Biol Fertil Soils (2004) 39:391–397
DOI 10.1007/s00374-004-0728-4
ORIGINAL PAPER
Hannan H. Youssef · Mohamed Fayez ·
Mohamed Monib · Nabil Hegazi
Gluconacetobacter diazotrophicus: a natural endophytic diazotroph
of Nile Delta sugarcane capable of establishing
an endophytic association with wheat
Received: 18 March 2003 / Accepted: 30 December 2003 / Published online: 2 March 2004

Springer-Verlag 2004
Abstract Gluconacetobacter- like diazotrophs were en-
countered as dense populations inside the root and stem
tissues of sugarcane cultivated in ancient agricultural
fields of the Nile Delta. Counts of >105 cells g-1 were
recorded in root and stem samples. The leaves contained a
smaller population (<103 g-1). The typical dark-orange
colonies which developed on LGIP agar plates were
purified. Identification was performed with the API
microtube systems: API 20E for Enterobacteriaceae
and API 20NE for non-Enterobacteriaceae. API profiles
of the local isolates were closely related to those of the
type culture Gluconacetobacter diazotrophicus (ATTC
49037). The isolates successfully reduced C2H2 and
produced appreciable amounts of ethylene in the presence
of cane juice. This suggested that the local isolates are
closely related to the type strain G. diazotrophicus. Wheat
seedlings were inoculated with a number of isolates under
gnotobiotic conditions. Both optical and scanning elec-
tron microscopy showed that endophytic Gluconaceto-
bacter spp. were present in all the samples tested. They
were observed in apparently intact and enlarged epider-
mal root cells, and also in stem tissues, indicating that the
bacterium was able to migrate upward into the shoot
tissues. Although Gluconacetobacter inoculation did not
stimulate the growth of the cereal plant, the results
obtained are particularly interesting because this bacterial
species was capable of colonizing the internal tissues of
wheat, not considered a natural host until now.
Keywords Gluconacetobacter diazotrophicus ·
Diazotorophs · Associative nitrogen-fixers · Sugarcane ·
Wheat
H. H. Youssef · M. Fayez · M. Monib · N. Hegazi ())
Environmental Studies and Research Unit (ESRU),
Faculty of Agriculture,
Cairo University, 12613 Giza, Egypt
e-mail: nhegazi@hotmail.com
Tel.: +20-2-5728483
Introduction
A new diazotroph among the a-subclass of the protobac-
teria, Gluconacetobacter diazotrophicus , was originally
isolated from surface-sterilized roots and stems of sugar
cane using a semi-solid N-deficient medium with cane
juice as the C source (Cavalcante and Dobereiner 1988;
Gillis et al. 1989). This bacterium is of special interest
because, besides fixing N2 in the presence of KNO3 and at
low pH values <3.0 (Stephan et al. 1991), it can excrete
almost half of the fixed N2 in a form potentially available
to plants (Cojho et al. 1993). Such an endophytic
diazotroph was also isolated from sugarcane in Mexico,
Cuba and Australia (Li and MacRae 1991, 1992; Fuentes-
Ramirez et al. 1993). The occurrence of the microorgan-
ism was reported in roots, tubers and stems of sweet
potato which confirm the endophytic nature of this
particular diazotroph (Dobereiner et al. 1988). In several
other instances, however, difficulties in finding this
bacterium may be related to methods used for surface
sterilization and isolation. Therefore, we report in the
present paper the most successful procedures of steriliza-
tion and isolation for this bacterium, in addition to tracing
the specific occurrence of the microorganism in Egyptian
sugarcane. The ability of tested isolates to establish an
endophytic association with wheat, as a non-legume, was
also investigated under gnotobiotic conditions and doc-
umented. So far, no information is available in the
literature on the natural and/or induced infection of
cereals in general, and in wheat in particular, with G.
diazotrophicus.
Materials and methods
Basic culture medium
Sugarcane plants were collected from different ancient agricultural
fields of Giza. Plant materials were separated into roots, stems and
leaves, then surface-sterilized by different procedures (Table 1).
Semisolid N-deficient medium was inoculated with serial
dilutions of root and stem samples. The medium used, a modified
LGIP medium (Cavalcante and Dobereiner 1988), contained: 100 g
392
Table 1 Most probable number Treatments
( MPN) of endophytic Glu-
conacetobacter in sugarcane
root and stem samples sterilized
by various procedures Tap water
Chloramine T (1.0%, 5 min)
H2O2 (3.0%, 5–10 min)
Mercuric chloride (0.1%, 5–10 min)
Sodium hypochlorite (3.0%, 3–5 min)
Sodium hypochlorite (5.0%, 30 min)
C2H5OH (95%, 5–10 s)+sodium hypochlorite
(3.0%, 30 min)
Mercuric chloride (0.2% in 50.0% C2H5OH,
4 min)
Sodium hypochlorite (6.0%, 2 min)+C2H5OH
(95.0%, 15 s)+flamed
sucrose l-1; 0.2 g K2HPO4 l-1; 0.6 g KH2PO4 l-1; 0.2 g MgSO4.7H2O
l-1; 0.02 g CaCl2.2H2O l-1; 0.002 g Na2MoO4.2H2O l-1; 0.01 g
FeCl3.6H2O l-1; 0.5% bromothymal blue in 0.2 M KOH, and 2.0 g
agar l-1. The pH was adjusted to 5.5.
Enumeration of Gluconacetobacter in sugarcane samples
Gluconacetobacter were isolated on modified semi-solid N-free
LGIP medium and the population densities were determined. Tubes
of culture medium were inoculated with aliquots of a series of serial
dilutions prepared for homogenates of plant parts in the medium
described by Reinhold et al. (1985) lacking C and N. Tubes were
considered positive when showing, after 5 days incubation at 30C,
the typical dark-orange surface pellicle of Gluconacetobacter and
the clear, colourless medium below. Most probable numbers (MPN)
were calculated according to Alexander (1982).
Representative isolates were obtained by single-colony isolation
on agar plates of the previous medium. After 7–10 days, pure
orange colonies were transferred onto LGIP medium. One hundred
and eighty-six pure isolates were examined for C2H2-reducing
activity according to Hegazi et al. (1980) and then screened to give
ten potential N2-fixing ones. Selected isolates were identified using
methods described by Krieg and Holt (1984). The API microtube
systems, API 20 E and API 20 NE, were used as a standardized
micromethod (Logan and Berkeley 1984) in addition to conven-
tional tests such as the Gram test and motility. A G. diazotrophicus
type culture (ATCC 49037) was used as a reference strain.
The isolates obtained, in addition to the type culture strain, were
grown in LGIP medium where sucrose was substituted by cane
juice at 10% concentration. Cultures were measured for C2H2
reducing activity.
Endophytic establishment of Gluconacetobacter in wheat plants
One of the local isolates (VIS 8) and the type culture strain
(ATCC 49037) were tested for their ability to invade the root
system of wheat cv. Gemaza 3 in a gnotobiotic culture. Tubes of
2.5 cm diameter and 14.5 cm length filled with 25 ml semi-solid
medium (see Reinhold et al. 1985) lacking C and N, and containing
fine sand (1 mm diameter, 20 g tube-1) were autoclaved and
thoroughly mixed.
Healthy seedlings which developed from surface-sterilized
seeds on the nutrient agar plates were transferred into tubes at
two seedlings per tube. They were top-inoculated with 1-ml
portions (108 cells) of either the local VIS 8 isolate or the ATCC
strain. Two sets of culture tubes were prepared. For the first set,
seedlings were vertically uprooted twice by completely raising the
root system then immersing it again into the same culture medium.
This procedure probably creates cracks on the root surface through
friction with sand particles, which should facilitate the invasion of
References Root Shoot
MPN (10 cells g-1)
4
Cavalcante and Dobereiner 12.6 315.0
(1988)
Paula et al. (1991) 21.6 24.3
Somasegaran and Hoben 820.0 1440.0
(1985) 3.0 1.6
2.2 15.3
Bilal et al. (1990) 24.3 24.3
Somasegaran and Hoben 1.0 1.6
(1985)
Gagne et al. (1987) 1.6 1.6
12.6 0.02
the introduced diazotrophs into the plant root. For the other set of
tubes, seedlings were not subjected to initial uprooting.
Tubes were incubated in a growth chamber with a day/night
cycle of 8/16 h with temperatures of 21/19C, respectively.
Seedlings were sampled when 21 days old, surface sterilized and
plant roots and stems were analysed for Gluconacetobacter
population densities using the MPN technique. To measure C2H2-
reducing activities, whole plant samples were aseptically trans-
ferred to test tubes, sealed with rubber stoppers and 10% of the gas
phase was replaced by C2H2. Then, tubes were incubated at 30C
for 24 h. The C2H4 produced was measured by injecting 0.5 ml of
the gases into a DELSI DN 200/250 gas chromatograph (Hegazi et
al. 1980). Plant biomass yield was determined after drying at 70C
to constant weights.
The presence of Gluconacetobacter as an endophyte was
studied by light and scanning electron microscopy. Roots and stems
were excised and cross-sections were carefully hand-prepared.
They were incubated overnight in tubes half filled with tetrazolium-
phosphate buffer solution (PBMT). This solution consisted of
0.05 M potassium phosphate buffer (pH 7.0) containing 0.625 g
malate l-1 and 1.5 g 2,3,5-triphenyltetrazolium chloride l-1. The
buffer-malate mixture was autoclaved and then tetrazolium added.
The PBMT-treated plant segments were placed in water under a
cover slip, examined microscopically under a bright field and
photographed (Patriquin and Dobereiner 1978). For scanning
electron microscopy (Harley and Fergusen 1990), root or stem
sections were cut, fixed in 2.5% glutaraldehyde for 24 h at 4C, and
then post-fixed in 1% osmium tetroxide for 1 h at room temper-
ature. The specimens were then dehydrated with increasing
concentrations of acetone, critical-point dried, and finally sputter-
coated with gold. The examination, measurements and photograph-
ing were done by using a Jeol scanning electron microscope (JSM-
T 330 A) equipped with an image recording and processing system
(SemAfore).
Results
Ecology of Gluconacetobacter spp.
The majority of the surface-sterilization procedures ap-
plied, except for the H2O2 treatment, were somewhat
similar in eliminating a great portion of root and stem
surface-colonizing microorganisms (Table 1). Among
them, treatment with 95% ethanol for 5–10 s followed
by 3% sodium hypochlorite (30 min) seemed to be the
most appropriate and successfully facilitated the gentle
isolation of the endophytic Gluconacetobacter dia-
zotrophs from the internal root and stem tissues of plants.
 393
Table 2 Physiological proper- Characters Gluconacetobacter Local isolates
ties of representative endophyt- (ATCC 49037)
ic local Gluconacetobacter-like IR VI R VI S IX R
isolates obtained from sugar
cane using API 20 E. Reading b-Galactosidase + + + + +
of API galleries extended for Arginine dihydrolase +   + +
36 h of the incubation. R Root, Lysine decarboxylase     
S stem origin Ornithine decarboxylase +  + + +
Citrate utilization + + + + +
H2S production     
Urease     
Tryptophan deaminase     
Indole production     
Acetoin production + + + + +
Gelatinase   +  
Fermentation of
Glucose + + + + +
Mannitol + + + + +
Inositol     
Sorbitol + + + + +
Rhamnose + + + + +
Sucrose + + + + +
Melibiose + +  + +
Amygdalin + + + + +
Arabinose + + + + +
Cytochrome-oxidase +    
NO2 production  + + + +
N2 production     
Motility + + + + +
MacConkey + + + + +
Fermentation of glucose + + + + +
Oxidation of glucose + + + + +

Table 3 Physiological charac- Characters Gluconacetobacter Local isolates

teristics of local Gluconaceto- (ATCC 49037)
bacter -like isolates based on IR VI R VI S IX R
their API 20 NE profile. Read-
ing of API galleries extended Reduction of NO3-to NO2- + + + + +
for 36 h of the incubation. For Reduction NO3- toN2 +    
abbreviations, see Table 2 Indole production  + + + +
Glucose (acidification) +   + +
Arginine dihydrolase, urease +    
Hydrolysis (b-glucosidase)  + + + +
Hydrolysis (protease)   €  €
b-Galactosidase + + + + +
Glucose + + + + +
Arabinose + + + + +
Mannose + + + + +
Mannitol + + + + +
N -Acetyl-glucosamine + + + + +
Maltose + + + + +
Gluconate + + + + +
Caprate    € +
Adipate    € 
Malate + + + + +
Citrate + + + + +
Phenylacetate    € +
Cytochrome oxidase     
Dinitrogenase activity 2.56 3 8 20 47
(nmoles C2H4 culture-1 h-1)

Gluconacetobacter spp. were encountered in dense pop-
ulations inside root and stem tissues of sugar cane with
population densities ranging from 1.0 to 820.0104 cells
g-1 root and from 0.02 to 1,440.0104 cells g-1 stem. This
indicates the endophytic nature of the organism in root
and stem tissues.
The typical dark-orange colonies developed on LGIP
agar plates were purified. The most active isolates for
C2H2 reduction (ten isolates) were selected for further
identification.
In general, cells of tested isolates were small, motile or
non-motile, Gram-negative, aerobic rods showing pellicle
394
Fig. 1 Cumulative C2H2-reduc-
ing activity of Glucanaceto-
bacter -like isolates and type
culture

Table 4 Population densities of endophytic Gluconacetobacter -like diazotrophs in roots, stems and leaves of sugar cane plants of various
ages determined by MPN and plate count techniques on LGIP. cfu Colony-forming units, ND not detected
Plant age Total endophytes Plate count Gluconacetobacter -like
(dark-orange colonies)
Total endophytes Gluconacetobacter -like (%) a
(dark-orange colonies)
MPN (103 cells g-1) (103 cfu g-1)
Roots Stems Leaves Roots Stems Leaves Roots Stems Leaves Roots Stems Leaves
1 Year old 2,000 7.0 0.6 8,600 54 09 390 9.0 1.0 4.53 16.7 11.1
2 Years old 290 3.0 ND 440 27 ND 50 10.0 ND 11.4 37.0 0.0
3 Years old
Site I 2,300 30.0 0.4 86,000 71 2.0 36,000 5.0 0.9 41.9 7.04 45.0
Site II 1,170 ND 0.03 5,900 710 300 2,750 280 3.0 46.6 39.4 1.00
Site III 7,400 120 1.1 340 200 69 80 90.0 30.0 23.5 45.0 43.5

formation (micro-aerobic aerotaxis) in semi-solid N-
deficient medium with 10% sucrose. They formed a thick
surface pellicle after 7–10 days of incubation at 30C.
Gluconacetobacter -like isolates exhibited C2H2-reducing
activities in the range of 3.2–59.9 nmoles C2H4 culture-1
h-1.
Further identification was performed with the API
microtube systems, API 20 E strip (Table 2) for Entero-
bacteriaceae and API 20 NE profile (Table 3) for non-
Enterobacteriaceae. The G. diazotrophicus type culture
(ATCC 49037) was found to be related to Enterobacter
cloacae and Pseudomonas luteala based on API 20 E and
20 NE respectively. The majority of local Gluconaceto-
bacter -like diazotrophs fitted the same description. This
indicates that such isolates are closely related to the type
culture G. diazotrophicus.
The comparative C2H2-reducing activity of Glu-
conacetobacter -like isolates and the type culture was
measured in LGIP medium containing 10% (v/v) cane
juice instead of sucrose. Figure 1 demonstrates that all
tested diazotrophs successfully reduced C2H2 and pro-
duced appreciable amounts of C2H4 in the presence of
cane juice. This presents additional evidence that the local
isolates are closely related to G. diazotrophicus. Among
all isolates, VIS 8 was found to be an active N2-fixer
(59.9 nmoles C2H4 culture-1 h-1) and therefore was used in
the inoculation experiment.
The occurrence of Gluconacetobacter spp. as an
endophyte was further checked in an additional number
of sugarcane plants of different ages taken from various
sites of the Giza fields. Treatment with C2H5OH followed
by sodium hypochlorite (treatment VII, Table 1) was
applied for surface sterilization of the plant parts. Pop-
ulation densities of the endophytic diazotrophs deter-
mined by MPN and plate count techniques are presented
in Table 4. Total endophytes were encountered in high
numbers of 102–>107 g-1. Roots hosted exceptional
densities compared to stem tissues. The microorganisms
 395
Table 5 Average root and stem dry weights and population of
Gluconacetobacter of 21-day-old wheat seedlings inoculated with
Gluconacetobacter spp. LSD Least significant difference
Treatments Dry weights Gluconacetobacter
population
(mg per six plants) (log no. per plant)
Root Shoot Root Shoot
Uprooting
Without uprooting (A) 41.9 115.9 1.46 1.75
With uprooting (B) 33.1 100.3 2.61 2.35
LSD (0.05) 12.8 12.49 0.22 0.56
LSD (0.01) 17.5 17.04 0.32 0.79
Inoculation
Non-inoculated (C) 53.5 136.4 0.00 0.00
Acetobacter type 27.6 78.4 3.59 4.16
culture (D)
-Local Acetobacter 31.5 109.6 2.52 1.99
isolate (E)
LSD (0.05) 10.5 10.19 0.18 0.68
0.01 14.3 13.91 0.26 0.97
Interactions
AC 53.5 136.4 0.00 0.00
BC 53.5 136.4 0.00 0.00
AD 36.0 79.1 2.69 3.45
BD 19.2 77.7 4.48 4.86
AE 36.2 132.3 1.69 1.79
BE 26.7 87.0 3.34 2.20
LSD (0.05) 18.5 17.7 0.32 0.96
LSD (0.01) 24.8 24.1 0.45 1.37

were rare in sugarcane leaves, present at densities of <103

g-1, or completely absent. In the majority of cases and
regardless of the sugarcane organ examined, Gluconace-
tobacter -like diazotrophs developing orange colonies on
LGIP agar plates represented up to 46.6% of the total
endophytes. There was a tendency towards an increasing
Fig. 2 Scanning electron micrograph of a 21-day-old wheat stem
population density of endophytes with increasing of plant inoculated with Gluconacetobacter diazotrophicus without ( A) and
age. with ( B) initial shaking with sand. A Cross-section illustrating the
xylem vessels filled with slime cells, B slime layers of bacteria
filling xylem parenchyma
Verifying the endophytic nature
of Gluconacetobacter spp. isolates
This was observed in apparently intact, enlarged epider-
mal cells of the root and also in stem tissues, indicating
Cells of Gluconacetobacter spp. were introduced into
the ability of the bacterium to migrate upward in the
seedlings of wheat grown in gnotobiotic cultures. In
transpiration stream to the shoot tissues. Figures 2, 3 and
general, inoculation did not alter the biomass production
4 show infection of the interior of parenchyma cells of the
of wheat plants and even their development was at the
cortex and vascular cylinder of wheat roots and stems.
expense of plant growth (Table 5). Initial shaking of the
Initial uprooting facilitated an intense colonization of
root system with sand particles decreased the dry weights
inner plant tissues.
of roots and stems.
Inoculated Gluconacetobacter did establish in the
gnotobiotic system (Table 5). The initial uprooting of
seedlings at the time of planting resulted in a significant Discussion
colonization of roots and shoots by the introduced
G. diazotrophicus, originally isolated by Cavalcante and
Gluconacetobacter. As for interactions, the highest and
Dobereiner (1988), has been further reported in sugarcane
statistically significant endophytic populations were
plants grown in Mexico, Cuba and Australia (Li and
recorded for initially uprooted seedlings inoculated with
MacRae 1992). Population densities ranged from 103 to
the type culture, followed by the local isolate. Examina-
107 colony-forming units g-1 fresh weight. They were
tions with both optical and scanning electron microscopy
traced in roots, basal and apical stems, leaves and in plant
showed that cells were endophytic in all samples tested.
debris (Dobereiner et al. 1988). This particular diazotroph
396
Fig. 3A–D Scanning electron
micrographs of 21-days-old
wheat roots, inoculated with the
local isolate VIS8 and initially
uprooted. A General aspect of
the whole section; B bacterial
cells and mucus-filled paren-
chyma in the cortex and vascu-
lar cylinder; C, D higher mag-
nification illustrating the in-
tense invasion of bacterial cells
into the parenchyma cells of the
vascular cylinder
has a number of interesting properties, some of which
make it a likely candidate for providing fixed N2 to
sugarcane. These characteristics include: (1) the ability to
grow on 10% sucrose as the sole C source, (2) a very low
pH (5.5) requirement for optimum growth, (3) no or little
inhibition of NO3- and NH4+ of the bacterial nitrogenase
activity, and (4) a capability for microaerobic N fixation
(Stephan et al. 1991; Burris 1994). These are a group of
properties that distinguish G. diazotrophicus from Azo-
spirillum spp.
The present paper presents previously unpublished
information on Gluconacetobacter-like bacteria associat-
ed with sugarcane traditionally cultivated in old Egyptian
fields. Among the tested methods of surface sterilization
for sugarcane parts, the combined treatment with C2H5OH
and sodium hypochlorite was efficient for the elimination
of contaminants and hence facilitated the isolation of the
diazotroph. Based on MPN estimates, the diazotroph was
found in all tested samples with as many as 105–107 cells
g-1 root or stem, indicating the internal colonization of
sugarcane by the bacterium. Similar high numbers were
encountered in sugarcane grown in Australia as assessed
with an indirect enzyme-linked immunosorbent assay (Li
and MacRae 1992). The endophytic nature of G. dia-
zotrophicus, reported previously by Dobereiner et al.
(1988) was confirmed, as this diazotroph was present in
roots, stems and aerial parts collected from various
cultivars at different stages of growth and maturation.
Population densities in all plant parts ranged from 103 to
106 cells g-1 fresh weight. The bacterium was also isolated
from xylem sap, indicating translocation of the microor-
ganism through the plant tissues in the xylem (Reis 1991).
An improved isolation method described by Reis et al.
(1994) was used to examine various samples representing
six different forage grasses, sorghum, maize and 11 weed
species collected within sugar cane fields. All were
negative for G. diazotrophicus. Therefore, an endophytic
nature and high specificity were proposed for this
bacterium on the basis of the observations showing its
exclusive occurrence in sugarcane, sweet potatoes and
Cameroon grass. All these plants are propagated vegeta-
tively, and contain high sugar concentrations (Dobereiner
et al. 1988).
Results of the present study and those of Dobereiner et
al. (1988), Dobereiner (1992), Reis et al. (1994), Paula et
al. (1991) and Li and MacRae (1992), who reported the
occurrence of G. diazotrophicus in roots, tubers and stems
of sweet potato, confirm the endophytic nature of G.
diazotrophicus in sucrose-rich plants.
In an attempt to test the ability of Gluconacetobacter
to endophytically colonize other non-legume plants, the
reported gnotobiotic culture experiment was designed
where wheat was inoculated with the diazotroph. Al-
though Gluconacetobacter inoculation did not exert
beneficial effects on the growth of the cereal plant, the
results obtained are of particular interest because the
tested isolate was capable of colonizing the internal
tissues of a non-sugar plant not considered a natural host
until now. Similar to the colonization in sugarcane, the
invading Gluconacetobacter cells were translocated to-
wards inner ground tissues, xylum and to various parts in
the wheat plants.

Fig. 4A. B Scanning electron micrographs of 21-day-old wheat
roots, inoculated with the G. diazotrophicus type culture together
with initial uprooting. A Bacterial cells and their mucus inside
xylem vessels, B a magnified portion of A
Acknowledgements Thanks are due to Prof. S. El-Deeb at the Plant
Pathology Department, the Faculty of Agriculture, Ain Shams
University for his unlimited support during the electron micro-
scopic examinations of plant samples. The very important botanical
information of Prof. K. El-Sahaar of the Faculty of Agriculture,
Cairo University is very much appreciated. This study was
supported by the research grant BLAFE /FC/31/3–94 offered by
the National Project on Agro-Technologies Based on Biological
Nitrogen Fixation for the Development of Sinai Agriculture.
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