In the name of Allah, the most Beneficent, the most Merciful
Isolation and Characterization of Azospirillum sp.
associated with wheat grown under different
moisture stress conditions.
Noshin Ilyas
Assistant Professor
Botany Department, PMAS AAUR
Ph.D. Supervisor: Prof Dr Asghari Bano
Department of Plant Sciences,
Quaid-i-Azam University, Islamabad
Soil
Sustainable
Agriculture
Plant
Microbes
Microbes …….
Play important role in the terrestrial ecosystem
Soil fertility
Wheat Facts in Pakistan
•
•
•
Area: 8.2 million hectares
Production: 22.0 million tons
Average grain yield: 2.5 tons/ha.
•
•
•
Dependence of 80 percent of the farmers
40 percent of the cropped area,
Contribute a quarter of the total crop sector value added
Azospirillum
Azospirillum are nitrogenfixing bacteria that live in a
symbiotic relationship in
the root cortex of several
tropical crops.
Stress ??????
An Environmental change that tends to inhibit
the normal cycle systems from functioning
Stresses may be
 Biotic
 Pests
 Diseases
 Abiotic
Water stress
 Salinity
 Soil nitrate
 Temperature
 Heavy metals
 Biocides
Situation in Pakistan
Out of total geographical area of Pakistan more than 70% (87.81million ha) is
arid or semi arid
Salt-Affected Soils
OBJECTIVES
Present investigation was aimed:
1) To isolate and characterize Azospirillum strains isolated
from different agroecological zones having different moisture
regimes
2) To determine the genotype differences in the
Azospirillum strains isolated and to evaluate the
phytohormones production of strains isolated
3) To select the strains which can be successfully used as
inoculant of wheat exposed to water stress.
Plan of Research
Isolation and biochemical characterization of efficient strains of Azospirillum from
rhizosphere and roots of wheat from semi-arid area of Attock (8% available soil
moisture) and Kallar Sayedan (14% available soil moisture) and irrigated National
Agricultural Research Center (25% available soil moisture).
Isolation and biochemical characterization of efficient strains of Azospirillum
isolates from plants of isolated from roots and rhizosphere soils of plants
grown under well watered (soil moisture 22 %) conditions and water stressed
( 8 % soil moisture) conditions in pots.
Stages of crops at which sampling was done:
Tillering
Anthesis
Methodology
Survival efficiency (CFU)
 Biochemical Techniques
QTS (Quick Test system) tests
Phytohormones production
 Molecular Techniques:
RAPD (Random Amplification
of Polymorphic DNA)
16srRNA Gene amplification
Reinocualtion studies
Isolates of Azospirillum obtained from rhizospheric soil and
roots of Wheat plants collected from water stressed and well
watered conditions.
AZOSPIRILLUM
From Roots
From Rhizosphere
Irrigated Area NARC (Tillering stage)
WRA1
WSA1
Attock (Tillering stage)
WRA2
WSA2
Kallar Sayedan (Tillering stage)
WRA3
WSA3
Irrigated Area NARC (Anthesis stage)
WRA4
WSA4
Attock (Anthesis stage)
WRA5
WSA5
Kallar Sayedan (Anthesis stage)
WRA6
WSA6
Well watered pot conditions (Tillering stage)
WRA7
WSA7
Water Stressed pot conditions (Tillering stage)
WRA8
WSA8
Well watered pot conditions (Anthesis stage)
WRA9
WSA9
Water stressed pot conditions (Anthesis stage)
WRA10
WSA10
5
a
4.5
a
b
a
b
b
b
a
4
c
c
b
c
b
c
3.5
d
c
d
d
f
Log cfu/g
3
d
2.5
2
1.5
1
0.5
Tillering stage
Anthesis
WSA10
WRA10
WSA9
WRA9
WSA6
WRA6
WSA5
WRA5
WSA4
WRA4
WSA8
WRA8
WSA7
WRA7
WSA3
WRA3
WSA2
WRA2
WSA1
WRA1
0
stage
Azospirillum isolated from wheat
Colony forming unit (cfu/g) of Azospirillum strains isolated from rhizosphere soil
and roots of wheat grown in field and in pots. The rhizosphere soil was collected
at a depth of 15 cm from soil surface under arid (soil moisture 8 %), semiarid (soil
moisture 14 %) and irrigated (soil moisture 25 %) conditions and from water
stressed (soil moisture 8 %) and well watered pot (soil moisture 22 %) conditions.
QTS (Quick Test system)
Morphological and biochemical characteristics (as determined by QTS) of Azospirillum strains
isolated from rhizosphere soil and roots of field wheat grown. The rhizosphere soil was
collected (at a depth of 15 cm from soil surface) from soil surface under arid (soil moisture 8
%), semiarid (soil moisture 14 %) and irrigated (soil moisture 25 %) conditions.
Tests
Isolates
Reactions
Colony Morphology
OPNG
CIT
MALO
LDC
ADH
ODC
H2S
URE
TDA
Group I
WRA1,WSA1, WRA4,WSA4
Irregular, wrinkled, pink
Group II
WRA3,WSA3, WRA6,WSA6
Irregular, wrinkled, pink
Group III
WRA2,WSA2, WRA5,WSA5
Irregular, wrinkled, pink
Gram Stain
-
-
-
Catalse and Oxidase
+
+
+
Ortho nitro phenyl β-D-galactopyranoside
Sodium citrate
Sodium malonate
Lysine decarboxylase
Arginine dihydrolase
Orthinine decarboxylase
H2S production
Urea hydrolysis
Tryptophane deaminase
Indole
+
+
+
+
+
+
-
+
+
+
+
+
-
+
+
+
+
+
-
+
+
+
+
+
+
+
+
Azospirillum brasilense
-
VP
VogerProskaur(Acetion)
GEL
Gelatin hydrolysis
GLU
Acid from glucose
MAL
Acid from maltose
SUC
Acid from sucrose
MAN
Acid from mannitol
ARA
Acid from arabinose
RHA
Acid from rhammose
SOR
Acid from sorbitol
INO
Acid from inositol
ADON
Acid from adontol
MEL
Acid from melibiose
RAF
Acid from raffinose
Organism identified
Morphological and biochemical characteristics (as determined by QTS) of
Azospirillum strains isolated from rhizosphere soil was collected and roots of pot
grown wheat. The rhizosphere soil was collected (at a depth of 15 cm from soil
surface) from wheat grown in water stressed (soil moisture 12 and 8 %) and well
watered
pot
conditions
(soil
moisture
22
%)
conditions.
Tests
Isolates
Reactions
Colony Morphology
Group I
WRA7, WSA7, WRA9, WSA9
Group II
WRA8, WSR8
Group III
WRA10, WSA10
Irregular, wrinkled, pink
Irregular, wrinkled, pink
Gram Stain
-
-
Irregular, wrinkled, pink
-
Catalse and Oxidase
+
+
+
OPNG
Ortho nitro phenyl β-D-galactopyranoside
+
+
+
CIT
MALO
LDC
ADH
ODC
Sodium citrate
Sodium malonate
Lysine decarboxylase
Arginine dihydrolase
Orthinine decarboxylase
+
+
+
-
+
+
+
-
+
+
+
-
H2S
URE
TDA
H2S production
Urea hydrolysis
Tryptophane deaminase
+
+
+
-
+
-
Indole
-
-
-
VP
VogerProskaur(Acetion)
-
-
-
GEL
GLU
MAL
SUC
MAN
ARA
RHA
SOR
INO
ADON
MEL
RAF
Organism identified
Gelatin hydrolysis
Acid from glucose
Acid from maltose
Acid from sucrose
Acid from mannitol
Acid from arabinose
Acid from rhammose
Acid from sorbitol
Acid from inositol
Acid from adontol
Acid from melibiose
Acid from raffinose
+
+
+
+
+
+
+
+
Azospirillum brasilense
-
Without Tryptophan
60
50
a
a
a
a
b
a
(µg/100 ml)
40
bc
a
30
b
b
ab
b
b
b
c
d
c
a
20
d
b
d
c
c
WRA2
WSA2
cd
c cb
a
b
c
bc
c
a
a
d d
e
b
d
b
c
c
WRA5
WSA5
d
cb
cb
e
10
0
WRA1
WSA1
WRA3
WSA3
WRA4
WSA4
WRA6
WSA6
Azospirillum isolates from Wheat field study
Phytohormone production (µg/100ml) by the Azospirillum strains isolated from
rhizosphere soil and roots of wheat grown in field without tryptophan. The
rhizosphere soil (collected at a depth of 15 cm from soil surface) was sampled under
arid (soil moisture 8 %), semiarid (soil moisture 14 %) and irrigated field (soil
moisture 25 %) conditions
IAA
GA
ABA
Zeatin
With Tryptophan
700
600
(µg/100ml)
500
a
b
a
a
b
b
a
b
d
d
400
a
b
b
c
c
c
c
e
c
e
d
d
300
a
c
ea
e
e
a
200
b
b
e
100
c
c
c
f
d
d
e
d
c
c
c
e
e
WRA5
WSA5
b
b
d
0
WRA1
WSA1
WRA2
WSA2
WRA3
WSA3
WRA4
WSA4
WRA6
WSA6
Azospirillum isolates from Wheat field study
Phytohormone production (µg/100ml) by the Azospirillum strains isolated from
rhizosphere soil and roots of wheat grown in field with tryptophan. The
rhizosphere soil (collected at a depth of 15 cm from soil surface) was sampled
under arid (soil moisture 8 %), semiarid (soil moisture 14 %) and irrigated field
(soil moisture 25 %) conditions
IAA
GA
ABA
Zeatin
Without Tryptophan
50
a
45
a
b
40
a
a
(µg/100ml)
35
b
b
ab
b
c
b
b
b
c
IAA
GA
ABA
Zeatin
a
b
c
20
15
b
a
a
a
30
25
a
a
a
b
b
c
c
c
b
b
b
10
5
0
WRA7
WSA7
WRA8
WSA8
WRA9
WSA9
WRA10
WSA10
Azospirillum isolates from Wheat pot study
Phytohormone production (µg/100ml) by the Azospirillum strains isolated from
rhizosphere soil and roots of wheat grown in pots without tryptophan. The
rhizosphere soil (collected at a depth of 15 cm from soil surface) was sampled under
water stressed (soil moisture 8 %) and well watered pot (soil moisture 22 %)
conditions.
With Tryptophan
700
600
500
aa
b
aa
b
b
a
c
400
a
a
a
b
(µg/100ml)
c
c
IAA
a
c a
b
300
c
GA
b
c
200
b
b
c
c
WRA8
WSA8
WRA9
WSA9
WRA10
Azospirillum isolates from Wheat pot study
WSA10
c
100
c
c
b
c
c
0
WRA7
WSA7
Phytohormone production (µg/100ml) by the Azospirillum strains isolated from
rhizosphere soil and roots of wheat grown in pots with tryptophan. The rhizosphere
soil (collected at a depth of 15 cm from soil surface) was sampled under water
stressed (soil moisture 8 %) and well watered pot (soil moisture 22 %) conditions.
Reinoculation studies
Effect of the inoculation of Triticum aestivum L with field isolated Azospirillum.
Treatments
Shoot Length
(cm)
Root Length
(cm)
Shoot Dry
Weight(g)
Root
Dry
Weight(g)
T0
Root to shoot
ratio
0.61
23e
17.5f
0.57b
0.45f
T1
1.7
17f
27.8d
0.22d
0.88g
T2
0.83
30a
26.3e
0.88a
0.57d
T3
2.6
24.7d
42.5a
0.49c
1.4b
T4
1.32
28.5b
31.8c
0.72b
0.7c
T5
3.65
26.5c
36.4b
0.53c
1.3a
Plants were harvested 45 days after sowing.T 0 not inoculated and kept well watered; T1 not inoculated and exposed to
induced water stress for 7 d; T2 inoculated with Azospirillum isolated from irrigated field and kept well watered; T 3
inoculated with Azospirillum isolated from irrigated field and exposed to water stress for 7 d; T 4 inoculated with
Azospirillum isolated from arid field and kept well watered; T5 inoculated with Azospirillum isolated from arid field and
exposed to water stress for 7 d.
Effect of the inoculation of Triticum aestivum L with pot isolated Azospirillum.
Treatments
Shoot Length
(cm)
Root Length
(cm)
Shoot Dry
Weight(g)
Root
Dry
Weight(g)
Root to shoot
ratio
T0
22.5e
18f
0.6c
0.46f
0.71
T1
18f
26.2d
0.54d
0.9d
1.6
T2
31a
28.3e
0.9a
0.79e
0.83
T3
23.9d
44.5a
0.51e
1.45b
2.7
T4
29.2b
32.6c
0.75b
0.93c
1.32
T5
27c
37.1b
0.55d
1.88a
3.65
Plants were harvested 45 days after sowing
T0; not inoculated and kept well watered;T1;not inoculated and exposed to induced water stress for 7 d; T2 inoculated
with Azospirillum isolated from well watered potted plants and kept well watered; T3 inoculated with Azospirillum
isolated from well watered potted plants and exposed to water stress for 7 d; T4 inoculated with Azospirillum isolated
from potted plants under induced water stressed conditions and kept well watered; T5 inoculated with Azospirillum
isolated from potted plants under induced water stressed and exposed to water stress for 7 d.
UPGMA Analysis of RAPD banding patterns by OP-01 primer of
Azospirillum strains isolated from rhizosphere soil and roots of wheat
grown under arid (soil moisture 8 %), semiarid (soil moisture 14 %) and
irrigated
(soil
moisture
25
%)
conditions.
UPGMA Analysis of RAPD banding patterns by OP-06 primer of
Azospirillum strains isolated from rhizosphere soil and roots of
wheat grown under arid (soil moisture 8 %), semiarid (soil
moisture 14 %) and irrigated (soil moisture 25 %) conditions
UPGMA Analysis of RAPD banding patterns by OP-01 primer of
Azospirillum strains isolated from rhizosphere soil and roots of wheat
plants grown in water stressed (soil moisture 12 and 8 %) and well
watered pot conditions (soil moisture 22 %) conditions.
UPGMA Analysis of RAPD banding patterns by OP-06 primer of
Azospirillum strains isolated from rhizosphere soil and roots of wheat
plants grown in water stressed (soil moisture 12 and 8 %) and well
watered
pot
conditions
(soil
moisture
22
%)
conditions.
PCR product of 16S rRNA gene of Azospirillum strains isolated from
rhizosphere soil and roots of wheat grown under arid (soil moisture 8 %),
semiarid (soil moisture 14 %) and irrigated (soil moisture 25 %) conditions
Lane M=1kb DNA ladder;
Lane1: Azospirillum isolate from roots of plants growing at NARC collected at tillering stage; Lane 2:
Azospirillum isolate from Rhizosphere Soil of plants growing at NARC at tillering stage; Lane 3: Azospirillum isolate from roots of
plants growing at Attock at tillering stage; Lane 4: Azospirillum isolate from Rhizosphere Soil of plants growing at Attock at
tillering stage; Lane 5: Azospirillum isolate from roots of plants collected from Kallar Sayedan at tillering stage; Lane 6:
Azospirillum isolate from rhizospheric soil of plants growing at Kallar Sayedan at tillering stage; Lane 7: Azospirillum isolate from
roots of plants growing at NARC collected at anthesis stage; Lane 8: Azospirillum isolate from Rhizosphere Soil of plants growing
at NARC at anthesis stage; Lane 9: Azospirillum isolate from roots of plants growing at Attock at anthesis stage; Lane 10:
Azospirillum isolate from Rhizosphere Soil of plants growing at Attock Lane 11: Azospirillum isolate from roots of plants collected
from Kallar Sayedan at anthesis stage Lane 12: Azospirillum isolate from rhizospheric soil of plants growing at Kallar Sayedan at
anthesis stage.
PCR product of 16S rRNA gene of Azospirillum isolates, from rhizospheric
soil and roots of wheat plants collected from water stressed conditions
(soil moisture 12 and 8%) and well watered conditions (soil moisture 22%)
Lane M=1kb DNA ladder; Lane1: Azospirillum isolates from roots of plants collected from pot study growing under well watered
conditions at tillering stage; Lane2: Azospirillum isolates from rhizospheric soil of plants collected from pot study growing under well
watered conditions at tillering stage; Lane3: Azospirillum isolates from roots of plants collected from pot study growing under water
stressed conditions at tillering stage; Lane 4: Azospirillum isolates from rhizospheric soil of plants collected from pot study growing
under water stressed conditions at tillering stage; Lane 5: Azospirillum isolates from roots of plants collected from pot study growing
under well watered conditions at reproductive stage; Lane 6: Azospirillum isolates from rhizospheric soil of plants collected from pot
study growing under well watered conditions at reproductive stage; Lane 7: Azospirillum isolates from roots of plants collected from
pot study growing under water stressed conditions at reproductive stage; Lane 8: Azospirillum isolates from rhizospheric soil of plants
collected from pot study growing under water stressed conditions at reproductive stage.
16S rRNA Sequencing
• On the basis of phytohormone production and tolerance to
water stress, WRA5 Azospirillum (isolated from rhizosphere soil
of wheat plants grown in arid field at anthesis stage) and WRA10
Azospirillum (isolated from roots of wheat plants growing
under induced water stress conditions in potted plants at
anthesis stage) were characterized by 16S rRNA gene
sequence analysis. Sequences were compared with standard
databases by BLAST (NCBI) software and deposited at NCBI
databank (with accession No.GQ144644 for WRA5, and for
WRA10 accession No. GQ144646).
Conclusions
 The present investigation has confirmed that association of
microbes depends on developmental stage and root architecture of
plants.
• Azospirillum spp. isolated from moisture stressed conditions
either from field or pot improved tolerance to water stress and thus
they can be used as inocula to promote plant growth on stressed
sites, such as semiarid and arid regions.
• The isolates from water unstressed conditions had less
production of IAA, GA and t-zr than isolates from water
stressed/arid field area but have higher ABA/t-zr ratio.
•Azospirillum isolates WSA5 and WRA10 (strains isolated at the
anthesis stage from rhizosphere soil of wheat plants grown in arid
field and from roots of wheat plants growing under water stress
conditions in potted plants) were the most efficient and had higher
ABA/trans zeatin riboside ratio (ABA is the phytohormones which
gives tolerance to plant under water stress and thus can be used as
biofertilizers in arid and semi-arid conditions.
Future perspectives and Recommendations
Efficiency of these isolates may be tested under field
conditions.
Rhizobium and Azospirillum spp. isolated from moisture
stressed conditions / arid region have potential for increased
tolerance to water stress if used as an inoculum to promote
plant growth on stressed sites particularly semiarid and arid
regions of Pakistan.