This
invasion process appears not to be detrimental to the
health of the plantlets and may even confer some growth
bene®ts upon them (Sevilla et al., 1998; Reis et al., 1999;
this study). At this early stage of the interaction, the
number of G. diazotrophicus within the surface-sterilized
plantlets appears to be quite high (107±108 g 1 fresh
weight; James et al., 1994; Reis et al., 1999; Olivares and
James, 2000; this study), although it should be noted that
this number almost certainly includes many surfacedwelling bacteria that have survived the disinfestation
process via tight adherence to plant surfaces within
`mucus' anduor a preference for colonizing cracks and
crevices (see James et al., 1994; Reis et al., 1999 for more
details). Moreover, previous studies have shown that
these high `internal' numbers are rarely sustained beyond
the early stages of the interaction. When the inoculated
plantlets are transferred to pots under greenhouse
conditions with high irradiances the concentration of
bacteria within them decreases considerably, being of
the order of only 104±105 g 1 fresh weight by 30 d
after inoculation (Paula et al., 1991; Reis et al., 1999;
Sevilla et al., 1998). Therefore, it would appear that
754
James et al.
Fig. 4. Light micrographs from Experiment 2 stained with toluidine blue showing colonization of 5-week-old sugar cane leaves by G. diazotrophicus at
7 d after inoculation: X, metaxylem; Pr, protoxylem; P, phloem; *, bundle sheath cells. (a) This TS was taken from the region immediately adjacent to
the inoculation point and shows that the metaxylem vessels are occluded with material (staining pink- anduor blue-green). The right hand vessel is the
one closest to the point of inoculation; note that, compared to the left hand vessel, it is distorted in shape. The intercellular spaces in this region are also
®lled with material (staining pink or blue) (small arrows). The left-hand vessel contains a small bacterial colony (large arrow). Bar ˆ 10 mm. (b) TS of
a vascular bundle containing bacteria that have been immunogold labelled (followed by silver-enhancement) with an antibody raised against
G. diazotrophicus. This section was taken from a region approximately 4 cm up the leaf from the inoculation point. The bacteria are present in the
metaxylem vessels, and can be seen as black particles adjacent to the vessel walls (arrows). Note the absence of bacteria within the protoxylem.
Bar ˆ 10 mm. (c) TS of a vascular bundle. This section was taken from a region approximately 4 cm up the leaf from the inoculation point. Bacteria can
be seen within both the meta- and the protoxylem (arrows). L, ligni®ed xylem parenchyma. Bar ˆ 10 mm. (d) This LS was taken from a region
approximately 4 cm up the leaf from the inoculation point. The lumen of the vessel is ®lled with material (stained blue-green) and bacteria can be seen
within it alongside the walls of the vessel (arrows). Bar ˆ 10 mm.
Sugar cane and endophytic bacteria
755
Fig. 5. TEMs from Experiment 2 showing colonization of 5-week-old sugar cane leaves by G. diazotrophicus at 7 d after inoculation. They are all
taken from a region approximately 4 cm up from the inoculation point. (a) Bacteria (arrows) in the protoxylem. TS. Note that there are distinct
electron transparent `haloes' separating the bacteria from the host-derived material surrounding them (*). The bacteria have been immunogold labelled
with an antibody raised against G. diazotrophicus. The labelling can be seen clearly both on the surface of the bacteria and within the haloes
surrounding them: W, cell wall; L, ligni®ed xylem parenchyma cell. Bar ˆ 500 nm. (b) Bacteria (arrows) in the metaxylem. TS. This section was treated
as for (a), except that the primary antibody was omitted. There is no gold-labelling of the bacteria. Note the host-derived material in the vessel (*) and
the close association of the bacteria with the vessel wall (W). One of the bacteria has disrupted cytoplasm and probably is senescent (large arrow).
Bar ˆ 1 mm. (c) Bacteria (arrows) in the metaxylem. LS. As with (a, b), the bacteria are surrounded by host-derived material (*) and there are electron
transparent haloes around them. W, vessel wall. Bar ˆ 1 mm. (d) Higher magni®cation of some of the bacteria in (c). A bacterium is dividing (large
arrow). The bacteria have been immunogold labelled with an antibody raised against G. diazotrophicus, and the labelling is visible on the surface of the
bacteria (small arrows). Bar ˆ 200 nm.
756
James et al.
G. diazotrophicus behaves similarly to most other nonpathogenic, endophytic bacteria (Hallmann et al., 1997),
i.e. its numbers are diluted as the plants grow and the
bacteria are dispersed.
The present study has shed further light upon the
invasion processes illustrated by previous studies (James
et al., 1994; Reis et al., 1999). These studies suggested that
G. diazotrophicus ®rst colonized the root surfaces and
then infected the roots via lateral root junctions anduor
root tips, and subsequently entered the root vascular
system from whence they were translocated to the lower
stem in the xylem. Although the present study has not
con®rmed the infection of roots by G. diazotrophicus, high
numbers of the bacteria were observed within the xylem
vessels in the stems of all the inoculated plantlets, thus
supporting the possibility of xylem translocation from the
roots. However, in addition to the possibility of infection
at lateral root junctions, the present study suggests that
there were at least two other potential sites of infection:
wounds caused when the plantlets were separated into
individuals at the start of the inoculation experiments
(Fig. 2), and stomata (Fig. 3). In both these locations the
bacteria elicited a localized host defence response in the
form of a polymeric matrix material that surrounded
them. As G. diazotrophicus is generally regarded as a nonpathogen (Baldani et al., 1997; Sevilla and Kennedy,
2000) this host defence response was particularly interesting, and it could be that the very low irradiances
used in micro-propagation (Reis et al., 1999), as well as
their very young physiological age, possibly so weaken
the plantlets that it makes them more susceptible to
aggressive infection, even from non-pathogens. Indeed,
it is possible that G. diazotrophicus from the wound sites
were able subsequently to infect healthy xylem vessels
deeper within the cortices of the stem bases (Fig. 2e, f ),
and these may actually be the source of the bacteria
illustrated in Fig. 1a±c, rather than (or in addition to)
bacteria translocated from the roots (James et al., 1994;
Reis et al., 1999; Olivares and James, 2000).
Finally, as with H. rubrisubalbicans in sorghum and
sugar cane leaves (James et al., 1997; Olivares et al.,
1997), some non-senescent G. diazotrophicus cells were
shown to express the Fe-protein of nitrogenase within the
xylem (Fig. 2f ). The fact that there was no acetylene
reduction activity with these plants however, suggests that
either the nitrogenase enzyme was not active or else
activity was too low to be detected by this assay. This
contrasts with the study of James et al. (James et al.,
1994) which showed some nitrogenase (acetylene reduction) activity with inoculated plantlets, although it was
impossible to determine if this activity was due to the
endophytic or the epiphytic bacteria (or a combination of
both). Therefore, although the present study has shown
some very limited expression of nitrogenase protein by
xylem-dwelling G. diazotrophicus, it is clear that further
studies are needed to determine if this bacterium can
actually ®x N2 within the plants (Fuentes-Ramirez et al.,
1999; James, 2000).
Infection of inoculated leaves
Experiment 2 has shown that direct inoculation of sugar
cane stems with G. diazotrophicus will result in the subsequent colonization of the leaves by the bacteria, without
any macroscopically visible symptoms, and without
eliciting a HR. This is similar to the response of sugar
cane cv. SP 70-1143 to inoculation with the diazotrophic
pathogen, Herbaspirillum rubrisubalbicans (Olivares et al.,
1997). As a HR is normally elicited after plants are infected with incompatible phytopathogens (Sequeira et al.,
1977; McKhann and Hirsch, 1994), this demonstrates
that G. diazotrophicus and H. rubrisubalbicans have compatibility with sugar cane leaves. This is not surprizing, as
both G. diazotrophicus (Li and MacRae, 1992; Reis et al.,
1999; Boddey et al., 2000; Muthukumarasamy et al.,
1999) and H. rubrisubalbicans (Olivares et al., 1996;
Boddey et al., 2000; Muthukumarasamy et al., 1999) have
been routinely isolated from sugar cane leaves in ®eld and
greenhouse studies. However, this contrasts with the
behaviour of another important sugar cane endophytic
diazotroph, Herbaspirillum seropedicae. Although this
non-pathogenic bacterium can be isolated from roots and
stems, it cannot be isolated from sugar cane leaves
(Olivares et al., 1996; Boddey et al., 2000), and it will elicit
a HR after being inoculated into them (Olivares et al.,
1997).
As with H. rubrisubalbicans (Olivares et al., 1997), the
present study suggests that the vascular tissue is the most
likely location for G. diazotrophicus within sugar cane
leaves. On the other hand, unlike leaves infected with
H. rubrisubalbicans, those infected with G. diazotrophicus
contained relatively few bacteria, and these were largely
con®ned to the walls of the metaxylem vessels. Moreover,
and again in contrast to H. rubrisubalbicans (Olivares
et al., 1997; James et al., 1997; James and Olivares, 1998),
the G. diazotrophicus cells generally did not form microcolonies within the leaf vessels (except in the damaged
vessels close to the points of inoculation; Fig. 4a), and
nor did they express the nitrogenase Fe-protein (although
see Fig. 2f of infected stems). Distinctive electrontransparent `haloes' separating the host defence gum
from the bacteria were a consistent feature of the leaf
colonization by G. diazotrophicus and they appeared to
mark the boundary between the bacteria and the host.
The fact that they were also often a site of accumulation
of immunogold particles suggests that the bacteria within
them were releasing immunoreactive material, most likely
exopolysaccharide (EPS) (James et al., 1994; James and
Olivares, 1998). A similar interaction was reported with
Herbaspirillum spp. in sugar cane and sorghum leaves,
Sugar cane and endophytic bacteria
where bacterial microcolonies that had formed within the
host defence material that ®lled the vessels had very
distinct boundaries between them and the plant gums
(Olivares et al., 1997; James et al., 1997; James and
Olivares, 1998). Electron-transparent haloes surrounding
xylem-colonizing bacteria are a common phenomenon,
for example, they have also been observed in sugar cane
infected by C. xyli subsp. xyli (Gillaspie and Teakle,
1989), and in tomato (Lycopersicon esculentum) infected
by Pseudomonas (Ralstonia) solanacearum (Vasse et al.,
1995).
Although it is known that H. rubrisubalbicans is
both compatible with sugar cane, and a mild pathogen,
causing symptoms of `mottled stripe disease' on leaves
of susceptible varieties (Pimentel et al., 1991; Olivares
et al., 1997), the observation from the present study
(Experiments 1, 2) that G. diazotrophicus may also be
slightly `pathogenic' when inoculated in high numbers
is new. It is also rather controversial, as this bacterium
has hitherto been regarded as a purely symptomless
endophyte, causing no harm to the host (James et al.,
1994; Dong et al., 1994; DoÈbereiner et al., 1995; Baldani
et al., 1997). On the other hand, there were no macroscopically visible symptoms, indicating that infection by
G. diazotrophicus is different from that by `genuine'
vascular pathogens of sugar cane, such as H. rubrisubalbicans, C. xyli subsp. xyli, X. albilineans, and X. campestris
pv. vasculorum. These pathogenic bacteria may accumulate within the vessels in very large numbers (up to
1010 CFU g 1 fresh wt. in the case of X. albilineans;
Rott et al., 1997), and consequently elicit a much more
aggressive host defence reaction. Also, in susceptible
varieties, they may cause long-term damage, or even plant
death (Kao and Damann, 1980; Harrison and Davis,
1988; Gillaspie and Teakle, 1989; Ricaud and Ryan, 1989;
Ricaud and Autrey, 1989; Pimentel et al., 1991; Olivares
et al., 1997; Rott et al., 1997). In contrast to these
pathogens, with G. diazotrophicus all evidence of `pathogenicity' was at the microscopical level, and mainly took
the form of gum production in the vessels. The only
evidence of actual damage to the plants was distorted
metaxylem vessels and the degraded bundle sheath cells
immediately adjacent to the points of inoculation. As the
latter damage was also seen with the control plants
inoculated with sterile water (not shown), much of it is
likely to be a simple wound response brought about by
the injection procedure. Similarly, the violent response
observed when cut sugar cane stalks were immersed in
liquid cultures of G. diazotrophicus (Dong et al., 1997) is
also likely to be largely the result of massive (fatal) wounding caused by the stems being cut (Fuentes-Ramirez et al.,
1999).
Therefore, although it was shown that G. diazotrophicus can provoke a mild, localized host defence response
when inoculated into sugar cane, it cannot properly be
757
termed a phytopathogen as all of the numerous studies
with ®eld-grown plants have isolated it only from
plants showing no macroscopically visible symptoms
(Cavalcante and DoÈbereiner, 1988; Li and MacRae,
1992; Fuentes-Ramirez et al., 1993; Muthukumarasamy
et al., 1999). Moreover, it does not produce a HR with
sugar cane (this study), or in a standard pathogenicity test
with tobacco leaves (FL Olivares, unpublished data). The
most likely reason for the limited `pathogenicity' shown
by G. diazotrophicus in the present study is that high
numbers of the bacteria were `forced' into the plants,
either by inoculation of wounded plantlets (Experiment 1)
or by leaf injection (Experiment 2). Indeed, the limited
host defence response observed is similar to that which
has been observed previously when plants have been
inoculated with high numbers (e.g. 108 cells ml 1) of nonpathogenic bacteria, for example, heat-killed pathogenic
Pseudomonas spp and live Escherischia coli (Sequeira
et al., 1977; McKhann and Hirsch, 1994), and hence
suggests that any bacteria, if inoculated into a plant in
suf®ciently high numbers, will elicit some host defence
response in order to control and suppress their numbers.
Does Gluconacetobacter diazotrophicus live in the
xylem or the intercellular apoplast of sugar cane?
Although Experiment 1 has con®rmed that G. diazotrophicus will readily colonize the xylem of inoculated sugar
cane plantlets, as these experiments were conducted with
very young plants and under unusual growth conditions
(e.g. low irradiances; James et al., 1994; Reis et al., 1999;
Sevilla et al., 1998) the pattern of localization may not be
representative of that in mature plants. On the other
hand, Experiment 2 has shown that the xylem is the
principal location of the bacteria in fully-expanded leaves
of inoculated greenhouse-grown plants (albeit with a
slight host-defence response), and this may be more
representative of the ®eld situation. This location is
strongly supported by a recent study of mature plants
infected by b-glucuronidase marked G. diazotrophicus, in
which it was reported that the xylem and the xylem
parenchyma (and possibly the phloem) were the only
observed sites of colonization (Fuentes-Ramirez et al.,
1999). Therefore, although the possibility of G. diazotrophicus living in other locations (e.g. the intercellular
apoplast; Dong et al., 1994; Figs 2b, c, 3a, b, this study) is
not ruled out, considering that Reis et al. (Reis et al.,
1994) and Caballero-Mellado et al. (Caballero-Mellado
et al., 1995) have also isolated the bacteria from the
xylem sap of ®eld-grown plants, it must be concluded that
at least part of the G. diazotrophicus population in sugar
cane resides in the xylem.
This is in strong contrast to Dong et al. (Dong et al.,
1994, 1997), who concluded that the intercellular apoplast
of the sucrose storage tissue was the only probable
758
James et al.
location for the bacteria in mature stems, and that it
would be `most unlikely' for them to live within the xylem
(see Introduction for more details). However, there are
a number of ¯aws in this argument. Firstly, there is
no convincing anatomical evidence for the presence of
G. diazotrophicus within the intercellular spaces of mature
stems. Dong et al. did not con®rm the identity of the
bacteria in their micrographs via immunological or
molecular methods (Dong et al., 1994) and, therefore,
given the diverse micro¯ora within sugar cane (James
and Olivares, 1998; Fuentes-Ramirez et al., 1999), it is
impossible to say with any certainty that the bacteria
were G. diazotrophicus. Secondly, the `apoplastic ¯uid'
obtained from stems by centrifugation also contained up
to 20% xylem sap (vuv) (Dong et al., 1994), and hence the
bacteria isolated from it may also have come from the
xylem as well as the intercellular spaces. Finally, although
Dong et al. (Dong et al., 1994, 1997) have insisted that the
sucrose-rich stem apoplast is where G. diazotrophicus
must live due to its `need' for a high concentration
(10±12%) of sucrose to support `symbiotic' N2 ®xation
(see Introduction), not only is this insistence not supported by convincing anatomical evidence (see above), it
is also based on two misconceptions.
(1) There is little evidence, as yet, that G. diazotrophicus is actually an N2-®xing symbiont of sugar cane, or
that it even expresses active nitrogenase in planta (James
and Olivares, 1998; Fuentes-Ramirez et al., 1999; this
study). Indeed, recent studies suggest that any bene®cial
effects it may have on plant growth are more likely to be
via mechanisms other than N2 ®xation, such as production of indole-acetic acid (IAA) (Fuentes-Ramirez et al.,
1993; Sevilla et al., 1998; BastiaÂn et al., 2000).
(2) Gluconacetobacter diazotrophicus does not have
a requirement for high levels of sucrose. Reis and
DoÈbereiner (Reis and DoÈbereiner, 1998) have shown that
it will grow and ®x N2 in media containing only 1%
sucrose, and therefore concentrations at the lower end
of the range commonly found within sugar cane
xylem (0±9%; Dong et al., 1997, and references therein)
could provide suf®cient carbon to support the relatively
low populations of the bacteria that are commonly isolated from the aerial parts of the plant (c.-102±104;
Paula et al., 1991; Dong et al., 1994; Reis et al., 1999;
Fuentes-Ramirez et al., 1999; dos Reis et al., 2000).
Acknowledgements
We thank RM Boddey, J DoÈbereiner, LE Fuentes-Ramirez,
C Kennedy, JA Raven, JI Sprent, and M Sevilla for helpful
discussions, and G Baeta da Cruz, M Gruber and M Kierans
for technical assistance. EK James was funded by the World
Bank through the Inter-American Institute for Co-operation in
Agriculture (IICA), and FL Olivares by the Brazilian National
Research Council (CNPq).
References
Baldani JI, Caruso L, Baldani VLD, Goi SR, DoÈbereiner J. 1997.
Recent advances in BNF with non-legume plants. Soil Biology
and Biochemistry 29, 911±922.
BastiaÂn F, Rapparini F, Baraldi R, Piccoli P, Bottini R. 2000.
Inoculation with Acetobacter diazotrophicus increases glucose
and fructose content in shoots of Sorghum bicolor (L.)
Moench. Symbiosis 27, 147±156.
Boddey RM, de Oliveira OC, Urquiaga S, Reis VM, Olivares FL,
Baldani VLD, DoÈbereiner J. 1995. Biological nitrogen ®xation
associated with sugar cane and rice: contributions and
prospects for improvement. Plant and Soil 174, 195±209.
Boddey RM, da Silva LG, Reis VM, Alves BJR, Urquiaga S.
2000. Assessment of bacterial nitrogen ®xation in grass
species. In: Triplett EW, ed. Prokaryotic nitrogen ®xation: a
model system for analysis of a biological process. UK: Horizon
Scienti®c Press, 705±726.
Bull TA, Gayler KR, Glasziou KT. 1972. Lateral movement of
water and sugar across xylem in sugarcane stalks. Plant
Physiology 49, 1007±1011.
Caballero-Mellado J, Fuentes-Ramirez LE, Reis VM,
Martinez-Romero E. 1995. Genetic structure of Acetobacter
diazotrophicus populations and identi®cation of a new genetically distant group. Applied and Environmental Microbiology
61, 3008±3013.
Cavalcante VA, DoÈbereiner J. 1988. A new acid-tolerant
nitrogen-®xing bacterium associated with sugarcane. Plant
and Soil 108, 23±31.
DoÈbereiner J, Baldani VLD, Reis VM. 1995. Endophytic
occurrence of diazotrophic bacteria in non-leguminous crops.
In: Fendrik I, del Gallo M, Vanderleyden J, de Zamaroczy M,
eds. Azospirillum VI and related microorganisms. Berlin,
Heidelberg: Springer-Verlag, 3±14.
Dong Z, Canny MJ, McCully ME, Roboredo MR, Cabadilla CF,
Ortega E, Rodes R. 1994. A nitrogen-®xing endophyte of
sugarcane stems. Plant Physiology 105, 1139±1147.
Dong Z, McCully ME, Canny MJ. 1997. Does Acetobacter
diazotrophicus live and move in the xylem of sugarcane stems?
Anatomical and physiological data. Annals of Botany 80,
147±158.
Franke IH, Fegan M, Hayward C, Leonard G, Stackebrandt E,
Sly LI. 1999. Description of Gluconacetobacter sacchari sp.
nov., a new species of acetic acid bacterium isolated from
the leaf sheath of sugar cane and from the pink sugar-cane
mealy bug. International Journal of Systematic Bacteriology
49, 1681±1693.
Fuentes-RamõÂrez LE, Caballero-Mellado J, SepuÂlveda J,
MartõÂnez-Romero E. 1999. Colonization of sugarcane by
Acetobacter diazotrophicus is inhibited by high N-fertilization.
FEMS Microbiology Ecology 29, 117±128.
Fuentes-RamõÂrez LE, Jimenez-Salgado T, Abarca-Ocampo IR,
Caballero-Mellado J. 1993. Acetobacter diazotrophicus, an
indoleacetic acid producing bacterium isolated from sugarcane cultivars of Mexico. Plant and Soil 154, 145±150.
Gillaspie AG, Teakle DS. 1989. Ratoon stunting disease. In:
Ricaud C, Egan BT, Gillaspie AG, Hughes CG, eds. Diseases
of sugarcane. Amsterdam: Elsevier, 59±80.
Hallmann J, Quadt-Hallmann A, Mahaffee WF, Kloepper JW.
1997. Bacterial endophytes in agricultural crops. Canadian
Journal of Microbiology 43, 895±914.
Harrison NA, Davis MJ. 1988. Colonization of vascular
tissues by Clavibacter xyli subsp. xyli in stalks of sugarcane
cultivars differing in susceptibility to ratoon-stunting disease.
Phytopathology 78, 722±727.
Sugar cane and endophytic bacteria
Hawker JS. 1965. The sugar content of cell walls and
intercellular spaces in sugar cane stems and its relation to
sugar transport. Australian Journal of Biological Science 18,
959±969.
James EK. 2000. Nitrogen ®xation in endophytic and associative
symbiosis. Field Crops Research 65, 197±209.
James EK, Olivares FL. 1998. Infection and colonization of
sugar cane and other graminaceous plants by endophytic
diazotrophs. Critical Reviews in Plant Sciences 17, 77±119.
James EK, Olivares FL, Baldani JI, DoÈbereiner J. 1997.
Herbaspirillum, an endophytic diazotroph colonizing vascular
tissue in leaves of Sorghum bicolor L. Moench. Journal of
Experimental Botany 48, 785±797.
James EK, Reis VM, Olivares FL, Baldani JI, DoÈbereiner J.
1994. Infection of sugar cane by the nitrogen-®xing bacterium
Acetobacter diazotrophicus. Journal of Experimental Botany
45, 757±766.
Kao J, Damann KE. 1980. `In situ' localization and morphology
of the bacterium associated with ratoon stunting disease of
sugar cane. Canadian Journal of Botany 58, 310±315.
Li R, MacRae IC. 1992. Speci®c identi®cation and enumeration
of Acetobacter diazotrophicus in sugar cane. Soil Biology and
Biochemistry 24, 413±419.
Lima E, Boddey RM, DoÈbereiner J. 1987. Quanti®cation of
biological nitrogen ®xation associated with sugar cane using a
15
N-aided nitrogen balance. Soil Biology and Biochemistry 24,
413±419.
McKhann HI, Hirsch AM. 1994. Does Rhizobium avoid the host
response? In: Dangl JL, ed. Bacterial pathogenesis of plants
and animals. Berlin, Heidelberg: Springer-Verlag, 139±162.
Muthukumarasamy R, Revathi G, Lakshminarasimhan C. 1999.
In¯uence of N fertilization on the isolation of Acetobacter
diazotrophicus and Herbaspirillum spp. from Indian sugarcane
varieties. Biology and Fertility of Soils 29, 157±164.
Olivares FL, Baldani VLD, Reis VM, Baldani JI, DoÈbereiner J.
1996. Occurrence of the endophytic diazotrophs Herbaspirillum spp. in roots, stems and leaves predominantly of
Gramineae. Biology and Fertility of Soils 21, 197±200.
Olivares FL, James EK. 2000. Endophytic establishment of
diazotrophic bacteria in sugar cane plants. In: Pedrosa FO,
Hungria M, Yates MG, Newton WE, eds. Nitrogen ®xation:
from molecules to crop productivity. Dordrecht: Kluwer,
413±414.
Olivares FL, James EK, Baldani JI, DoÈbereiner J. 1997.
Infection of mottled stripe disease susceptible and resistant
varieties of sugar cane by the endophytic diazotroph
Herbaspirillum. New Phytologist 135, 723±737.
Patriquin DG, Gracioli LA, Ruschel AP. 1980. Nitrogenase
activity of sugar cane propagated from stem cuttings in sterile
vermiculite. Soil Biology and Biochemistry 12, 413±417.
Patriquin DG, DoÈbereiner J, Jain DK. 1983. Sites and processes
of association between diazotrophs and grasses. Canadian
Journal of Microbiology 29, 900±915.
Paula MA, Reis VM, Dobereiner J. 1991. Interactions of Glomus
clarum with Acetobacter diazotrophicus in infection of sweet
potato (Ipomoea batata), sugarcane (Saccharum sp.) and sweet
sorghum (Sorghum vulgare). Biology and Fertility of Soils 11,
111±115.
Pimentel JP, Olivares FL, Pitard RM, Urquiaga S, Akiba F,
DoÈbereiner J. 1991. Dinitrogen ®xation and infection of grass
leaves by Pseudomonas rubrisubalbicans and Herbaspirillum
seropedicae. Plant and Soil 137, 61±65.
Reinhold-Hurek B, Hurek T. 1998a. Interactions of gramineous
plants with Azoarcus spp. and other diazotrophs: identi®cation, localization, and perspectives to study their function.
Critical Reviews in Plant Sciences 17, 29±54.
759
Reinhold-Hurek B, Hurek T. 1998b. Life in grasses: diazotrophic
endophytes. Trends in Microbiology 6, 139±144.
Reis dos FB, Silva da LG, Reis VLM, DoÈbereiner J. 2000.
OcorreÃncia de bacteÂrias diazotro®cas em diferentes genoÂtipos
de cana-de-acËuÂcar. Pesquisa Agropecuaria Brasileira 35,
985±994.
Reis VM, Olivares FL, DoÈbereiner J. 1994. Improved methodology for isolation of Acetobacter diazotrophicus and
con®rmation of its endophytic habitat. World Journal of
Microbiology and Biotechnology 10, 101±104.
Reis VM, DoÈbereiner J. 1998. Effect of high sugar concentration
on nitrogenase activity of Acetobacter diazotrophicus. Archives
of Microbiology 171, 13±18.
Reis VM, Olivares FL, de Oliveira ALM, Reis dos FB, Baldani JI,
DoÈbereiner J. 1999. Technical approaches to inoculate micropropagated sugar cane plants with Acetobacter diazotrophicus.
Plant and Soil 206, 205±211.
Ricaud C, Autrey LJC. 1989. Gumming disease. In: Ricaud C,
Egan BT, Gillaspie AG, Hughes CG, eds. Diseases of
sugarcane. Amsterdam: Elsevier, 21±38.
Ricaud C, Ryan CC. 1989. Leaf scald. In: Ricaud C, Egan BT,
Gillaspie AG, Hughes CG, eds. Diseases of sugarcane.
Amsterdam: Elsevier, 39±58.
Rott P, Mohamed IS, Klett P, Soupa D, de Saint-Albin A,
Feldmann P, Letourmy P. 1997. Resistance to leaf scald
disease is associated with limited colonization of sugarcane and wild relatives by Xanthomonas albilineans. Phytopathology 87, 1202±1213.
Ruschel AP. 1981. Associative N2-®xation by sugar cane.
In: Vose PB, Ruschel AP, eds. Associative N2-®xation,
Vol. II. Boca Raton: CRC Press, 81±90.
Sequeira L, Gaard G, de Zoeten GA. 1977. Interactions
of bacteria and host cell walls: its relation to mechanisms of
induced resistance. Physiological Plant Pathology 10, 43±50.
Sevilla M, Kennedy C. 2000. Genetic analysis of nitrogen
®xation and plant-growth-stimulating properties of Acetobacter diazotrophicus, an endophyte of sugar cane. In:
Triplett EW, ed. Prokaryotic nitrogen ®xation: a model system
for analysis of a biological process. UK: Horizon Scienti®c
Press, 737±760.
Sevilla M, de Oliveira A, Baldani I, Kennedy C. 1998. Contributions of the bacterial endophyte Acetobacter diazotrophicus to sugarcane nutrition: a preliminary study. Symbiosis 25,
181±191.
Silva da LG. 1999. Estudos de colonizacËaÄo em cana de acËuÂcar
(Saccharum spp.) por Acetobacter diazotrophicus e Herbaspirillum spp. utilizando teÂcnicas imunoloÂgicas. MSc thesis,
Universidade Federal Rural do Rio de Janeiro.
Sprent JI, James EK. 1995. N2-®xation by endophytic bacteria:
questions of entry and operation. In: Fendrik I, del Gallo M,
Vanderleyden J, de Zamaroczy M, eds. Azospirillum VI and
related microorganisms. Berlin, Heidelberg: Springer-Verlag,
15±30.
Teakle DS, Appleton JM, Steindl DRL. 1978. An anatomical
basis for resistance of sugar cane to ratoon stunting disease.
Physiological Plant Pathology 12, 83±91.
Urquiaga S, Cruz KHS, Boddey RM. 1992. Contribution of
nitrogen ®xation to sugar cane: Nitrogen-15 and nitrogenbalance estimates. Soil Science Society of America Journal 56,
105±114.
Vasse J, Frey P, Trigalet A. 1995. Microscopic studies of
intercellular infection and protoxylem invasion of tomato
roots by Pseudomonas solanacearum. Molecular PlantMicrobe Interactions 8, 241±251.
Welbaum GE, Meinzer FC, Grayson RL, Thornham KT. 1992.
Evidence for and consequences of a barrier to solute diffusion
760
James et al.
between the apoplast and vascular bundles in sugarcane stalk
tissue. Australian Journal of Plant Physiology 19, 611±623.
Yamada Y, Hoshino K, Ishikawa T. 1997. The phylogeny of
acetic acid bacteria based on the partial sequences of 16S
ribosomal RNA: the elevation of the subgenus Gluconoacetobacter to the generic level. Bioscience, Biotechnology and
Biochemistry 61, 1244±1251.
Yamada Y, Hoshino K, Ishikawa T. 1998. Gluconacetobacter
nom. corrig. (Gluconoacetobacter wsicx). In: Validation of
publication of new names and new combinations previously
effectively published outside the IJSB, List no. 64. International
Journal of Systematic Bacteriology 48, 327±328.