Symbiosis of nodule bacteria with perennial xerophyte leguminous plants of Central Asia

doi:10.4236/as.2010.11004

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Vol.1, No.1, 24-38 (2010)s

doi:10.4236/as.2010.11004

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:

zair@dostlink.net

Received 27 April 2010; revised 3 May 2010; accepted 5 May 2010.

ABSTRACT

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

1. INTRODUCTION

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

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. MATERIALS AND METHODS

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

Microvegetation/Vegetation

Experiments

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

bacteria.

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

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

Activity

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

105°C.

2.8. Drying of Plant Biomass

Drying of plant biomass for determination of biomass

was conducted at 70°C during day.

2.9. PCR Ampliﬁcation of the 16S rRNA

Gene

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 ampliﬁed using universal primers

1070f (59-ACGGGCGGTGTG- TAC-39) and 1392r

(59-CGCCCGCCGCGCCCCG- CGCCCGGCCCGCCG-

CCCCCGCCCC-ACGGGCGGTGTGTAC-39) [6]. Each

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 ﬁnal reaction volume of 50 l. The DNA thermal cy-

cler used for PCR ampliﬁcation 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 ﬁnal 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-

samplings.

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

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-

volts.

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. RESULTS

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

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)

AC8-1(1)

AC11

AC15

AC21

AU7(2)

AU17-1

AU30-1

AU30-2

AV1(3)

AV3

AV6-1

AV8-1

AV9

AV9-1

AV30

0.82

1.31

0.72

0.68

0.84

0.77

1.08

0.81

0.76

0.93

1.27

0.75

0.84

0.81

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

limit(b)

(M)

Plasmid profile

(kbp) Antibiotic

Resistance Growth

temp., oC Growth

pH Melanin pro-

duction Colony

Type

AC8-1

AC11

AC15

AC21

AU7

AU17-1

AU30-1

AU30-2

AV1

AV3

AV6-1

AV8-1

AV9

AV9-1

AV30

0.82

1.31

0.72

0.68

0.84

0.77

1.08

0.81

0.76

0.93

1.27

0.75

0.84

0.81

1.75

2.00

1.75

1.50

1.75

1.50

1.75

2.00

1.00

370, 515

118, 370, 515

370, 515

118, 370, 515

370, 515

118, 370, 515

370, 515

Amp

Amp, Km, Str, Tc

Amp, Tc

Amp, Km, Str, Tc

Amp, Tc

Amp, Cm

Amp, Cm, Km, Str

8-40

12-40

8-40

4-11

6-10

4-11

4-10

4-11

5-11

4-11

5-11

4-11

–

weak

–

+++

–

III

(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

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-

peratures.

3.2. Germination of Plant Seeds, Nodulation

Test (Direct Inoculation and

Cross-Inoculation)

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-

lation”).

During search of optimal conditions for plants grow-

ing up the following types of potting substrates were

used:

1) Microvegetation experiments:

Strips of filter paper in agronomic tubes (60 ml) with

ascending flow of nutritive solution for plants in sterile

conditions.

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

(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

(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

type.

0.02

Z. S. Shakirov et al. / Agricultural Sciences 1 (2010) 24-38

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

3,8

4,32

4,86

5,48

6,41

200

400

600

800

1000

1200

Dry biomass, mg

Moistu re, %

Astraga lus

un ifoliola tus

Ammodendron

conollyi

Astraga lus

villossimus

Figure 3. The influence of drought on dry biomass of perennial desert

leguminous plants.

6.41 5.48 4.86

4.32

3.8

Z. S. Shakirov et al. / Agricultural Sciences 1 (2010) 24-38

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

100

200

300

500

100

Survavility, %

Salinity, mM NaCl

Ammodendron

con ollyi

Astragalus

unifoliolatus

Astragalus

villossimus

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

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

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

Z. S. Shakirov et al. / Agricultural Sciences 1 (2010) 24-38

4. DISCUSSION

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

feature.

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

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

nodules

number

ARA,

ppm

C2H4

tube/hour

Efficiency

symbiosis,

% Strain Average

nodules

number

ARA,

ppm

C2H4

tube/hour

Efficiency

symbiosis,

% Strain Average

nodules

number

ARA,

ppm

C2H4

tube/hour

Efficiency

symbiosis,

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

ecosystem.

5. ACKNOWLEGDEMENTS

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),

USAID/CDR/CAR Program.

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