Isolation and Characterization of Azospirillum sp. associated with wheat grown under different moisture stress conditions.

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Isolation and Characterization of Azospirillum
sp. associated with wheat grown under different
moisture stress conditions.
In the name of Allah, the most Beneficent, the
most Merciful

• Noshin Ilyas
• Assistant Professor
• Botany Department, PMAS AAUR
• Ph.D. Supervisor Prof Dr Asghari Bano
• Department of Plant Sciences,
• Quaid-i-Azam University, Islamabad

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Soil
Sustainable Agriculture
Plant
Microbes
3
Microbes .

• Play important role in the terrestrial ecosystem
• Soil fertility

4
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

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Azospirillum

• Azospirillum are nitrogen-fixing bacteria that
  live in a symbiotic relationship in the root
  cortex of several tropical crops.

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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

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Situation in Pakistan
Out of total geographical area of Pakistan more
than 70 (87.81million ha) is arid or semi arid
Salt-Affected Soils
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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.

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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

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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

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Isolates of Azospirillum obtained from
rhizospheric soil and roots of Wheat plants
collected from water stressed and well watered
conditions.
AZOSPIRILLUM AZOSPIRILLUM 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
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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.
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QTS (Quick Test system)
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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 Reactions Group I Group II Group III
Isolates WRA1,WSA1, WRA4,WSA4 WRA3,WSA3, WRA6,WSA6 WRA2,WSA2, WRA5,WSA5
Colony Morphology Irregular, wrinkled, pink Irregular, wrinkled, pink Irregular, wrinkled, pink
Gram Stain - - -
Catalse and Oxidase
OPNG Ortho nitro phenyl ß-D-galactopyranoside
CIT Sodium citrate
MALO Sodium malonate - - -
LDC Lysine decarboxylase
ADH Arginine dihydrolase
ODC Orthinine decarboxylase - - -
H2S H2S production - - -
URE Urea hydrolysis
TDA Tryptophane deaminase - -
Indole - - -
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 Organism identified Azospirillum brasilense Azospirillum brasilense Azospirillum brasilense
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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 Reactions Group I Group II Group III
Isolates WRA7, WSA7, WRA9, WSA9 WRA8, WSR8 WRA10, WSA10
Colony Morphology Irregular, wrinkled, pink Irregular, wrinkled, pink Irregular, wrinkled, pink
Gram Stain - - -
Catalse and Oxidase
OPNG Ortho nitro phenyl ß-D-galactopyranoside
CIT Sodium citrate
MALO Sodium malonate - - -
LDC Lysine decarboxylase
ADH Arginine dihydrolase
ODC Orthinine decarboxylase - - -
H2S H2S production - - -
URE Urea hydrolysis
TDA Tryptophane deaminase - -
Indole - - -
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 Organism identified Azospirillum brasilense Azospirillum brasilense Azospirillum brasilense
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• 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

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• 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

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• 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.

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• 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.

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Effect of the inoculation of Triticum aestivum L
with field isolated Azospirillum.
Reinoculation studies
Treatments Shoot Length (cm) Root Length (cm) Shoot Dry Weight(g) Root Dry Weight(g) Root to shoot ratio
T0 23e 17.5f 0.57b 0.45f 0.61
T1 17f 27.8d 0.22d 0.88g 1.7
T2 30a 26.3e 0.88a 0.57d 0.83
T3 24.7d 42.5a 0.49c 1.4b 2.6
T4 28.5b 31.8c 0.72b 0.7c 1.32
T5 26.5c 36.4b 0.53c 1.3a 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 irrigated field and kept well watered T3
inoculated with Azospirillum isolated from
irrigated field and exposed to water stress for 7
d T4 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.
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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 wateredT1not
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.
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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.
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• 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

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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.
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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.
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Lane M1kb 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
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
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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 M1kb 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.

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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).

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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.

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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.

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