Vol.1, No.1, 24-38 (2010)s
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/as/
Agricultural Science
Symbiosis of nodule bacteria with perennial xerophyte
leguminous plants of Central Asia
Zair S. Shakirov*, Sardor A. Khakimov
Institute of Microbiology of Uzbekistan Academy of Sciences, Tashkent, Republic of Uzbekistan; *Corresponding Author:
Received 27 April 2010; revised 3 May 2010; accepted 5 May 2010.
From nodules of perennial xerophyte desert
leguminous plants – Ammodendron conollyi,
Astragalus villossimus, Astragalus unifoliolatus
– 151 bacterial isolates have been isolated. The
study of nodulation showed that AC8-1, AC11,
AC21, AC1-1, AC12-1 isolates (from Ammoden-
dron conollyi) , AV1 , AV8 - 1, AV 9, AV2 6 -1 , AV 3 6- 1
isolates (from Astragalus villossimus) and AU17-1,
AU30-1, AU30-2, AU20-1, AU23 isolates (from
Astragalus unifoliolatus) formed an effective
nitrogen-fixing symbiosis with the host plants.
As a result of 16S rRNA gene study of the
salt-resistant nodule bacteria it has been de-
termined that bacteria were related to Rhizo-
bium, Burkholderia and Achromobacter genera.
The study of isolates growth has revealed that
there were fast-growing and moderately-grow-
ing isolates that possessed with doubling-time
varying from 20 to 45 min. Their examination for
antibiotic-resistance showed that the number of
bacterial colonies of selected strains decreased
to some extent in the presence of chloram-
phenicol, but in all strains the resistance to an-
tibiotics was detected. The further investiga-
tions of resistance of the formed symbiosis to
stresses (drought, salinity) showed that at 6.41%
of moisture the maximal height and biomass of
inoculated plants of Ammodendron conollyi we re
21 cm and 2320 mg, but at 3.8% moisture the
height reduced by 4 times (up to 4.5 c m) and th e
biomass – by 11 times (203 mg). The analogous
effect was observed in Astragalus villossimus
and Astragalus unifoliolatus symbiosises. The
salinity equal to 100-200 mM NaCl did not affect
practically on normal growth and development
of desert leguminous plants symbiosis, while
for Astragalus villossimus such affecting con-
centration comprised up to 100 mM NaCl. The
light microscopy and electron microscopy of
Astragalus villossimus nodule sections showed
that V1 nodule bacteria strain efficiently colo-
nized the internal space within nodules, where
they were transformed into bacteroids. At 100 mM
NaCl salinity concentration the colonization of
nodule bacteria within nodule plant cells re-
duced in comparison with control nodules of
plant s grown in non-salted conditions.
Keywords: Ammodendron con ollyi; Astragalus
villossimus; Astragalus unifoliolatus; Nodulation;
Nitrogen Fixation; Salinity; Bacteroid; Rhizobium;
Burkholderia; Achromobacter
Deserts occupy one of third of dry land and they are un-
claimed potential for conducting human’s economical
activity. In many regions the desertification process with
its spreading to arable lands is going on, so the problem
of combating desertification is actual for many countries.
Desert legumes are represented with annual plants and
perennial trees and shrubs, which serve as a frame basis
for desert ecosystem. There are literature data about
symbiosis of relatively hygrophilous acacia trees with
their nodule bacteria [1,2], but there is a few data about
development of nitrogen-fixing symbiosis of xerophyte
desert legumes, their nodule bacteria and nodulation.
Nodulation in hygrophilous Acacia auriculiformis and
Acacia ampliceps reduced during the change of moistur e
content from 0.008 МРа to 0.08 МРа twice and totally
disappeared at 0.8 МРа [1]. Under inoculation of salt-
resistant Acacia ampliceps species by salt-tolerant strain
of nodule bacterium the nitrogen-fixing activity of
formed symbiosis considerable increased in the presence
of salt in comparison with nitrogen-fixing activity of
symbiosis caused with inoculation by salt-intolerable
strain [1]. In 20 Acacia species nodules formed after 5
months of growth, but in Leucaena leucocephala the
formation of nodules was stimulated by low doses of
nitrogen fertilizers (30-50 kg/Ha) and suppressed with
Z. S. Shakirov et al. / Agricultural Sciences 1 (2010) 24-38
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/as/
high doses (100 kg/Ha and more) [1]. More humid sands
promoted the increase of nodulation frequency in Pro-
sopis glangulosa (honey mesquite), desert perennial le-
guminous shrub/tree. Rhizobia, isolated from Acacia,
Prosopis and Leucaena were resistant to 500-850 mM
NaCl [2]. For the most of rhizobia the optimal tempera-
ture for growth in culture varies within range 28-31°C,
however, some strains of rhizobia isolated from woody
legumes of Acacia and Prosopis species grew well at
40-44°C [2]. The growth of plants, their nodulation and
nitrogen fixation (nitrogen content) in Acacia redolens
and Acacia cyclops, inoculated with salt-resistant nodule
bacteria, grown in vegetation experiments within sand,
were observed up to salinity value 80 mM NaCl, but in
Acacia ampliceps plants during growing within sand —
up to 200 mM NaCl [2]. Bacterial cells of rhizobia fro m
Acacia nilotica displayed the stable growth up to 850
mM NaCl and formed effective nitrogen-fixing nodules
in Acacia trees at 150 mM NaCl [2].
The objects of the present research were microbiologi-
cal and symbiotic properties of nodule bacteria isolated
from Ammodendron conollyi (“sandy acacia”, tree), As-
tragalus villossimus (shrub) and Astragalus unifoliolatus
(sandy semi-shrub), xerophyte perennial leguminous
plants of Kyzylkum desert (Uzbekistan) that grow at av-
erage annual rainfall norm 60 mm and temperature 45-50°C
[3], and also the host specificity of their nitrogen-fixing
symbiosis towards to host plant as well as resistance of
their symbiosi s to st resses (salinity, drought).
2.1. Description of Ammodendron conollyi,
Astragalus villossimus, Astragalus
unifoliolatus plants
Ammodendron connollyi Bge. (“sandy Acacia”) belongs
to Leguminosae family and Ammodendron genus. It
looks like a shrub (in juvenile state), later (after 3-4
years) it turns into tree that in mature state can reach of
height 2-3 m and maximal height – up to 8 m [3].
Astragalus villossimus Bge. belongs to Leguminosae
family and Astragalus genus, Cercidothrix subgenus,
Ammodendron Bge section. It is a branched shrub with
height up to 70 cm and surface with wh ite hairs. Woody
branches are long and thick; they are covered with light
splintered bark. Annual stems (branches) are either very
short or long (6-23 cm length), they twine round and,
they are fluffy and white [3].
Astragalus unifolio latus Bge. b elong s to Legumino sae
family, Astragalus genus, Cercidothrix subgenus, Am-
modendron Bge. section. It is a shrub with woody stem
(trunk). Annual branches are long, branchy, 13-22 cm
length, cylindrical, closely twisted, white [3].
2.2. Nodule Sampling and Isolation of
Nodule Bacteria
Nodules were sampled during intense blossoming and
repeatedly rinsed with sterile distilled water, then they
were sterilized in 96% ethanol with followed th eir burn-
ing in open flame. Bacterial isolates of nodule bacteria
were isolated from nodules and purified in correspon-
dence with common methods [4,5], then they were
re-sowed to medium of the following co mposition (g/L):
glucose – 5, sucrose – 5, К2НРО4 – 0.5, КН2РО4 – 0.5,
MgSO4•7H 2O – 0.5, CaSO4 – 0.2, pea – 50, agar – 20,
water distilled – up to 1 L, pH – 6.8-7.0 (pea was boiled
during 1 hour and th e medium w as pr epared on th e basis
of pea’s broth).
2.3. Treatment and Germination of Seeds
Seeds of plants were treated with 96% H2SO4 during
40 min for Ammodendron conollyi and 20 min for both
Astragalus villossimus and Astragalus unifoliolatus,
whereupon the seeds were repeatedly rinsed by sterile
distilled water and transferred to 1 % water agar with
moisture enough for swelling of seeds and their germi-
nation in Petri dishes. Next 1-2 da ys after treatment with
acid the seeds swelled and scarification of seed’s coat
was conducted, then opened appeared roots of seeds
were submerged into sterile agar for fast development of
seeds` roots in Petri dishes that were introduced into
thermostat at 30°C. After 2-3 days from the starting of
seed germination the seedlings with developed roots
were transplanted in potting substrate for their further
development and growth.
2.4. Preparation of Potting Substrate for
The ability of desert leguminous plants seedlings to
grow in ascending flow of nutritive solution in tubes
(microvegetation experiments) was tested. Tubes were
filled for one third volume by sterile nutritive solution
and filter paper strips were introduced into the tubes, the
upper end of which was fixed on the height 1-2 cm from
the surface of nutritive solution in tubes, but the lower
end of strips was submerged into the nutritive solution
for formation of ascend ing flow of th e nutritive solution .
Composition of the nutritive solution for plant growing
was following: MgSO4•4 H 2O – 5 mM, K2SO4 - 10 mM,
CaCl2•2H2O – 1 mM, phosphate buffer (NaH2PO4 +
Na2HPO4, pH 6.5) – 15 mM, microelements –0.05 ml/L
of medium, Fe source –5 mM; microelements composi-
tion (g/L) – H3BO3 – 17.16, MnSO4 – 7.2, ZnSO4 – 1.32,
CuSO4 – 1.65, Na2MoO4 – 0.12 [4]. The seedlings were
put on the upper end of strips and inoculated by nodule
As vessels for potting substrate 2-Litre volume black
perforated bags (vegetation experiments) were used.
Z. S. Shakirov et al. / Agricultural Sciences 1 (2010) 24-38
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/as/
They were filled by combinations of substrates (ver-
miculite, peat, sand, soil) that were sterilized at 1 at-
mosphere during 30 min.
2.5. Nodulation Test
Ten strains of every nodule bacteria isolated from Am-
modendron conollyi (AC1-1, AC2, AC4-1, AC8-1, AC 11,
AC12-1, AC13-1, AC15, AC18-1, AC21), Astragalus
villossimus (AV1, AV2, AV3, AV6-1, AV8-1, AV9,
AV9-1, AV26-1, AV30, AV36-1) and Astragalus unifoli-
olatus (AU2-1, AU3-1, AU4, AU7, AU17-1, AU20-1,
AU23, AU28, AU30-1, AU30-2) were selected for in-
oculation. Strains of Azorhizobium caulinodans ORS571
(University of Nottingham, Centre for crop nitrogen
fixation) and CXM1 (the Russian industrial strain for
alfalfa inoculation) were also used. The bacteria were
grown in 2 % hard (agar-containing) medium with pea’s
broth during 3 days at 28°C. The plant seedlings were
sowed into bags on the depth of substrate 2-3 cm and
further there was done an inoculation of planted seed-
lings with 5 ml of culture nodule bacterial suspension
(109 cells/ml) per each variant and simultaneous irriga-
tion with nutritive solution.
2.6. Determination of Nitrogen-Fixing
Nitrogen-f ixing activity was estimated by the acety-
lene-reductase activity (ARA) assay described by Hardy
[5]. The plant samples (with root nodules) were washed
with sterile water and transferred into 60 ml capacity tube
fitted with airtight rubber stoppers. Acetylene (10 volume
%) was injected and the tube s were incubated at 30°C for
24 hours. The data was the mean of three replicates. The
samples without acetylene were used as control. The
quantitative estimation of ethylene gas produced in the
samples was measured on a gas chromatograph (Hewlett
Packard-5890) using a “Porapak-N” column and a
H2-flame ionization detector (FID). The acetylene-
reductase activity of the plants was expressed as ppm
C2H4 tube/hour.
2.7. Soil Moisture
Soil moisture was measured with help of both
TDR-method (electrical conductivity of soil) and gra-
vimetric method (weighing of soil samples). The soil
was taken from 0-30 cm horizons and was dried twice at
2.8. Drying of Plant Biomass
Drying of plant biomass for determination of biomass
was conducted at 70°C during day.
2.9. PCR Amplication of the 16S rRNA
The 16S rRNA gene from nodule bacteria of Ammoden-
dron conollyi (АС1-1, АС8-1, AC11, AC15, АС21), As-
tragalus villossimus (AV1, AV3, AV6-1, AV8-1, AV9)
and Astragalus unifoliolatus (AU3-1, AU7, AU17-1,
AU30-1, AU30-2) was amplied using universal primers
1070f (59-ACGGGCGGTGTG- TAC-39) and 1392r
PCR mixture contained the following: 10 pmol each
primer, 200 M dNTPs, 1U Tag DNA polymerase,
100-200 ng genomic DNA and Taq polymerase buffer in
a nal reaction volume of 50 l. The DNA thermal cy-
cler used for PCR amplication was programmed as
follows: an initial extensive denaturation step at 94°C
for 5 min; 30 cycles of 94°C for 1 min, 53°C for 1 min
and 72°C for 1.5 min; and a nal extension step at 72°C
for 10 mi n.
2.10. Phylogenetic Analysis
The complete 302-343-bp 16S rRNA gene sequences
were compared with the sequences available in the
GenBank database using the standard Basic Local
Alignment Search Tool, BLASTn [6], at the National
Center for Biotechnology Information (NCBI) (http://blast.
ncbi.nlm.nih.gov/Blast.cgi). From the aligned sequences,
neighbor-joining dendrograms [8] were constructed with
the software MEGA version 4.0.2 [9]. The robustness of
the inferred trees was evaluated by 1000 bootstrap re-
2.11. Molecular-Genetic Investigations
Symbiotic NIF- and NOD-genes were determined in
nodule bacteria with help of correspondent probes -
Nif-probe (from Klebsiella pneumoniaea) and Nod-ABC
probe (from Sinorhizobium fredii) – by means of
dot-(Southern)-hybridization in nylon membranes [10].
Plasmids were visualized by the in-gel lysis method of
Eckhardt [11] as modified by Priefer [12].
2.12. Salinity Experiments
Plants were irrigated by the nutritive medium containing
100 mM, 200 mM, 300 mM and 500 mM NaCl,
with/without addition of 1 mM NH4NO3, up to satura-
tion of potting su bstrate with the medium and its leaking
from the bottom of perforated bags every 5th day.
2.13. Doubling Time of Bacterial Cells of
Nodule Bacteria
Nodule bacteria were grown in liquid ТУ medium at 30о
C in orbital shaker at 140 rpm/min and the increase of
bacterial biomass was measured on optical density
(photometrically). Obtained results were treated with
special computer program supplied by Sevilla University.
Composition of ТУ medium was as followed (g/L):
bactotryptone – 4 ; yeast extract – 2; CaCl2•6H2O – 1.3
or CaCl2•2H2O – 0.87; phosphate buffer (Na2HPO4 – 2.4;
Z. S. Shakirov et al. / Agricultural Sciences 1 (2010) 24-38
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/as/
KH2PO4 – 1.0), H2O – up to 1 L, pH – 6.8-7.0 [13].
2.14. Light and Electron Microscopy
Nodules of Ammodendron conollyi, Astragalus villos-
simus, Astragalus unifoliolatus were fixed in 2.5% glu-
taric aldehyde and 0.025 M potassium phosphate pH 7.2
during 4 hours at room temperature with further
post-fixation in buffered 1% osmium tetraoxide. Then
nodules were dehydrated by acetone and in a series of
alcohols with increased alcohol concentration and after
this they were embedded into araldite. Mid-sections of
nodules and ultra-thin sections were done in LKB-ultra-
microtome (Sweden), they were contrasted with 1%
uranyl-acetate and 0.8% led citrate prepared according
Reynhold and we re v iew ed in electron microscope “JEM
(Jeol)-10” (Japan) at accelerating voltage 60-80 kilo-
For study of sections in the light microscope the sec-
tions were fixed in 2% osmium tetraoxide. After this
they were coloured by mixture of equal parts of fuchsin
and methylene blue in 1% acetic acid. It should colour
preparations during 3-5 seconds, otherwise they will be
re-coloured and colour palette will change. In prepara-
tions nodule plant tissues are coloured in blue colour and
bacteria – in red colour. Bacterial preparations were
grown in TY hard medium (as mentioned above) during
18-20 hours. After growing up the bacterial cells 3-4
times were rinsed by 0.025 M potassium phosphate
buffer (pH 7.2) and centrifuged at 6,000 rpm during 20
min, and then they were fixed by 3% formalin solution
(preliminarily neutralized by K2CO3 beforehand 24
hours). Before their transfer to special metallic grids the
bacterial cells were fixed (contrasted) by 2% phospho-
rous-tungstic acid (pH 7.2) during 15 min and with help
of sprinkler the cells were dusted to metallic grids. All
electron microscopic samples were viewed in electron
microscope “JEM (Jeol)-10” (Japan) in Central Asian
Pediatric Institute (Tashkent, Uzbekistan).
3.1. Isolation and Screening of
Microbiological and Symbiotic
Properties of Bacterial Isolates
of Nodule Bacteria
151 bacterial isolates of nodule bacteria were isolated
from perennial leguminous plants of Kyzylkum Desert.
During growth on hard pea’s medium there were col-
ourless, whitish, greyish, faintly-grey and yellowish-
reddish, transparent, semi-transparent and muddy with
different degree of slime, convex, conical and spherical
colonies, but rough colonies occurred also. They differed
on growth rate – fast-growing bacteria prevailed and
slowly-growing isolates occurred also. The time of col-
ony appearance varied in average from 2 to 4 days (in
slowly-growing ones — from 4-5 days and more). Bac-
terial isolates, isolated from Astragalus villossimus, were
mainly represented by fast-growing bacteria, while bac-
terial isolates, isolated from from Ammodendron conollyi
and Astragalus unifoliolatus, were represented by equal
halves of both fast-grow ing bacteria and slowly-growing
bacteria. During analysis of colonies and their slime pro-
duction ability when their growing on the pea’s agar it
was noted that there was some correlation between ap-
pearance of colonies and their size; fast-growing bacte-
rial isolates formed bigger colonies with middle slime
production ability. The most of colonies, that had typical
(characteristic features for nodule bacteria), they were
greyish-white, semi-transparent, with abundant slime
production. Bacterial colonies were mainly conical and
they had convex profiles with even colony edges, that
probably allows to suppose that they are S-forms of
nodule bacteria. But, at the same time, there were also
rough colonies, semi-transparent with uneven edges that
perhaps can be considered like as R-формы [14]. After
study of morphological-physiological properties and
selection for growth rate (appearance of colonies in Petri
dishes with medium for cultivation of bacteria) about 50
perspective isolates were selected. Analysis of cells
growth of selected strains showed that fast-growing and
moderately-growing strains occurred among them that
had doubling time from 20 to 45 min. The presence of
salt in TY medium increased doubling time of strains -
by в 1.4-1.9 times in the presence of 0.75 M NaCl in
comparison with control and by 1.6-4.0 times in the
presence of 1 M NaCl (Ta b le 1 ). At higher salt concen-
trations in medium composition the differences in
salt-resistance became even more expressed: AV9-1 and
AV30 strains lost an ability to grow, doubling time of
AV3 and AV8-1 were by more than 26 times higher than
control, while AV9 strain was less sensitive to salt and it
had doubling time that exceeded control one only by 2
times. At salt content 1 .75 M NaCl AU7 and AV 3 strains
lost a growth ability and in the rest of strains the increase
of doubling time was observed. Finally, only AC11,
AC15 and AV9 strains grew at 2 M NaCl, where AV9
strain showed doubling time that exceeded control only
by 3 times, but growth of C21 strain was extremely slow.
All strains were able to grow in the range of tempera-
tures 12-40°C, and AV9 strain – at 45°C. Except for AV9
strain, all the rest strains grew in agar at 8°C (Ta ble 2).
During study of growth ability at different values of pH
(from 4.0 to 11.0), many strains were able to grow ex-
cept AV8-1 and AV9-1 strains that lost growth ability at
pH 4.0. AС11 strain grew only starting from pH 6.0, and
AU30-2 strain was able to grow on ly at pH values lower
than 10.0.
Testing of strains for resistance to antibiotics showed
Z. S. Shakirov et al. / Agricultural Sciences 1 (2010) 24-38
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/as/
Tabl e 1. Effect of salt on growth. Bacteria were cultivated in TY medium containing 0.04 (control), 0.75, 1.0, 1.5, 1.75 or 2.0 M
NaCl. Cultures were maintained in an orbital shaker at 30oC.
Isolate Control
td (h)(a) 0.75 M
td (h) 1.0 M
td (h) 1.5 M
td (h) 1.75 M
td (h) 2.0 M
td (h)
1.29 (1.6)(b)
2.51 (1.9)
1.06 (1.5)
1.00 (1.5)
1.29 (1.5)
1.46 (1.7)
1.08 (1.4)
1.66 (1.5)
1.31 (1.6)
1.44 (1.9)
1.64 (1.8)
1.98 (1.6)
1.13 (1.5)
1.44 (1.7)
1.29 (1.6)
1.72 (2.1)
3.17 (2.4)
1.72 (2.4)
1.51 (2.2)
2.01 (2.4)
2.08 (2.5)
2.52 (3.3)
2.6 (2.4)
1.75 (2.2)
2.94 (3.9)
2.32 (2.5)
5.04 (4.0)
1.20 (1.6)
3.16 (3.8)
2.12 (2.6)
4.27 (5.2)
10.33 (7.9)
5.45 (7.6)
5.12 (7.5)
15.57 (18.5)
8.85 (10.5)
11.92 (15.5)
12.04 (11.1)
7.46 (9.2)
20.19 (26.6)
7.04 (7.6)
37.62 (29.6)
1.50 (2.0)
11.58 (14.1)
13.68 (10.4)
19.11 (26.5)
10.38 (15.3)
33.44 (39.8)
42.13 (54.7)
45.60 (42.2)
24.27 (30.0)
11.04 (11.9)
150.48 (118.5)
NG(c )
19.8 (15.1)
66.88 (92.9)
2.26 (3.0)
(a) Doubling time; (b) td: td of control; (c) No detectable growth.
AС(1) – nodule bacteria isolated from Ammodendron connollyi nodules;
AU(2) – nodule bacteria isolated from Astragalus unifo liolatus nodules;
AV(3) – nodule bacteria isolated from Astragalus villossimus nodules.
Table 2. Relevant characteristics of the isolates described in previous.
Isolate td(a)
(h) NaCl
Plasmid profile
(kbp) Antibiotic
Resistance Growth
temp., oC Growth
pH Melanin pro-
duction Colony
370, 515
370, 515
118, 370, 515
370, 515
370, 515
370, 515
370, 515
370, 515
118, 370, 515
118, 370, 515
370, 515
118, 370, 515
118, 370, 515
118, 370, 515
370, 515
Amp, Km, Str, Tc
Amp, Tc
Amp, Km, Str, Tc
Amp, Tc
Amp, Tc
Amp, Tc
Amp, Cm
Amp, Cm, Km, Str
(a) Doubling time in TY liquid medium at 30°C.
(b) Highest concentration of NaCl in TY liquid medium at which growth was observed (see Table 1).
that amount of colonies decreased somewhat in the
presence of chloramphenicol (and in some cases in the
presence of other antib iotics), but in all strains the resis-
tance to antibiotics was observed. The ability to grow in
dishes with ampicillin was the most widespread in all
strains, it was followed further with ability to grow in
the presence of tetracycline. AU7 and AU30-1 strains
grew in the presence of four of the antib iotics (ampicillin ,
kanamycin, streptomycin, tetracycline, Table 2).
During identification of Nod- and Nif-genes in se-
lected bacterial isolates with help of dot-hybridization
and specific probes the positive hybridization reflexes
were obtained. In this study of selected bacterial isolates
for salt-resistance about 30 perspective strains of nodule
bacteria were revealed which were able to grow in the
presence of 1 M NaCl and more (up to 2 M NaCl). Dur-
ing study of plasmid profile of strains from 2 (in nodule
bacteria strains of Ammodendron conollyi and Astraga-
Z. S. Shakirov et al. / Agricultural Sciences 1 (2010) 24-38
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/as/
lus unifoliolatus) to 3 megaplasmids (in Astragalus vil-
lossimus nodule bacteria strains) with molecular masses
118, 370 and 515 kB correspondingly (Table 2).
Melanin formation is a phenotypic feature which can
also help in identification of closely-related rhizobia.
Screening of isolates on this feature was conducted in a
way of detection of diffusing dark-brown pigment that is
produced with cells grown in Petri dishes containing TY
medium with added tyrosine and copper [15]. The fast
and abundant formation of melanin was observed in
U30-1 strain and almost the same like it took place in
V30 strain; lesser formation of melanin was observed in
AU7, AU17-1, AU30-2, AV1 and AV6-1 strains. The rest
of strains did not produce diffusing dark pigment and
they were considered lik e as Mel–strains (Table 2).
All strains grew in Luria-Bertani medium [12]. Test-
ing of their growth in TY medium showed at least 6 dif-
ferent ty p es of colonies:
Type 1: Middle size, round, convex, light and white.
Type 2: Young colonies like as in type 1, they b ecome
yellow later.
Type 3: Young colonies like as in type 1, they b ecome
yellow-brown later.
Type 4: As type 1 , but smaller in sizes.
Type 5: Middle size, round, dim and curly.
Type 6: Young colonies like as in type 1, later they
become darker than type 3.
All above-mentioned investigations have confirmed
that the selected isolates are nitrogen-fixing nodule bac-
teria which are able in the wide ranges of pH and tem-
3.2. Germination of Plant Seeds, Nodulation
Test (Direct Inoculation and
After study of microbiological properties of bacterial
isolates of nodule bacteria next stage of investigations
was a study of their symbiotic properties. It was neces-
sary for this to conduct a search of optimal conditions
for seed germination of wild desert leguminous plants
(Ammodendron conollyi tree, Astragalus unifoliolatus
and Astragalus villossimus shrubs), their growth and
development as well as nodulation process of these
plants roots upon inoculation with selected isolates
(strains) of nodule bacter ia.
No prolonged treatment of plant seeds with concen-
trated sulphuric acid (Ammomodendron conollyi during
3 hours) nor their moistening in water during several
days did not lead to increase of seed germination output
of desert xerophyte plants higher than 10-15% (in natu-
ral conditions 5-10% output of seed germination is ob-
served) [16]. In literature there are data about efficiency
of scarification of only hygrophilous foot-hill species of
acacia [17].
As a result of search the combination of treatment
with sulphuric acid and scarification of root part of seeds
was found when almost total (about 90-95%) seed ger-
mination of xerophyte desert wild leguminous plants in
sterile conditions was obtained. The tasks of investiga-
tions included growing up of seedlings up to mature state
and study of root nodul ation of t he grown up plant s.
First of all it was necessary to obtain normal vegeta-
tion of plants (that would be favourable for their nodula-
tion). During study of nodulation it was important to
clear up two matters:
1) to determine the belonging (biological test) of iso-
lated nodule bacteria to their maternal host-plants and,
thus, to confirm their originality (“direct inoculation”);
2) to examine a host specificity of nodule bacteria to-
wards other (non-maternal) host plants (“cross-inocu-
During search of optimal conditions for plants grow-
ing up the following types of potting substrates were
1) Microvegetation experiments:
Strips of filter paper in agronomic tubes (60 ml) with
ascending flow of nutritive solution for plants in sterile
Veg etation experiments:
2) Sterile vermiculite impregnated with nutritive solu-
tion for plants in pots (0.7 L) in greenhouse.
3) “Light structure” in bags (sterile, 2 L) – 1 part peat:
1 part vermiculite: 1 part soil: 2 parts sand.
4) “Structure close to natural conditions” (in bags) – 3
parts sand: 1 part vermiculite.
5) Natural conditions (fine-grained sand).
6) Field conditions (soil).
In microvegetation experiments during growing and
inoculation of plants seedlings with nodule bacteria on
filter paper strips after 1 month both suppression and
stoppage of their growth and development were ob-
served. In vegetation experiments at growing on the 2nd
type of substrate the height of plants was good, but after
45 days from the starting of experiment the stoppage of
growth and development was observed also. In both pre-
vious experiments there was no nodulation. Probably,
plants being xerophytes and in natural conditions having
only low moisture in the upper soil horizon (0-30 cm),
they did not “manage” with moisture excess in two pre-
vious variants, because the presence of available mois-
ture exceeded the transpiration of water by desert plants.
In this connection it was necessary to create the condi-
tions close to natural co nditions. To achieve this aim the
3rd type with sand prevalence was taken together with
“light” potting structures (vermiculite wou ld retain more
moisture) which would accelerate drainage and removal
of water from potting substrate, during this the moisture
was supported within range 15-20 %. After 2.5 months
(from moment of seedling planting up to the mature state)
of growing in the 3rd type the height and development of
plants corresponded to 1-2 yearly plants (15-30 cm) that
were observed in nature. But, there was not nodulation
Z. S. Shakirov et al. / Agricultural Sciences 1 (2010) 24-38
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/as/
(there were only single nodules). During use of the 4th
type of substrate, where sand was dominating part of
substrate and conditions were close to natural ones, the
intense growth and development of plants was observed.
After 2 months of growing the nodulation was found in
all plants, and thus, the search of optimal conditions for
total nodulation finally has crowned with success. But,
nodulation after inoculation with Azorhizobium cauli-
nodans ORS571 and CXM1 was not observed. Further,
all experiments on study of plant nodulation were con-
ducted in sand, the 5th type of potting substrate.
As it is seen from Figure 1(a) and Ta b le 3 the nod-
ules of Ammodendron cono llyi, formed after inoculation,
were pink and large (up to 7 mm in diameter). In vari-
ants of Ammodendron conollyi plant inoculation the av-
erage nodule number per plant was 1-5 and their bio-
mass comprised 4.5-27.5 mg. At the same time the ni-
trogen fixation intensity did not influence essentially on
the height of shoot part and length of roots. High nitro-
gen-fixing activity was recorded in direct inoculation
with own nodule bacteria, isolated from Ammodendron
conollyi (AC1-1, AC2, AC12-1, AC18-1, AC21). The
cross-inoculation of Ammodendron conollyi plants by
nodule bacteria isolated from Astragalus villossimus, has
led to visibly low nitrogen-fixing activity in comparison
with results of direct inoculation of the same plants with
own nodule bacteria. It should be noted that cross-
inoculation of Astragalus villossimus plants by Am-
modendron conollyi nodule bacteria (AC8-1, AC11,
AC15) gave comparatively higher values of nitrogen
fixation than their direct inoculation with own Astraga-
lus villossimus nodule bacteria, during this there was an
increase of nodules amount in Astragalus villossimus
plants. As it is seen from Table 3, AС1-1 strain was
more specific towards maternal Ammodendron conollyi
plants during direct inoculation than Astragalus villos-
simus plants during their cross-inoculation. It is interest-
ing to note that, on the whole, nitrogen-fixing activities
of Ammodendron conollyi plants inoculated with direct
inoculation and cross-inoculation were in several times
higher than nitrogen-fixing activities of inoculated As-
tragalus villossimus plants and Astragalus unifoliolatus
plants. Highly-efficient symbiosis (under term “symbio-
sis efficiency” is meant the increase of biomass of in-
oculated plants in comparison with biomass of non-in-
oculated plants) in Ammodendron conollyi plants was
observed during inoculation with such nodule bacteria as
AC1-1, AC8-1, AC11, AC12-1, AC18-1, AV1, AV8-1,
AV9-1, AV30, when efficiency (biomass) of shoot part
increased from 143.2 to 168.6 % in comparison with
control. At observation of Astragalus villossimus plants
(Table 3) during direct inoculation with AV1, AV2, AV3,
AV9, AV26-1 and cross-inoculation by AC11, AC15,
AC21 and AU30-1 numerous nodules were formed (av-
erage nodule number 10-29 and 1-2.5 mm size, their
colour varied from weakly-reddish to white and greenish)
(Figure 1(b)). High nitrogen-fixing activ ity of symbiosis
of inoculated Astragalus villossimus plants was observed
during direct inoculation with their own nodule bacteria
AV2, AV3, AV9, AV30 and AV36-1. At the same time
during cross-inoculation of Astragalus v illossi mus plan ts
with Astragalus unifoliolatus nodule bacteria the com-
paratively low nitrogen-fix ing activity was obtained th an
during direct inoculation. Efficiency from 121.5 to
128.4% was obtained during inoculation with AV1, AV3,
AV8-1, AV9 and AC8-1 strains.
During inoculation of Astragalus unifoliolatus plants
the highest nodule number – up to 41 – was recorded,
nodules were numerous and small (0.5-1.5 mm) (Figure
1(c)). Astragalus unifoliolatus plants, inoculated with
their own nodule bacteria (direct inoculation) had con-
siderably low nitrogen fixation than under cross-inocu-
lation by nodule bacteria of Astragalus villossimus. Hi gh
efficiency of Astragalus unifoliolatus symbiosis was
observed at direct inoculation with AU23, AU28, AU30-1
and cross-inoculation by AV1, AV2, AV6-1, AV8-1 and
AV9-1 strains.
3.3. Phylogenetic Analysis of the 16S rRNA
Gene of Nodule Bacteria Strains
For taxonomic identification and creation of phyloge-
netic tree 15 salt-tolerant bacteria were re-isolated again
from nodules formed on the roots of desert leguminous
plants which were grown in sterile vegetation experi-
ment. Results of BLAST-analysis for sequence of 16S
rRNA gene of bacteria showed that studied bacteria were
related to Alphaproteobacteria and Betaproteobacteria
classes. The nucleotide sequence of 16S rRNA gene of
АС1-1, АС8-1, АС21, AV1, AV3, AU3-1, AV8-1, AV9
and AU30-1 strains matched by 96-97% together with
analogous genes of Rhizobium sp. GGNM 66; the genes
of AC15, AU 17-1 , A U30 -2 and AU 7 bacteria w er e id en-
tical by 89-97% to nucleotide sequ ences of Burkholderia
cepacia NBRAJG97 species and the genes of AC11 and
AV6-1 strains were identical by 95% and 98% to genes
of Achromobacter xylosoxidans species. On phyloge-
netic tree the studied bacteria could be grouped into 4
clusters (Figure 2): AU7 strain (Astragalus unifoliolatus)
formed the 1st cluster, the 2nd cluster included AC11
(Ammodendron conollyi) and AV6-1 (Astragalus villos-
simus) strains, the 3rd group was created by AC15 (Am-
modendron conollyi), AU17-1 and AU30-2 (Astragalus
unifoliolatus) strains, and, at last, the 4th big group was
formed by АС1-1, АС8-1, АС21 (Ammodendron co-
nollyi); AV1, AV3, AV8-1, AV9 (Astragalus villossimus),
Astragalus unifoliolatus (AU3-1; AU30 -1) n odule bacte-
ria strains. It may conclude from obtained results that
nucleotide sequences of 16S rRNA gene of studied bac-
teria that were highly identical between themselves
within group and also bacteria isolated from each cor-
Z. S. Shakirov et al. / Agricultural Sciences 1 (2010) 24-38
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/as/
(a) (b) (c)
Figure 1. Nodulation of desert leguminous perennial plants: (a) Inocu-
lation of Ammodendron сonollyi plant with AС11 strain; (b) Inoculation
of Astragalus villossimus plant with AV8-1 strain; (c) Inoculation of
Astragalus unifoliolatus plant with AU30-2 strain.
Figure 2. Phylogenetic tree based on the 16S rRNA gene strains of nodule bacteria
from desert leguminous perennial plants. The branching pattern was produced by the
neighbour-joining method. The GenBank accession numbers for the sequences used
are indicated in parentheses. Symbionts of desert legume plants are shown in bold
Z. S. Shakirov et al. / Agricultural Sciences 1 (2010) 24-38
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/as/
respondent leguminous host plant were related to both
Alpha class and Bet aproteobacteria class.
3.4. Study of Influence of Drought and
Salinity on Symbiosis of Desert
Leguminous Plants
Experiment on study of influence of drought on xero-
phyte desert leguminous plants was carried out in
greenhouse in soil (field conditions, the 6th type) during
2.5 months. Plants, inoculated by correspondent nodule
bacteria (AC21-1, AV1, AU30-1), were planted in rows
into the soil and the first irrigation was done to equalize
of soil moisture under the plants and one week later the
soil moisture was within range 15-20%. Afterwards the
further irrigation of soil (the area which did not include
the square which was not occupied by plants) was done
from one side across the rows of planted plants. For
whole period of plants gr owing there w ere 3 (three) such
irrigations in order to obtain a gradient of soil moisture.
The gradient of so il moisture was obtained due to that
irrigation was conducted from the one side (side of the
first plants from each row), but on the contrary side of
the last plants (according to order of row) there were
constantly dried dehydrated soils. As a result the gradi-
ent of soil moisture in the range from 4 to 7% was ob-
tained. During growing of plants the gradient of plants
height – from the side of conducted irrigation (the be-
ginning of row) to the contrary side (end of row) – was
obtained. Thus, model system for study of plants drought
resistance was created. From Figure 3 it is seen that
from the moment of starting of experiment up to plants
harvesting the gradient of soil moisture comprised from
3.8 to 6.41%. The height and biomass of inoculated
plants also decreased during this. Maximal height and
biomass of inoculated plants of Ammodendron conollyi
were 21 cm and 2320 mg (at 6.41% soil moisture), but at
3.8% moisture the height decreased by 4 times (up to
4.5 cm) and biomass - by 11 times (203 mg). Analogous
situation was observed in Astragalus unifoliolatus. For
mation of nodules in this experiment was not observed.
As our experiments showed, even under insignificant
increase of soil moisture from 3.8 to 4.32% (by 0.52%),
the biomass of all studied plants increased by 2-3 times,
and under further increase from 3.8 to 6.41% (by 2.61%)
the biomass of all plants increased by 10-15 times. At
the same time it should be noted that under increase of
moisture from 5.48 to 6.41% (by 0.93%) the biomass of
all plants increased only by 1.5 times. From here one
may conclude that 6.41% of soil moisture for these
plants is emergent value which is close to stationary
level of moisture that is optimal for growth and devel-
opment of these plants (Figure 3). Analogous results
were received for plants Astragalus villossimus as well.
For conducting of exp eriment on salin ity 20-da ily sap-
lings of these plants were taken, that further were sub-
jected to salt stress under their irrigation with nutritive
medium for plants that contained 100 mM, 200 mM, 300
mM and 500 mM NaCl for each irrigation. Experiment
was conducted in two modifications: a) without added
nitrogen, and b) on the background of added nitrogen (1
mM NH4NO3). Plant inoculation was done with the cor-
respondent nodule bacteria during seedling plantings
into the bags: Ammodendron conollyi – by AC8-1 strain,
Astragalus villossimus – by AV9 strain and Astragalus
unifoliolatus – by AU30-1 strain. As it is seen from Fig-
ure 4, under salinity treatments with 100 and 200 mM
NaCl both in the absence and in the presence of added
nitrogen (1 mM NH4NO 3) as a sole nitrogen source, the
survival of Ammodendron conollyi inoculated plants
remained almost entirely – 75-100%, and average green
biomass of 1 plant increased by 16.2% in comparison
with control. Further increase of NaCl concentration up
Dry biomass, mg
Moistu re, %
Astraga lus
un ifoliola tus
Astraga lus
Figure 3. The influence of drought on dry biomass of perennial desert
leguminous plants.
6.41 5.48 4.86
Z. S. Shakirov et al. / Agricultural Sciences 1 (2010) 24-38
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/as/
to 500 mM negatively influenced on yieldness and sur-
vival of Ammodendron conollyi plants under both pres-
ence and in the absence of nitrogen source, the survival
decreased up to 25 and 50% accordingly (Figure 4).
Study of salinity influence on Astragalus species
showed that at 100 mM NaCl the survival of Astragalus
villossimus was 46.1% and for Astragalus unifoliolatus
66.7%. It is interesting to note that in spite to decrease of
survival of Astragalus villossimus in the presence 100
mM NaCl, the average green biomass (yieldness) of one
plant increased by 40% in comparison with control, and
at 300 mM NaCl the yieldness got equalized with con-
trol. At salinity from 100 to 500 mM NaCl both in the
absence and in the presence of nitrogen the survival of
Astragalus villossimus and Astragalus unifoliolatus
regularly decreased. Nodulation was observed in Am-
modendron conollyi in amount 1-2 nodules /1 plant at
100 mM NaCl, while in Astragalus villossimus (11.0 -
0.25 nodules/ plant) – up to 200 mM NaCl and for
Astragalus unifoliolatus (12.5 – 1 nodules/1 plant) – up
to 300 mM NaCl.
Individual plants of Ammodendron conollyi and As-
tragalus unifoliolatus were resistant to 100-200 mM
NaCl, these salinity values did not influence practically
on normal growth and development, while in Astragalus
villossimus such concentration of salt-resistance was up
to 100 mM NaCl. This testified about individual adapta-
tion of studied plants subjected to salt stress. Further
study of influence of NaCl on growth and development
of Ammodendron conollyi, Astragalus villossimus and
Astragalus unifolilatus showed that salinity up to 500 mM
considerably decreased yieldness and survival of plants,
but it also indicated to the fact that th e last concentration
of NaCl was not critical value for salt-resistance of stud-
ied plants.
3.5. Light/Electron Microscopy of Nodule
Bacteria and Transections of Nodules
During electron-microscopic study of typical samples of
Survavility, %
Salinity, mM NaCl
con ollyi
Figure 4. The influence of salinity on survival of perennial desert legu-
minous plants (in the presence of nitrogen source).
(a) (b) (c)
Figure 5. The desert nodule bacteria in free-living state: (a) – Astragalus
villossimus’s nodule bacterium AV1 strain (magnification 5,000); (b) –
Ammodendron conollyi’s nodule bacterium AC8-1 strain (magnification
5,000); (c) – Astragalus unifoliolatus’s nodule bacterium AU30-1 strain
(magnification 6,000).
0.17 mk
0.17 mkm 0.2 mk
Z. S. Shakirov et al. / Agricultural Sciences 1 (2010) 24-38
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selected samples of nodule bacteria the unipolar mono-
trichous mobile rod-shaped forms of bacterial cells were
revealed (Figure 5). Under differential colouring of
nodule transections (preparations of light microscopy) of
desert leguminous plant – Ammodendron conollyi tree or
“sandy acacia”, Astragalus villossimus and Astragalus
unifoliolatus shrubs it was possible to observe the simi-
larity in organization and deve l opm ent of speci fi c cel l ular
structures, and also some difference between them. So,
for example, under inoculation of Astragalus villossimus
plants with AV1 strain it took place a wide and intensive
colonization of internal plant cells of nodule (located to
the nodule’s center) by bacterial cells of this strain. As it
is shown in Figure 6, after branchy (coloured in pink
colour) bacterial net in o uter covering cells of nodule th e
zone of formation and development of centres of infec-
tion threads followed further which finally (at least) led
to formation of completely colonized (densely-colonized)
deep internal plant cells of Astragalus villossimus nodule
by bacterial cells of nodule bacteria.
Under higher amplifications it was possible to see a
formation of “stairs-like” structures that seemingly are
intracellular infection threads from which nodule bacteria
further released into plant nodule cells and are trans-
formed into non-mobile symbiotic forms – “bacteroids”
within plant cells. It is important to note that infection
threads move through and develop along perimeter of
plant cells, i.e. along membranes of internal cells of plant
nodule cells [18,19]. The microscopy results concerning
to influence of salinity effect on the formed symbiosis
showed that number of nodule bacteria cells internally-
colonizing plant nodule decreased already at 100 mM
NaCl in comparison with control (Figure 6(g)), and un-
der the further increase of NaCl salinity concentration u p
to 200 mM the nodule bacteria colonization of nodule
internal spaces reduced practically (Figure 6(h)).
Figure 6. Light microscopy of nodule transection of Astragalus villossimus
plant, inoculated with its own Astragalus villossimus AV1 nodule bacteria
strain: (a) – General picture of longitudinal part of nodule (magnification
148); (b) – Part of general picture taken under higher magnification (magni-
fication x 315); not only central plant cells of nodule densely-colonized by
bacterial cells, but also outer covering plant cells of nodule are visible, the
zone of formation and development of infection threads centres follows
further; (c) – Higher magnification of the same part (magnification 530); the
outer bacterial net on plant bark (cortical) part is visible; (d) and (e) –
Higher magnification of the infection thread zone (magnification 974);
“stairs-like” intracellular infection threads are visible (they are shown by
arrows) and perhaps some elements of inter-cellular infection threads
(spherical-like black-red structures) ; (f) – The highest magnification (mag-
nification 2835); development and distribution of thick infection threads
along plant membranes of nodule and through nodule cells with releasing
bacterial cells of nodule bacteria; (g)-(h) – The light microscopy of Astra-
galus villossimus nodule section of plant grown at salinity 100 mM NaCl (g)
and 200 mM NaCl (h).
(a) (b) (c) (d)
(e) (f) (g) (h)
Z. S. Shakirov et al. / Agricultural Sciences 1 (2010) 24-38
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For study and observations of more fine structural prop-
erties of legume-rhizobial symbiosis of desert legumi-
nous plants and selected nodule bacteria the elec-
tron-microscopic preparations were used. In the whole,
bacteroids were represented by polymorphic structural
forms – from globular (spherical) up to mace-like and
many other random-shaped forms (Figures 7(a)-(c)). As
to infection threads, the use of electron microscopy en-
abled to observe them more in detail. Infection threads
formed in both inter-cellular free spaces between plant
nodule cells (Figure s 7(d) and (e)) and within plant nodule
cells (Figures 7(f)-(i)). The form of infection threads also
varied – from roundish to rectangular (Figures 7(d)-(i)).
Thus, as a result of legume-rhizobial symbiosis the
structural reconstructions of both nodule bacteria and
legume host plant took place (occurred). Under this not
only penetration of bacteria into plant nodule cells and
their colonization by bacteria occurs, but also transfor ma-
tion (transition) of free-living uniform mobile cells of
nodule bacteria into symbiotic polymorphic non-motile
forms (bacteroids). Penetration of nodule bacteria into
correspondent host plant is realized through special struc-
ture – infection thread – that moves forward and distribu-
tion of which occurs through inter-cellular free spaces
between plant nodule cells within thei r volum es [20,21].
(a) (b) (c)
(d) (e) (f)
(g) (h) (i)
Figure 7. Electron microscopy of nodules sections for desert legume plant
Ammodendron conollyi: (a) (AV1), (b) (AC8-1 ) and (c) (AU30-1) – poly-
morphism of bacteroids, (d) (AV1) and (e) (AC8-1 ) – intercellular infec-
tion threads, (f)-(i) – intracellular infection threads. Localization of infec-
tion threads is indicated by arrows.
0.7 mkm 0.5 mkm 0.5 mkm
2 mkm 2 mkm 2.5 mkm
2 mkm 2 mkm 2.5 mkm
Z. S. Shakirov et al. / Agricultural Sciences 1 (2010) 24-38
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From obtained results one can conclude that there was
correlation between nitrogen-fixing activity and effi-
ciency of symbiosis by nodule bacteria AС1-1, AC12-1,
AC18-1, AC8-1, AV1, AV3, AV9, AU23 and AU30-1.
On the basis of this data one can estimate the specificity
of nodule bacteria towar ds legum inous host plant. Duri ng
this it is necessary to consider both direct host nodula-
tion specificity (belonging of nodule bacterium to defi-
nite maternal host plant from which this bacterium
originates from) and wide host/cross host nodulation
specificity (ability of this bacterium to form nitro-
gen-fixing symbiosis with other non-maternal host
plants). So, under analysis of nitrogen-fixing activity and
nodulation in Ta ble 3 one can note that nodule bacteria
of Ammodendron conollyi have the highest host specific-
ity towards both their own maternal host plant and
also to other host plants (wide host specificity), and in
this connection they are the most perspective inoculants
for obtaining of efficient symbiosis in perennial desert
plants. Nodule bacteria of Astragalus villossimus have
the middle specificities towards to its matern al ho st plan t
and other plants. Nodule bacteria of Astragalus unifoli-
olatus have lower specificity towards own maternal host
plant and other plants. Thus, one can suppose that there
is some regularity between attaching of nodule bacteria
to concrete habitat and their specificity to come into
symbiosis with different host plants. Since the collection
of nodules of Ammodendron conollyi (they were sam-
pled from Kara-Kata, Kyzylkum Desert), Astragalus
villossimus (they were taken from Kyzylkum Desert
Biostation) and Astragalus unifoliolatus (they were
gathered from Yamandjar, Kyzylkum Desert) was con-
ducted in separate places which were distant from each
other on the distance 80-100 km, probably there were
separate populations of local nodule bacteria although
during expeditions the common habitats of Ammoden-
dron conollyi with Astragalus villossimus as well as the
areas of common inhabiting of Ammodendron conollyi
with Astragalus unifoliolatus occurred. But it was nec-
essary to collect nodules in places distant from each
other in order to have distinct groups of nodule bacteria
for their study. If Ammodendron conollyi plant can grow
in pure sands and semi-sand soils, then Astragalus vil-
lossimus plant grows in poorer dried soils and semi-sand
soils, while Astragalus unifoliolatus plant - mainly in
pure sands. Therefore, Astragalus unifoliolatus plant has
comparatively narrower area of habitat and maybe its
low specificity to other plants is determined with this
According to observations of some authors, Astraga-
lus genera form nodules by the 2nd year of their vegeta-
tion in natural conditions. At the same time, from expe-
ditional observations of both our botanists and us, the
nodules and roots of Ammodendron conollyi were brown
and black colors correspondently in natural conditions
(in our microvegetation experiments after 2.5 months of
growth the initially pink-colored fragile nodules and
white roots became dark-brown color; the appearance
and development of 2.5 monthly plants of Ammodendron
conollyi and Astragalus looked like as 1-2 yearly natur al
plants). Probably, due to increase of requirements in ni-
trogen by the 2nd year in natural conditions the plants
begin to form nodules that are necessary for intense
growth and development of young plants as well as their
adaptation to surroun ding environ mental conditions. Th e
studied strains of desert rhizobia are fast-growing rhizo-
bia, but they are slower than Azorhizobium caulinodans
(its generation time is 40-45 min [22]. They have 2
similar megalpasmids and some of them have additional
3rd megalpasmid, they do not form nodules in alfalfa.
Microbiological and molecular-genetic characteristics of
rhizobial symbiont of Sesbania revealed the availability
of 2 megaplasmids (300 and 450 MDa), generation
(doubling-time) time 2 hours 15 min and capability for
growth at 2% NaCl, sensitivity to antibiotics and capa-
bility for nodule formation in Vigna ung uiculata [23]. In
literature the rhizobia isolated from Astragalus are re-
lated to Mezorhizobium [24-26], but at the same time the
authors noted that the most of studied rhizobia differed
on DNA-homological groups from all known species of
rhizobia and one subgroup was a unique genetic line
[27]. Other authors on the basis of fingerpriniting noted
that rhizobia of Astragalus adsurgens were related to
Agrobacterium, Mezorhizobium, Rhizobium and Si-
norhizobium genera [28]. During study of rhizobia (Si-
norhizobium, Mezorhizobium), which cause nodulation
in leguminous tress of Africa and South America, it was
noted that there were both similarity and differences in
symbiotic determinants of rhizobia [29]. In our investi-
gations it was determined that nucleotide sequence of
16S rDNA gene of АС1-1, АС8-1, АС21, AV1, AV3,
AU3-1, AV8-1, AV9 and AU30-1 nodule bacteria strains
matched by 96-97% together with analogous genes of
Rhizobium sp. GGNM 66 species; the genes of AC15;
AU17-1; AU30-2 and AU7 89-97% were identical to
genes of Burkholderia cepacia NBRAJG97 species and
genes of AC11 and AV6-1 strains were identical by 95%
and 98% to the genes from Achromobacter xylosoxidans
species. In whole, according to phylogenetic tree the
evolutionary origin of studied bacteria is close each
other and they belong to both Alpha and Betaproteobac-
teria classes.
Thus, as a result of conducted work, the conditions for
study of growth and nodulation of desert leguminous
perennial plants have been chosen and found, the inter-
actions between nodule bacteria and perennial xerophyte
tree and shrubs have been determined. On the basis of
this it may conclude that model system of efficient ni-
trogen-fixing symbiosis “nodule bacterium-perennial
Z. S. Shakirov et al. / Agricultural Sciences 1 (2010) 24-38
Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/as/
Table 3. Influence of nodule bacteria on nodulation, ARA and yieldness of desert leguminous tree Ammodendron conollyi, legumi-
nous shrub Astragalus villossimus and leguminous semi-shrub Astragalus unifoliolatus.
Ammodendron conollyi Astragalus villossimus Astragalus u nifoliola tus
Strain Average
% Strain Average
% Strain Average
AC1-1 5±2.64 6.91±0.44 147.7 AV1 18±2.161.72±0.99121.5 AU2-1 19±2.0 0.47±0.21 62.5
AC2 5±3.0 5.59±0.25 108.9 AV2 17±2.162.52±0.73102.5 AU3-1 41±5.29 3.33±0.96 103.5
AC4-1 2±1.73 2.81±0.63 98.5 AV3 18±3.262.95±0.77122.4 AU4 19±4.58 1.5±0.51 62.5
AC8-1 3±1.0 2.5±0.43 143.2 AV6-1 5±1.0 0.61±0.2795.6 AU7 19±2.64 1.44±0.78 65.1
AC11 4±1.0 2.34±0.19 158.2 AV8-1 11±2.0 0.73±0.35122.4 AU17-120±7.54 1.81±0.71 116.0
AC12-1 5±3.0 6.91±0.32 143.2 AV9 25±10.02.87±0.55123.2 AU20-123±4.58 1.58±0.45 109.8
AC13-1 3±1.0 2.82±0.63 108.9 AV9-1 10±1.631.31±0.65106.8 AU23 32±8.54 4.02±0.52 136.6
AC15 4±1.73 3.2±0.75 89.7 AV26-117±2.641.93±0.86104.3 AU28 24±6.55 1.87±0.68 136.6
AC18-1 4±2.64 4.67±0.38 147.7 AV30 19±3.262.25±0.6899.1 AU30-120±2.64 4.15±0.92 151.7
AC21 5±1.0 4.67±0.67 119.4 AV36-120±2.643.13±0.99114.6 AU30-221±3.46 1.42±0.64 121.4
AV1 4±1.0 4.77±0.57 153.7 AC8-1 15±5.295.64±0.78128.4 AV1 32±4.35 4.41±0.99 151.7
AV3 2±1.0 2.0±0.55 134.3 AC11 20±3.6 3.04±0.85114.6 AV2 35±5.29 3.07±0.4 139.2
AV6-1 4±1.73 3.93±0.45 132.8 AC15 29±5.295.44±0.91111.2 AV3 27±4.0 3.26±0.45 116.0
AV8-1 4±2.64 2.43±0.7 150.7 AC21 15±4.0 0.7±0.44 121.5 AV6-1 28±4.0 2.46±0.41 154.4
AV9 1±0.0 1.24±0.32 134.3 AC1-1 10±5.0 1.19±0.6194.8 AV8-1 35±9.84 4.66±0.68 157.1
AV9-1 3±1.0 1.77±0.39 168.6 AU17-112±5.560.86±0.47103.4 AV9 26±3.6 2.77±0.24 125
AV30 3±1.0 3.03±0.79 140.2 AU30-118±5.0 1.42±0.6980.1 AV9-1 25±6.08 3.54±0.54 145.5
AV36-1 3±1.73 1.06±0.59 92.5 AU30-212±2.641.16±0.8277.5 AV30 23±6.55 3.74±0.55 130.3
Control - - 100.0 Control - - 100 Control - - 100
Values are the ±SE, n = 3; ARA – acetylene-reductase activity.
leguminous plant” has been created. As components of
such ecosystem it is necessary to support the definite
moisture 5-7% and sand availability, scarifying and
drainaging agent. Implementation of these conditions
approaches functioning of created model ecosystem in
order that the latter would be included into desert natural
Authors express a deep gratitude to Prof. Herman Lips, Dr. M. Ines M.
Soares (Institute for Desert Research Ben-Gurion University of the
Negev Sede Boqer), Prof. Eduardo Santero Santurino (Laboratorio
Andaluz de Biología Facultad de Ciencias Experimentales, Universi-
dad Pablo de Olavide), Dr. Maria-Hoce and Dr. Luis Carlos (Sevilla
University, Department of Genetics and Department of Microbiology)
for help in carrying out of microbiological-physiological and molecu-
lar-genetic experiments, and also to V.S. Bulatnikov (Central Asia
Pediatric Institute, Tashkent) for help in microscopic investigations.
These investigations were carried out owing to grant support of
Inco-Copernicus Program (4th program of the European Union),
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