Você está na página 1de 64

Screening and characterization of PGPR

60

Chapter 3 Screening and Characterization of PGPR on Their Plant
Growth Promoting Attributes
3.1 Introduction
Of all the variables that impact upon plant growth, soil microbial activity is arguably the
very complex but plays a very important role in agricultural (or conservation)
management. The importance of the microbiota to biogeochemistry has long been
appreciated (Conrad 1996). Interactions between plants and microbes have long been
known and we are increasingly aware of inter-kingdom communication signals across a
broader range of ecological interactions than simple two-species mutualisms. The point
that the microbiota are an intimate part of the plant ecosystem and that understanding
their roles will lead to new management opportunities. Through describing patterns of
variation in soil microbiota, and explaining the basis of their ecological interactions with
plants, soil microbial ecologists aim to develop new management tools for plant systems.
Plant growth promoting rhizobacteria (PGPR) can have an impact on plant growth and
development in two different ways: indirectly or directly. The indirect promotion of plant
growth occurs when bacteria decrease or prevent some of the deleterious effects of a
phytopathogenic organism by one or more mechanisms.
On the other hand, the direct promotion of plant growth by PGPR generally entails
providing the plant with a compound that is synthesized by the bacterium or facilitating
the uptake of nutrients from the environment (Glick 1995; Glick et al. 1999).
Rhizosphere bacteria multiply to high densities on plant root surfaces where root
exudates and root cell lysates provide ample nutrients. Sometimes, they exceed 100 times
to those densities found in the bulk soil (Campbell and Greaves 1990). Certain strains of
these plant associated bacteria stimulate plant growth in multiple ways: (1) they may fix
atmospheric nitrogen, (2) reduce toxic compounds, (3) synthesize phytohormones and
siderophores, or (4) suppress pathogenic organisms (Bloemberg and Lugtenberg 2001).



Screening and characterization of PGPR
61

Research on the biocontrol activity of rhizobacteria has seen considerable progress in
recent years. Disease suppression of soilborne pathogens includes competition for
nutrients and production of antimicrobial compounds or lytic enzymes for fungal cell
walls or nematode structures (Persello-Cartieaux 2003). By contrast, systemic resistance
can also be induced by rhizosphere-colonizing Pseudomonas and Bacillus species where
the inducing bacteria and the challenging pathogen remained spatially separated
excluding direct interactions (Ryu et al. 2004). PGPR has been reported not only to
improve plant growth but also to suppress the plant pathogens, of which Pseudomonas
spp. and Bacillus spp. are important as these are aggressive colonizers of the rhizosphere
of various crops and have broad spectrum of antagonistic activity against many pathogens
(Weller et al. 2002). Biocontrol bacterial species generally employ an array of
mechanisms such as antibiosis, competition, production of hydrocyanic acid, siderophore,
fluorescent pigments and antifungal compounds to antagonize pathogens (Singh et al.
2006).
It is a well known fact that actively growing microbes are greater in number in the
rhizosphere as crop plants release root exudates that contribute, in addition, to simple and
complex sugars and growth regulators, contain different classes of primary and secondary
compounds including amino acids, organic acids, phenolic acids, flavonoids, enzymes,
fatty acids, nucleotides, tannins, steroids, terpenoids, alkaloids and vitamins (Uren 2000).
Researchers around the world attempted to isolate PGPR organisms from the
rhizospheres of crop plants and the compost (Khalid et al. 2004). Plant growth promoting
bacterial strains must be rhizospheric competent, able to survive and colonize in the
rhizospheric soil (Cattelan et al. 1999). Unfortunately, the interaction between associative
PGPR and plants can be unstable. The good results obtained in vitro cannot always be
dependably reproduced under field conditions (Chanway and Holl 1993; Zhender et al.
1999). The variability in the performance of PGPR may be due to various environmental
factors that may affect their growth and exert their effects on plant. The environmental
factors include climate, weather conditions, soil characteristics or the composition or
activity of the indigenous microbial flora of the soil.


Screening and characterization of PGPR

62
Several factors play a role in developing the rhizosphere effect (Table 3.2). The three
most important factors which alter the biochemical activity in the vicinity of the plant
root are the soluble organic materials that are secreted or exuded from the plant root cells,
the debris derived from the root-cap cell, dying root hairs and cortical cells, and the lysis
of plant root cells. The increased availability of organic carbon in the rhizosphere
provides a habitat which is highly favorable for the proliferation of microorganisms. This
microbial community brings about further change by altering various chemical and
biological properties of the rhizosphere. Beneficial microbes are often used as inoculants
(Bloemberg and Lugtenberg 2001). They can be classified according to the goal of their
application: biofertilizers, phytostimulators, rhizoremediators and biopesticides. PGPR
and their applications will significantly reduce the use chemical fertilizers and pesticides.
However, their application will be essential for achieving sustainable crop responses
(Table 3.1) in agriculture.
To achieve the maximum growth promoting interaction between PGPR and nursery
seedlings it is important to discover how the rhizobacteria exerting their effects on plant
and whether the effects are altered by various environmental factors, including the
presence of other microorganisms (Bent et al. 2001). Therefore, it is necessary to develop
efficient strains in field conditions. One possible approach is to explore soil microbial
diversity for PGPR having combination of PGP activities and well adapted to particular
soil environment.




63
PGPR Crops Responses References
Azotobacter sp. Maize Inoculation with strain efficient in IAA production had significant growth
promoting effects on maize seedlings.
Zahir et al.(2000)
Azospirillum brasilense A10,
CDJA
Rice All the bacterial strains increased rice grain yield over uninoculated control Thakuria et al.(2004)
Azospirillum lipoferum strains 15 Wheat Promoted development of wheat root system even under crude oil contamination in
pot experiment in growth chamber
Muratova et al. (2005)
Azotobacter sp. Sesbenia Increasing the concentration of tryptophane from 1 mgml-1to 5 mgml-1 resulted in
decreased growth in both crops
Ahmad et al. (2005)
Alcaligenes sp. ZN4 Rice Strain of Bacillus sp., proved to be efficient in promoting a significant increase in
the root and shoot parts of rice plants
Beneduzi et al. (2008)
Bacillus circulans P2 Wheat Promoted development of wheat root system even under crude oil contamination in
pot experiment in growth chamber
Muratova et al. (2005)
Bacillus licheniformis Spinach All bacterial strains were efficient in indole acetic acid (IAA) production and
significantly increased growth of wheat and spinach
akmaki et al. (2007a)

Bacillus sp. Rice Strain of Bacillus sp., proved to be efficient in promoting a significant increase in
the root and shoot parts of rice plants
Beneduzi et al. (2008)

Pseudomonas fluorescens Groundnut Involvement of ACC deaminase and siderophore production promoted nodulation
and yield of groundnut
Dey et al. (2004)
Pseudomonas denitrificans

Wheat Both the bacterial strains had been found to increase plant growth of wheat and
maize in pot experiments
Egamberdiyeva (2005)



Screening and characterization of PGPR
Table 3.1 plant growth promoting rhizobacteria and their crop responses to the respective plants



Screening and characterization of PGPR
64

_________________________________________________________
Release of soluble organic compounds by plant roots
Sloughed off root cell debris and dying root hairs
Plant root cell lysis
Higher concentration of carbon dioxide
Lower concentration of oxygen
Lower concentration of nutrient ions
Partial desiccation of soil due to absorption of water by roots
________________________________________________________
Table 3.2 Factors responsible for the development of the soil-plant root rhizosphere.

Microbes being an integral component of any soil ecosystem provide life to the soil.
Native soils minus microbes are merely dead material. It is now widely being recognized
that the presence and abundance of microbial wealth provide soils richness in terms of
making available slow-release nutrients, continuous breaking down of complex macro-
molecules and natural products into simpler ones to enrich beneficial substances,
maintaining physicochemical properties of the soils and most essentially, providing
support to the plants in terms of growth enhancement and protection against diseases and
pests through their metabolic activities that go on in the soil along day and night.
In Indian context, the important issue is to grow oilseed trees on wasteland, which can
also fulfill the future energy requirement. Jatropha curcas (Euphorbiaceae family)
plantation on wastelands of the country, not only provides rich biomass for various
applications (mainly biodiesel production) but also checks degradation of land. Although
this plant can grow on wastelands but its growth is limited. Inoculation of beneficial
microbes to these lands may improve plant growth by enhancing plant resistance to
adverse environmental stresses, e.g. water and nutrient deficiency and heavy metal
contamination (Shen 1997).


Screening and characterization of PGPR
65

Relevance of using bioinoculants individually as well as in consortia, lies in their ability
to enhance biomass yield by increasing stress tolerance, nutrient recycling, uptake of
nutrients, and synthesis of growth hormones, vitamins, antibiotics, and by improving soil
conditions. The renewed interest, in fuels of biological origin particularly bio-diesel, to
ensure energy security, cleaner environment and sustainable development has drawn
research attention on non-edible oils along with other sources. One of such feedstock is
the non-edible oil of Jatropha curcas. It is a multipurpose large shrub or small tree of
Latin American origin which has got adjusted throughout arid and semiarid tropical
region of the world (Gubitz et al. 1999). Exploitation of Jatropha for various purposes is
described several workers (Kumar and Sharma 2008). The recent interest in the
plantation of Jatropha is gaining momentum for bio-diesel production on wastelands.
However, there is a concern for increasing its productivity in some ways, which at the
same time will take care of soil ecology too. The advantages of using PGPR are that it
reduces pollution levels and hence preserves ecological balance, enhances productivity
and ensures sustainable agriculture by keeping the soil fertile (Meelu 1996). PGPR helps
in soil maintenance by improving soil aeration, water holding capacity and stimulates
microorganisms in the soil that make plant nutrients readily available leading to higher
yield and better quality of plants .
Considering the above, pot experiments were conducted to evaluate the efficacy of MS1,
MS2, MS3, MS4 and MS5 individually to increase the germination (%), survival and
other growth related characters of Jatropha curcas at different interval of time.

3.2 Materials and methods
3.2.1 Screening of rhizosphere isolates
All the isolates obtained from Jatropha rhizosphere soil were inoculated in their
respected basal medium and incubated for 24 h at 37
o
C. Growth of all isolates was then
measured spectrometrically. The fast growers were then selected for further studies.


Screening and characterization of PGPR
66

These five isolates were selected from the five different sites MS1 from GS1, MS2 from
GS5, MS3 from GS4, MS4 from GS2 and MS5 from GS3.
3.2.2 Phosphate solubilization (P) by selected isolates
P solubilization was checked using tricalcium phosphate as insoluble phosphate. Spot
inoculation of the isolates was done in the center of the Pikovaskays medium amended
with bromophenyl blue. These plates were then incubated at 37
o
C for 48 to 72 h.
Phosphate solubilization was checked in the form of a clear yellow colour halo formed
around the colony representing the production of organic acids as a possible mechanism of
the phosphate solubilization. Quantitative phosphate solubilization was carried out in
liquid Pikovaskay's medium in 250 ml flasks for 14 d. The concentration of the soluble
phosphate in the supernatant was estimated every 7 d by Stannous Chloride (SnCl
2
. 2H
2
O)
method (Gaur 1990). A simultaneous change in the pH was also recorded in the
supernatant on systronics digital pH meter ( pH system 361).

3.2.3 Indole acetic acid production by selected isolates
Auxin production was checked in trypton yeast medium. Bacteria were grown in 50 ml
yeast extract broth supplemented with 50 mgl
-1
of L-Tryptophan and incubated in dark on
orbital shaker at 200 rpm for 72 h. IAA production was checked in supernatant using
Salkowskys reagent method (Sarwer and Kremer 1995). One ml of culture supernatant
was mixed with 1 ml of Salkowskys reagent and incubated in dark for 30 min for
development of pink colour, which was then estimated on spectrophotometer at 536 nm.
The amount of IAA produced was calculated from the standard graph of pure indole acetic
acid. Study was carried out every 24 h for up to 120 h and the pattern of IAA production
was recorded.





Screening and characterization of PGPR
67

3.2.4 Exopolysaccharide (EPS) production by selected isolates
Normally EPS production is studied in basal medium of all different organisms. As
carbohydrate source 5% of sucrose is to be added as polysaccharide in to the medium
(Modi et al.1989). 10 ml of culture suspension was collected after 5-6 days and centrifuge
at 30,000 rpm for 45 mins. add thrice the volume of chilled acetone. EPS will be
separated from the mixture in the form of a slimy precipitates. Precipitates were collected
on a predried filter paper. Allow the precipitates to dry overnight at 50
0
C. reweigh the
dried filter paper after overnight drying. Note the increase in the weight of filter paper, is
the EPS produced.
3.2.5 Siderophore production by selected isolates
Siderophore production was checked on solid CAS universal blue agar plates (Schwyn
and Neilands 1987). Actively growing cultures were spot inoculated on the CAS blue
agar plate and incubated at 30
o
C for 48 h. Formation of yellow-orange halo around the
colony indicated production and release of the siderophores on the agar plate.
Quantitative Estimation
One ml actively growing isolates with 0.5 OD at 600 nm were inoculated in 50 ml of
MM9 medium in 250 ml EM flasks. All flasks were incubated at 30
o
C for 30 h on orbital
shaker. After 30 h, all cultures were centrifuged at 5,000 rpm for 20 min. Supernatant
was collected and tested for pH, fluorescence and siderophore production. A
simultaneous change in growth pattern of the isolates was also carried out. Catecholate
types of siderophores were checked by Arnows method (Arnows 1937) and for
Hydroxymate type of siderophores Csakys method (Csakys 1948) was used.
On the basis of results obtained from these characterization five different isolates MS1,
MS2, MS3, MS4 and MS5 were finally selected for ACC deaminase enzyme production,
antibiotic resistance studies, Carbon utilization profile, Biochemical tests, FAME analysis
and 16S rRNA as well as their influence on growth of Jatropha curcas plant.


Screening and characterization of PGPR
68

3.2.6 Ammonia and HCN production by selected isolates
Each strain was tested for the production of ammonia in peptone water. Overnight broth
cultures (100 l inoculum with approximately 3 x 10
8
c.f.u. ml
-1
) were inoculated in 10
ml peptone water and incubated at 30
o
C for 4872 h. Nesslers reagent (0.5 ml) was
added to each tube. Development of brown to yellow color was recorded as a positive test
for ammonia production (Cappucino and Sherman 1992). Production of hydrocyanic acid
(HCN) was checked on nutrient agar slants streaked with the test isolates. Filter paper
strips dipped in picric acid and 2 % sodium carbonate were inserted in the tubes. HCN
production was checked on the basis of changes in colour from yellow to light brown,
moderate brown or strong brown of the yellow filter paper strips (Morrison and Askeland
1983).

3.2.7 ACC deaminase production by selected isolates
The bacteria were first cultured in rich medium and then transferred to minimal medium
with ACC as sole source of nitrogen. Bacterial cells were grown to mid- up to late log
phase in 15 ml Trypton Soy Broth. Cultures were incubated over night in a shaking water
bath at 200 rpm at 30
o
C. Bacterial cell mass was then harvested by centrifugation at
8000 g for 10 min at 4
o
C. The supernatant was then removed and the cells were washed
with 5 ml DF (Dworkin and Foster 1958) salts medium. Following an additional
centrifugation for 10 min at 8000 g at 4
o
C, the cells were suspended in 7.5 ml of DF
medium in a fresh culture tube. Just prior to incubation, the frozen 0.5 M ACC solution
was thawed and an aliquot of 45 l was added to the cell suspension to obtain a final
ACC concentration to 3.0 mM. The bacterial cells were then again incubated in shaking
water bath to induce the activity of ACC deaminase. The cells were then harvested by
centrifugation as mentioned above and were washed twice in 5 ml of 0.1 mM Tris-HCl at
pH 7.6 so as to ensure that the pellet is free of the bacterial growth medium. The bacterial
cells were suspended in1.0 ml of 0.1 M Tris-HCl and transferred to 1.5 ml micro-
centrifuge tubes and centrifuged at 16,000 g for 5 min.


Screening and characterization of PGPR
69

The supernatant of the tube was then discarded and pellet was suspended in 600 l of 0.1
M Tris-HCl pH 8.5. Thirty micro-liters of toluene was added to the suspended cells and
vortexed at highest speed for 30 s. 200 L of the toluenized cell suspension was then
placed in 1.5 ml micro-centrifuge tubes. 20 l of 0.5 M ACC was then added to the
suspension, briefly vortexed and then incubated at 30
o
C for 15 min. Following the
addition of 1 ml of 0.56 M HCl, the mixture was vortexed and then centrifuged for 5 min
at 16,000 rpm. One ml of this supernatant was then vortexed with 800 l of 0.56 M HCl.
Thereupon, 300 l of 2, 4- dinitrophenylhydrazine reagent was added to the glass tube,
the content vortexed and then incubated at 30
o
C for 30 min. Thereafter 2 ml of 2 N
NaOH was added and the absorbance was measured at 540 nm. Production of ACC
deaminase was then measured as the amount of -ketobutyrate produced when the
enzyme cleaves ACC (Penrose and Glick 2003). The more details regarding ACC
deaminase were studied and reported in chapter 4.
3.2.8 Antibiotic resistance
Antibiotics discs with different concentration of different antibiotics on different discs
were used to check the antibiotic resistance of the isolates. Various antibiotic discs used
are as listed below.
1. OD 007 G 3 minus
2. OD 042 G Vl minus
3. OD Combi X
Inoculate 0.1 ml of culture suspension to cooled melted agar medium. Pour the inoculated
melted medium in sterile plates and allow them to solidify. Place different antibiotics
discs in the center of the basal agar plates aseptically. Incubate for 24 h at 37
o
C and next
day check for the clear zone of inhibition of the growth of the test isolates. Note down the
results. Measure the diameter of the zone of inhibition of growth and record the results in
a tabular form.




Screening and characterization of PGPR
70


3.2.9 Morphological and biochemical analysis of the selected isolates
All the isolates were once again studied for their morphological characteristics by
performing gram staining. Biochemical tests of the selected PGPR isolates were carried
out to authenticate and identify them according to the Bergey's Manual of Systematic
Bacteriology (Kreig and Holt 1984).

3.2.10 Carbohydrate utilization profile of selected isolates
The catabolic activity and functional diversity of soil microbial communities was
assessed by their ability to utilize 21 different carbohydrates. The medium used was 1 %
Peptone water with Phenol red as indicator and amended with various carbohydrates at
0.5 % (w/v) final concentrations. The list of carbohydrates is as given in table. The
medium tubes also contained Durhams vials. Five ml of medium was filled in tubes with
inverted Durhams vials and autoclaved at 15 lbs pressure for 20 min. Individual
carbohydrates in form of sterile disc containing 25 mg respective carbohydrates procured
from Hi-media were added after medium sterilization. Tubes were then inoculated with
100 l of actively growing respective cultures. Control tube was also inoculated which
did not contain any carbon source. Tubes were incubated at room temperature under
sterile conditions for 3 days. The positive results ie. acid production were identified by
color change of medium from red to yellow and recorded. The results in terms of gas
production and alkali production (pink color) were also noted. Intensity of acid produced
was noted as 0, +1, +2, and +3.

3.2.11 FAME (Fatty acid methyl ester) analysis and 16S rRNA sequencing
All the finally screened five isolates MS1, MS2, MS3, MS4 and MS5 were identified by
fatty acid methyl ester analysis and 16S rRNA. FAME analysis and 16S rRNA
sequencing was done by Disha life sciences Ahmedabad, India for confirmation.



Screening and characterization of PGPR
71

Sequence data has been deposited in the GenBank nucleotide sequence database under
the specific accession number.
3.2.12 Seed bacterization
Jatropha seeds (Jatropha curcas SDAU J1 Chhatrapati) collected from Regional research
station S.D. Agriculture University, Sardarkrushinagar, Gujarat, were soaked in 0.02%
sodium hypochlorite for 2 min. and washed five times with sterilized distilled water.
Seeds were coated with 1% carboxymethylcellulose as adhesive. Then seeds were treated
with bacterial strain for 30 min. Each bacterial strain was inoculated in 150 ml flask
containing 60 ml medium and incubated at 28 1
0
C for three days. An optical density of
0.5 recorded at 535 nm was achieved by dilution to maintain uniform cell density (10
8
-
10
9
CFU/ml) (Gholami et al. 2009)
3.2.13 Seed germination testing during nursery condition
Daily record of seed that had emerged out of the surface of soil was kept. Recording of
germination was continuing for 21 to 28 days. At the end of 28 days all the seeds that had
not germinated are taken out and ungerminated seeds were counted and they were cut
open to find whether they are still viable or not. Under germination parameter:
germination percent, germination energy, germination capacity, and seedling vigor were
calculated (Abdul-Baki and Anderson 1973).
3.2.14 Pot experiments
Ten inoculated seeds of Jatropha were sown in each earthen pot filled with sandy loam
soil and watered regularly. For each treatment, three such pots were maintained.
Uninoculated seeds were sown in pot served as control. Jatropha plants were harvested
after every 30, 60, 90, and 120 days of seed sowing through separating of plants from
soil. For each observation, two plants were randomly selected from each treatment and
the mean of two plants was used as one replication. The plants were washed through
dipping into a vessel. Plant height (cm plant
-1
) and root length (cm plant
-1
) of each plant
were recorded.


Screening and characterization of PGPR
72

Dry weights of shoot and root were recorded after drying in an oven for 1 day at 70C.
The experiment was repeated twice. Observations were also recorded on rate of seedling
emergence, Chlorophyll content, leaf area, and total plant drawing random samples at 30,
60 , 90 and 120 days after showing (DAS) (Tank and Saraf 2008).
3.2.15 Statistical Analyses
Statistical analysis of all tests was carried out using SPSS 15.0 design. Data was analyzed with
ANOVA at P<0.05 level. Analyses were also carried out using t-test between varieties of
treatments. All tests were conducted in triplicates.

3.3 Results and discussion
3.3.1 Growth profile study of the selected isolates
Growth curve of these five isolates were determined by spectrophotometric method. Growth
profile (fig. 3.1) of these five isolates was determined by inoculating early exponential phase
culture in 50 ml of nutrient broth under aseptic condition. Samples were withdrawn after every
4 hour. Mean growth rate constant (K) was calculated using the formula: K = 3.322 (logZt
logZ0) / Dt; where Z0 and Zt are the initial and final cell populations, while Dt is difference in
culture time. All isolates were fast growing. K value of MS1, MS3, MS4, MS2, and MS5 was
1.17 0.02, 1.21 0.03, 0.83 0.02, 0.92 0.05 and 1.19 0.04 h
-1
respectively, in single-
species cultures. According to the results MS1 and MS3 were found to the fastest grower and
on the basis of their growth profile other plant growth promoting parameters were designed.

3.3.2 Phosphate solubilization by selected isolates
Results show that all the five isolates were good P solubilizer and they showed zone of
phosphate solubilization on solid Pikovskyayas medium after 3 days of incubation at 30 2
o
C (Pic. 3.1). Maximum zone was observed in isolate MS5 (24 mm). Significant zones were
also seen in MS1 (22 mm), MS2 (15 mm), MS3 (23 mm) and MS4 (18 mm) after 120 hour of
incubation (fig. 3.3).


Screening and characterization of PGPR
73

Maximum TCP (Tricalcium phosphate) solubilization in liquid medium was observed in MS3
(49 g/ml) followed by MS1 (47 g/ml), MS2 (37 g/ml), MS4 (18.2 g/ml) and MS5 (18
g/ml) in descending order of solubilization (fig. 3.2). A noticeable result observed was that
though MS5 showed maximum zone of solubilization on solid medium, MS3, MS1 and MS2
gave maximum solubilization in liquid medium. The pH of the medium also showed a decrease
from 7.2 to a maximum of 3.33 after 21 d in MS3 (Table 3.3).
However, from the observations it is clear that no correlation could be established between the
degree of P-solubilization and final pH of the medium. In many isolates tested here, the final
pH was same but their respective P-solubilization was different. Similar results showing no
correlation between P-solubilization and pH reduction are also published by many researchers
(Tank and Saraf 2003). This drop in pH may also be an attribute of glucose utilization by the
isolates (Arora et al. 2008). Plant growth is frequently limited by an insufficiency of
phosphates, an important nutrient in plants next to nitrogen. Although all isolates showed
similar decline in pH, 3.3 -4.5, amount of phosphate solubilization was different in different
PGPR's isolated. This indicates that there is no relation between degree of phosphate
solubilized and change in pH of the (Gaur 1990). Jeon et al. (2003) also reported that although
phosphate solubilization observed in Pseudomonas fluorescens and B. megaterium was higher
than 360 mg l
-1
from tricalcium phosphate, final pH did not reach strong acidic level during the
studies. Though it is known that production of organic acids by soil microorganisms is the
major mechanism of inorganic phosphate solubilization among soil bacteria, chelation of metal
ions by gluconic acid may also be a mechanism of phosphate solubilization (Whitelay et
al.1999). Some other mechanism in addition to change in pH may be responsible for phosphate
solubilization. Sinorhizobium meliloti TR1 was also reported to solubilize TCP in both liquid
and solid pikovskyayas medium with a decline in pH (Tank and Saraf 2003)







Screening and characterization of PGPR

0
1
2
3
4
5
6
7
8
9
10
4 8 12 16 20 24
Duration (h)
L
o
g
1
0

C
F
U
/
m
l
MS1 MS2 MS3 MS4 MS5


Figure 3.1 Logarithmic growth studies of selected PGPR strains


-10
0
10
20
30
40
50
60
0 7 14
Duration (Day's)
P
h
o
s
p
h
a
t
e

s
o
l
u
b
i
l
i
z
a
t
i
o
n

g
/
m
l
21
MS1 MS2 MS3 MS4 MS5

Figure 3.2 Phosphate solubilization by selected PGPR strains
74


Screening and characterization of PGPR


Isolate 0 day 7
th
day 14
th
day 21
st
day
MS1 7.2 4.34 4.24 3.37
MS2 7.2 4.17 4.07 4.01
MS3 7.2 3.90 3.84 3.33
MS4 7.2 4.49 4.05 3.47
MS5 7.2 5.30 4.55 4.51

Table 3.3 Change in pH during P solubilization up to 21
st
day after inoculation





0
5
10
15
20
25
30
MS1 MS2 MS3 MS4 MS5
Isolates
Z
o
n
e

o
f

P

s
o
l
u
b
i
l
i
z
a
t
i
o
n

(
m
m
)
24h 48h 72h 96h 120h

Figure 3.3 Zone of P solubilization during qualitative study by the selected PGPR


75


Screening and characterization of PGPR



Picture 3.1 Phosphate solubilization by the selected isolates


-10
0
10
20
30
40
50
60
0 72 96
Duration (h)
I
A
A

p
r
o
d
u
c
t
i
o
n

g
/
m
l
120
MS1 MS2 MS3 MS4 MS5


Figure 3.4 Indole acetic acid productions by selected PGPR strains.
76


Screening and characterization of PGPR
77

3.3.3 IAA Production by selected isolates
No detectable IAA like substances were determined in un-inoculated control broths. All
the five selected isolates showed significant production of IAA. Highest IAA production
was reported in MS1 (52 g/ml) after 96 h of incubation in dark followed by MS3 (47
g/ml), MS4 (39 g/ml), MS5 (32 g/ml) and MS2 (27 g/ml) (fig. 3.4). All the isolates
showed a continuous increase and decrease in the IAA production potential along with
increase in incubation time. Different isolates showed different optimum incubation time
for highest IAA production. It is estimated that about 80 % of soil bacteria possess IAA
producing potential (Patten and Glick 2002).Though reports reveal that IAA production
reaches maximum after 120 h (5 d) of incubation (Zimmer and Bothe 1988) many of our
isolates did not follow this pattern and showed maximum IAA production even after 240
h (10 d). However reports of other researchers (Bhattacharya and Pati 1999) showed that
IAA production was not detected after 5 d. Though it is reported that there is continuous
decrease in IAA production after reaching the peak production, this pattern was also
followed by our isolates. IAA production curves of the isolates showed continuous
increase and decrease up to 12 d. These types of curves are in agreement with the IAA
production curves reported by Rubio et al. (2000). The reason for such fluctuations could
be the utilization of IAA by the cells as nutrient during late stationary phase or
production of IAA degrading enzymes by the cells which are inducible enzymes in
presence of IAA (Bhattacharya and Pati 1999).
Holguin and Glick (2003) reported that IAA may be involved in the epiphytic fitness of
PGPR. The secretion of IAA by the bacterium may modify the micro-habitates of
epiphytic bacteria by increasing nutrient leakage of plant cells; enhanced nutrient
availability may better enable IAA producing bacteria to colonize the rhizosphere. Rubio
et al. (2000) reported a production of 34.24 g/ml of IAA by A. vinelandii where as
Chandra et al. (2007) reported a production of 24 g/ml of IAA by M. loti after 48 h of
incubation which is in correlation to our results. Tien et al. (1979) reported that presence
of 0.01g/ml of IAA significantly increased the weight of plant. Moreover, he revealed
that root system is more sensitive to auxin than shoot.


Screening and characterization of PGPR
78

He also reported that auxin especially promotes the growth of lateral roots on the main
root of oat seedling. Zimmer and Bothe (1988) also reported that roots of wheat seedlings
respond positively to addition of exogenous IAA by increase in wet weight and by
formation of lateral roots.
3.3.4 Exopolysaccharide (EPS) production by selected isolates
Maximum amount of EPS production was observed in isolate MS3 (33.6 mg/ml)
followed by MS1 (28 mg/ml), MS4 (27 mg/ml), MS2 (23 mg/ml) and MS5 (12 mg/ml)
(fig. 3.5) after five days of incubation. Mannitol and sucrose gives better production of
EPS as compared to other carbon sources. Maximum of EPS production occurs during
early stationary phase than in the late stationary of culture (Modi et al. 1989). Borgio et
al. (2009) reported three bacterial strains, Bacillus subtilis NCIM 2063, Pseudomonas
aeruginosa NCIM 2862 and Streptococcus mutans MTCC 1943 were examined for their
exopolysaccharide (EPS) producing ability at the laboratory level. The highest EPS
production was recorded in P. aeruginosa (226 g/ml) grown in nitrogen free medium
followed by S. mutans and B. subtilis (220 and 206 g/ml respectively) in nitrogen free
medium after 7 days of incubation at 37C. Kloepper et al. (1980) reported that
production of EPS by Burkholderia gladioli IN-26 a strain of PGPR reduced bacterial
speck on tomato. Similarly, Alami et al. (2000) reported that EPS produced by root
associated saprophytic bacterium (rhizobacterium) Pantoea agglomerans YAS34 was
associated with plant growth promotion of sunflower. Haggag (2007) reported that
Paenibacillus polymyxa produces a large amount of polysaccharide possessing high
activity against crown rot disease caused by Aspergillus niger in peanut plants.

3.3.5 Siderophore Production by selected isolates
Siderophore production by the isolates carried out on solid CAS blue agar showed a clear
zone of decolorization representing iron chelation by the isolate in the medium. Highest
zone of dye decolorization was observed in MS1 (24 mm), MS3 (22 mm), MS5 (21 mm)
where as MS4 and MS2 showed a zone of 20 mm and 18 mm after 120 h respectively


Screening and characterization of PGPR
79

(Table 3.4 and Pic. 3.2). The isolate MS5 showed siderophore production (32 g/ml)
followed by MS2 (28 g/ml), MS1 (25 g/ml), whereas MS3 and MS4 produced (22
g/ml) after 96 h of incubation (fig. 3.6). Siderophore production reduced thereafter on
further incubation up to 144 h. Qualitative and quantitative estimation of siderophore
production by Csakys method showed that all the five isolates produced hydroxamate
type of siderophore production. Increase in pH was observed with increase in siderophore
concentration. The pH increased from 6.8 to a maximum of 10 along with siderophore
production. Sarode et al. (2009) reported that A. calcoaceticus produced optimum
siderophore at 36 h of incubation period. Catechol type of siderophore was isolated from
supernatant of A. calcoaceticus and purified (60 mg/l) by using HP-20. Purified
siderophore of A. calcoaceticus showed positive CAS test, Csakys and Arnows test
confirming that it contains both of hydroxamate and catechol group. A. calcoaceticus has
also ability to synthesize IAA from tryptophan and solubilize tri-calcium phosphate.
Huddedar et al. (2002) have described plasmid pUPI126 mediated indole 3 acetic acid
(IAA) production in Acinetobacter strains from rhizosphere of wheat.
Chandra et al. (2007) reported production of 32 g/ml of hydroxamate type of siderophore
by M. loti after 48 h of incubation. Production of siderophore results in siderophore
mediated competition among the bacteria which further results into exclusion of
siderophore non producer pathogens from the rhizosphere due to lack of iron depletion for
sclerotia germination and hyphal growth. This was supported by Singh et al. (2008) who
showed that rhizosphere isolate Bacillus subtilis BN1 inhibited the growth of M.
phaseolina up to 60 %. Dileepkumar et al. (2001) reported that although all isolates
showed inhibition of phytopathogens, strains RBT 13 showed biocontrol potential even in
presence of iron while other isolates lost their biocontrol efficiency. This shows that
although siderophore acts as biocontrol agent there can be other mechanisms of biocontrol
by PGPR, like HCN, phenazines, chitinase, cellulose, -1,3 glucanase etc. The change in
pH in the medium during siderophore production was also shown by Budzikiewicz (1993)
who reported that alkalinity is important to avoid siderophore destruction showing that


Screening and characterization of PGPR

pyoverdins are labile in presence of acids or O
2
. On the contrary Sharma et al. (2003)
showed that higher pH is rather destructive to siderophores.

0
5
10
15
20
25
30
35
40
MS1 MS2 MS3 MS4 MS5
Isolates
E
P
S

p
r
o
d
u
c
t
i
o
n

(
m
g
/
m
l
)

Figure 3.5 Exopolysaccharide (EPS) production by selected PGPR strains


0
5
10
15
20
25
30
35
0 48 96 144 192
Duration (h)
S
i
d
e
r
o
p
h
o
r
e

p
r
o
d
u
c
t
i
o
n

g
/
m
l
MS1 MS2 MS3 MS4 MS5

Figure 3.6 Siderophore productions (Quantitative estimation) by selected isolates
80


Screening and characterization of PGPR



Picture 3.2 Siderophore productions (Qualitative test) by selected isolates



-20
0
20
40
60
80
100
0 10 11 12 1
Days
A
m
m
o
n
i
a

p
r
o
d
u
c
t
i
o
n


(

g
/
m
l
)
3
MS1 MS2 MS3 MS4 MS5

Figure 3.7 Ammonia productions by the selected PGPR

81


Screening and characterization of PGPR
82

Isolate 48 h (mm) 72 h (mm) 96 h (mm) 120 h (mm)
MS1 10 15 20 24
MS2 8 10 13 18
MS3 8 10 15 22
MS4 8 10 15 20
MS5 10 12 15 21

Table 3.4 Zone size produced during the qualitative test of Siderophore production by the
selected PGPR

3.3.6 Ammonia and HCN production by the selected isolates
Ammonia production was studied from 10
th
to 13
th
days of incubation as per method
given by Dye (1968). Maximum concentration of ammonia production was observed in
isolates MS5 and MS3 was 42 g/ml (10
th
d) and 42 g/ml (11
th
d) followed by MS1 41
g/ml (12
th
d), MS4 39 g/ml (11
th
d) and MS2 32 g/ml (12
th
d) (fig. 3.7). Consecutive
reading after 11
th
days of incubation showed that there was a decrease in ammonia
production in all isolates. This continued till 14 days. Maximum ammonia production
was observed at 11
th
day after that there is decrease in ammonia production. Ammonia
released by diazotrophs is one of the most important traits of PGPRs which benefits the
crop (Kundu 1987). This accumulation of ammonia in soil may increase in pH creating
alkaline condition of soil at pH 9-9.5. It suppresses the growth of certain fungi and
nitrobacteria due to it potent inhibition effect. It also upset the microbial community and
inhibits germination of spores of many fungi (Martin 1982). Christiansen et al. (1991)
have reported that level of oxygen in aerobic conditions was same as the level of
ammonia excretion under oxygen limiting conditions. However, Joseph et al. (2007)
reported ammonia production in 95% of isolates of Bacillus followed by Pseudomonas
(94.2%), Rhizobium (74.2%) and Azotobacter (45%).


Screening and characterization of PGPR
83

HCN production was checked in all isolates which showed significant results in
phosphate solubilization and IAA production potential. Out of these 5 isolates only 3
isolates showed HCN production after 48 and 72 h of incubation. Maximum HCN
production was observed in MS5 (Table 3.5) isolate followed by MS3 and MS1.
Presence or absence and intensity of HCN production can play a significant role in
antagonistic potential of bacteria against phytopathogens. Similar results were also
reported by Cattelan et al. (1999) who reported that production of cyanide was an
important trait in a PGPT in controlling fungal diseases in wheat seedlings under in-vitro
conditions. Chandra et al. (2007) reported production of HCN by the PGPR which was
inhibitory to the growth of S. sclerotium. Kumar et al. (2008) also reported in vitro
antagonism by HCN producing PGPR against sclerotia germination of M. phaseolina.
Production of HCN along with siderophore production has been reported as the major
cause of biocontrol activity for protection of Black pepper and ginger (Diby 2004).

Isolate 24h 48h 72h
MS1 nd + +
MS2 nd nd nd
MS3 nd + ++
MS4 nd nd nd
MS5 nd ++ +++

Table 3.5 HCN productions by the selected PGPR (+ low; ++ medium; +++ good; nd not
detected)




Screening and characterization of PGPR
84

3.3.7 ACC deaminase production by selected isolates
Study of ACC deaminase enzyme production by the selected 5 isolates MS1, MS2, MS3,
MS4 and MS5 showed that maximum ACC deaminase was produced by MS3 which was
82 nm -ketobutyrate/mg/h

followed by MS1 (79 nm -ketobutyrate/mg/h), MS5 (72 nm
-ketobutyrate/mg/h), MS2 (52 nm -ketobutyrate/mg/h) and MS4 (48 nm -
ketobutyrate/mg/h) (fig. 3.8). ACC deaminase enzyme production is considered as the
most important and highly desired trait for any rhizobacteria to act as a plant growth
promoting rhizobacteria.
Many researchers have reported the presence of this enzyme in all the effective PGPR
candidates. Shah et al. (1998) reported the presence of ACC deaminase activity in
different bacteria like E. coli, Pseudomonas and Enterobacter where maximum ACC
deaminase activity (507 M/mg/ml) was reported in P. putida ATCC 17399/pRK-ACC.
Belimov et al. (2007) observed ACC deaminase activity in P. brassicacearum and
reported that this activity is not reduced when P. brassicacearum were made resistant to
rifampicin. Yet the activity was influence by tagging the wild type isolates to different
types of stress adapters or resistances, probably due to increased metabolic load caused
by tagging. A. brasiliense mutants A. brasiliense Cd/pRKLACC produced 16 M/mg/ml
of -ketobutryic acid where as A. brasiliense Cd/pRKTACC mutant produced only 11
M/mg/ml of -ketobutryic acid (Holgiun and Glick 2003). Grichko and Glick (2001)
and Grichko et al. (2000) have also found that transgenic plants expressing ACC
deaminase were protected from different stresses like flooding and heavy metals. This
enzyme facilitates plant growth as a consequence of the fact that it sequesters and cleaves
plant produced ACC, thereby lowering the level of ethylene in the plant. In turn,
decreased ethylene levels allow the plant to be more resistant to a wide variety of
environmental stresses, all of which induce the plant to increase its endogenous level of
ethylene; stress ethylene exacerbates the effects of various environmental stresses (Saraf
et al. 2010).



Screening and characterization of PGPR


0
10
20
30
40
50
60
70
80
90
MS1 MS2 MS3 MS4 MS5
Isolates
A
C
C

d
e
a
m
i
n
a
s
e

a
c
t
i
v
i
t
y








(
n
m

-
k
e
t
o
b
u
t
y
r
a
t
e

m
g
-
1

h
-
1
)

Figure 3.8 ACC deaminase productions by selected isolates















85


Screening and characterization of PGPR
86

3.3.8 Antibiotic Resistance
Study of antibiotic resistance pattern of selected isolates showed that MS5 was most
resistant organism against the tested antibiotics. It showed resistance against 15 different
antibiotics sensitivity/resistance assay of isolate revealed that this strain is sensitive to
amikacin, ampicillin, chloramphenicol, ciprofloxacin, colistin, gentamicin, netillin,
norfloxacin, tobramycin, piperacillin, where as resistant to carbenicillin, ceftazidime and
cephoxitin.
Higher sensitivity of strain to clinical antibiotics is consistent with the fact that this is a
rhizosphere isolate. Where as MS4 and MS3 showed resistance towards 14 and 13
different antibiotics. Isolates MS1 and MS2 showed resistance towards 9 and 11 different
antibiotics respectively. Thus MS1 was the most antibiotic sensitive isolate where as
MS5 was the most resistant isolate (Table 3.6). Tetracycline, Ciprofloxicin, Nalidixic
acid and Gentamycin were the most effective antibiotics amongst all where as Ampicillin
was the least effective for all isolates except MS4.

















Screening and characterization of PGPR
87


Antibiotic Conc.
(g)
MS1 MS2 MS3 MS4 MS5
Tetracycline 30 0 12 13 14 17
Ampicillin 10 0 7 0 11 9
Ciprofloxacion 10 23 33 34 17 20
Colistin 10 0 0 0 0 10
Cotrimazole 25 0 10 0 18 20
Gentamycin 10 20 14 22 20 15
Nitrofurantoin 300 0 0 0 0 0
Streptomycin 10 18 21 15 10 0
Cephaloxime 30 0 21 23 8 12
Cephalexin 30 0 0 0 0 18
Chloramphenicol 30 0 0 10 13 15
Nalixidic acid 30 10 0 11 12 9
Furazolidene 50 0 0 8 0 0
Norfloxacin 10 12 0 14 22 15
Oxytetracycline 30 16 20 30 10 13
Ticarcillin 75 12 22 13 18 11
Gentamycin 10 21 22 30 12 12
Trimethoprim 1.25 0 0 0 0 0
Sulphametho
Xazole
25 10 13 11 12 17

Table 3.6 Zone diameter (mm) of antibiotic sensitivity pattern of selected strains

3.3.9 Morphological and biochemical study of the selected isolates
The results of gram staining reveal that all the five selected isolates were having
following characteristics.




Screening and characterization of PGPR
88

MS1: Gram positive rods in chains, colonies on agar opaque with glistering surface, and
stained with the color of crystal violet during grams staining and identified as
Brevibacillus brevis
MS2: Gram negative short rods, imtermediate in size of mucoid and glistering colony,
motile by peritricate flagella, facultative anaerobic strain was identified as Enterobacter
cloacae.
MS3: Gram positive rods in chains, colonies on agar opaque with rough surface, strongly
attached with agar and stained with the color of crystal violet during grams staining and
identified as Bacillus licheniformis.
MS4: Gram positive coccus, colonies were yellow have a granular surface with matt
appearance, growing in irregular clusters of tetrads, spheres 0.9-1.8 m in diameter and
identified as Micrococcus sps.
MS5: Gram negative short rods, non motile, forming smooth, colorless colony on nutrient
agar, were identified as Acinetobacter calcoaceticus.
All the five strains showed presence of enzymes like catalase, oxidase and dehydrogenase
except MS5 which shows oxidase negative. All isolates showed utilization of citrate
except MS4 where as MS1, MS2, MS3 and MS4 showed hydrolysis of gelatin but urea as
well as phenylalanine utilization was observed only in MS1. Formic acid fermentation
was observed in only MS4 whereas acetoin production was reported only in MS3. None
of the isolates showed indole production whereas hydrolysis of casein was observed in
MS1 and MS5 and starch utilization was observed in MS3 and MS5 both (Table 3.7). No
H
2
S production was reported in lead acetate strip kept in peptone water. Results of Triple
sugar iron slant show that all isolates could utilize sugars aerobically thereby turning
slant pink due to increased pH. Anaerobic fermentation was observed only in MS3 and
MS5 making the butt acidic thereby turning it yellow. None showed H
2
S production or
gas production in the butt region (Table 3.8).



Screening and characterization of PGPR
89



Test MS1 MS2 MS3 MS4 MS5
Lactose Fermentation -ve +ve -ve -ve -ve
Gelatine hydrolysis +ve + ve +ve +ve -ve
Starch hydrolysis -ve -ve +ve -ve +ve
Caesin hydrolysis +ve -ve -ve -ve +ve
Formic acid fermentation -ve -ve -ve +ve -ve
Acetoin Detection -ve -ve +ve -ve -ve
Indole production -ve -ve -ve -ve -ve
Urea utilization +ve -ve -ve -ve -ve
Nitrate reduction -ve -ve +ve -ve -ve
Ammonia production -ve -ve +ve +ve -ve
Catalase test +ve -ve +ve +ve +ve
Oxidase test +ve +ve +ve +ve -ve
Dehydrogenase test +ve +ve +ve +ve +ve
Citrate Utilization +ve +ve +ve -ve +ve
Phenyle alanine utilization +ve -ve -ve -ve -ve

Table 3.7 Biochemical characteristics of selected strains


Test MS1 MS2 MS3 MS4 MS5
Slant Alkaline Alkaline Alkaline Acidic Alkaline
Butt Alkaline Alkaline Acidic Alkaline Acidic
H
2
S production -ve -ve -ve -ve -ve
Gas production -ve -ve -ve -ve -ve

Table 3.8 Results of TSI test







Screening and characterization of PGPR
90


3.3.10 Carbohydrate utilization profile of selected isolates
Carbon source utilization pattern of the selected isolates showed that some of the isolate
released only acid during the utilization of different carbon sources. Carbon source
utilization profile of MS2 shows that the isolate was able to easily utilize Mannitol,
Rhamnose, Cellobiose, Mellibiose, Sorbitol and Arabinose where as produced only acid
but not gas when utilized carbon sources like Rhamnose and Salicin. MS2 utilized all the
C sources used during experiment accept Inuline, Dulcitol, Adonitol, and Inositol.
MS4 and MS5 shows that it readily utilized sugars like Mannitol, Trehalose, Galactose,
Xylose, Sucrose, Maltose, Fructose, Arbinose and Dextrose where as it could not use
sugars like Inositol, Adonitol, Lactose, Dulcitol and Inulin (Table 3.9). MS5 showed
utilization of fewer carbon sources like Mannitol, Mellibiose, Sorbitol, Sucrose, Salicin
and Dextrose where as it lately utilized Mannose and Lactose followed by other carbon
sources mentioned in the list. While both the isolates produced only gas when utilize the
carben source salicin.
MS1 and MS3 showed almost similar carbon source utilization pattern. MS1 and MS3
showed utilization of Mannitol, Rhamnose, Cellobiose, Sorbitol, Raffinose, Dulcitol,
Inulin, Galactose, Xylose, Sucrose, Salicin, Lactose, Maltose, Fructose, Arabinose and
Dextrose at higher rate along where as it could not utilize C sources like Lactose,
Inositol, adonitol and Dulcitol. The pattern showed that it is the only isolate which could
use maximum number of different Carbon sources with acid and gas production. (Table
3.9). Results show that Dextrose, Sucrose, Fructose, Mannitol and Maltose were used by
all isolates where as C sources like Lactose were utilized only by MS2. Ability to utilize
different carbon sources helps the organism to survive under deficiency conditions of
their conventional carbon sources.





Screening and characterization of PGPR
91


Sugar MS1 MS2 MS3 MS4 MS5
Mannitol +/+ +/+ +/+ +/+ +/+
Trehalose +/+ +/+ +/+ +/+ +/+
Rhamnose +/+ +/- +/+ +/+ +/+
Cellobiose +/+ +/+ +/+ +/+ +/+
Inositol -/- -/- -/- -/- -/-
Mannose +/+ +/+ +/+ +/+ +/+
Melibiose +/+ +/+ +/+ +/+ +/+
Sorbitol +/+ +/+ +/+ +/+ +/+
Adonitol -/- -/- -/- -/- -/-
Raffinose +/+ +/+ +/+ +/+ +/+
Dulcitol -/- -/- -/- -/- -/-
Inuline -/- -/- -/- -/- -/-
Galactose +/+ +/+ +/+ +/+ +/+
Xylose +/+ +/+ +/+ +/+ +/+
Sucrose +/+ +/+ +/+ +/+ +/+
Salicin +/- +/- -/- -/+ -/+
Lactose -/- +/+ -/- -/- -/-
Maltose +/+ +/+ +/+ +/+ +/+
Fructose +/+ +/+ +/+ +/+ +/+
Arabinose +/+ +/+ +/+ +/+ +/+
Dextrose +/+ +/+ +/+ +/+ +/+

Table 3.9 Carbohydrate utilization profile of selected strains (+/+ acid/gas both positive; -
/- acid/gas both negative)

3.3.11 FAME (Fatty acid methyl ester) analysis and 16S rRNA sequencing
FAME analysis of the isolate MS1 showed presence of major fatty acids peaks C
15:0 anteiso

(34.11 %), C
15:0 iso
(28.97 %) and C
17:1 iso 10c
(6.35 %). Other fatty acids separated by
GLC with the MIDI system are shown in Table 3.3.11.1 and Figure 3.3.11.1.


Screening and characterization of PGPR
92

On the basis of standard database of mini API the isolate showed maximum similarity
index (0.381) with Bacillus flexus which was further confirmed as Brevibacillus brevis by
16S rRNA gene sequencing.
FAME analysis of the isolate MS3 showed presence of major fatty acids peaks C
15:0 anteiso

(39.96 %), C
15:0 iso
(29.67 %), C
17:1 iso
(8.10 %) and C
17:1 anteiso
(10.29 %). Other fatty acids
separated by GLC with the MIDI system are shown in Table 3.3.11.2 and Figure
3.3.11.2. On the basis of standard database of mini API the isolate showed maximum
close similarity index with Bacillus subtilis (0.721) and Bacillus licheniformis (0.692)
which was further confirmed as Bacillus licheniformis by 16S rRNA gene sequencing.
FAME analysis of the isolate MS5 showed presence of fatty acids peaks C
12:0
(9.00 %),
C
14:0
(8.32 %), C
16:0
(19.66 %) and C
17:0 cyclo
(7.98 %). Other fatty acids shared the major
cellular fatty acid could not be separated by GLC with the MIDI system. This may belong
to the groups of two or three fatty acids and has been reported as summed feature 3
(16.18 %), summed feature 2 (12.37 %) and summed feature 8 (11.64 %) (Table 3.3.11.3
and Figure 3.3.11.3). On the basis of standard database of mini API the isolate showed no
match with any organism and so by 16S rRNA gene sequencing it was confirmed as
Acinetobacter calcoaceticus.
Phylogenetic analysis based on 16S rRNA gene sequences available from the European
Molecular Biology Laboratory data library constructed after multiple alignments of data
by ClustalX. Distances and clustering with the neighbor-joining method was performed
by using the software packages Mega version 4.0. Bootstrap values based on 500
replications are listed as percentages at the branching points.
The strain MS1 formed a separate branch in neighbor-joining (fig. 3.9) and was grouped
most closely to a cluster containing to Brevibacillus brevis B15 [AY591911] with 93 %
sequence similarity.



Screening and characterization of PGPR

93



The strain MS5 showed a separate branch in neighbor-joining (fig. 3.13) and was
grouped most closely to a cluster containing to Acinetobacter baumannii N1 [FJ887883]
and Acinetobacter calcoaceticus [EU921468] with 85 % and 100 % of sequence
similarity so they confirmed as Acinetobacter calcoaceticus.
The strain MS3 showed a separate branch in neighbor-joining (fig. 3.11) and was
grouped most closely to a cluster containing to Bacillus licheniformis SB 3131
[GU191917] and Bacillus licheniformis [AY479984] with 70 % and 100 % of sequence
similarity so they confirmed as Bacillus licheniformis. Based on nucleotide homology
and phylogenetic analysis the microbe, which was labeled as MS4 was detected to be
Micrococcus sp. CTSP34 (GenBank Accession Number: EU855211.1) (fig. 3.12).
Alignment view (Table 3.12) using combination of NCBI GenBank databases and
distance Matrix Table (Table 3.13) generated using Sample MS4 with ten closest
homolog microbes. Diagonal in the table indicates nucleotide similarity and below
diagonal distance identities.
The strain MS2 formed a separate branch in neighbor-joining (fig. 3.10) and based on
nucleotide homology and phylogenetic analysis the bacteria, which was labeled as MS2
was detected to be uncultured bacterium clone N4.5 sp. (GenBank Accession Number:
EF179835.1). Nearest homolog species was found to be Enterobacter cloacae sp.
(Accession No. AY335554.1) and so they confirmed as Enterobacter cloacae. Alignment
view for MS2 (Table 3.10) using combination of NCBI GenBank databases and distance
Matrix Table (Table 3.11) generated using Sample MS2 with ten closest homolog
microbes. Diagonal in the table indicates nucleotide similarity and below diagonal
distance identities.




94
min 0.5 1 1.5 2 2.5 3 3.5 4
pA
15
20
25
30
35
40
F ID1 A, (E 08826.588\A0041226.D)

0
.
7
3
4

1
.
5
8
4

1
.
6
1
1

1
.
6
2
9

1
.
6
7
4

1
.
8
4
2

1
.
8
6
7

1
.
9
2
2

2
.
1
2
9

2
.
1
6
1

2
.
2
3
9

2
.
3
4
3

2
.
3
7
2

2
.
4
3
7

2
.
4
6
7

2
.
4
8
5

2
.
5
0
4
2
.
5
5
2

2
.
6
0
8

2
.
6
8
6

2
.
7
1
6

2
.
7
5
6

2
.
7
8
2

2
.
8
0
3

2
.
8
2
2

2
.
8
7
3

2
.
9
3
1

3
.
0
0
7

3
.
0
3
8
3
.
0
7
7

3
.
1
0
9

3
.
1
2
5

3
.
1
6
2

3
.
1
9
1

3
.
2
2
6

3
.
2
6
6

3
.
3
1
5

3
.
3
5
6

3
.
3
7
2

3
.
3
9
8

3
.
4
2
3

3
.
4
4
7

3
.
4
6
1

3
.
4
9
8

3
.
5
1
3

3
.
5
3
3

3
.
5
6
0

3
.
5
7
7
3
.
6
1
3

3
.
6
3
6

3
.
6
5
3

3
.
7
3
4

3
.
7
8
0

3
.
7
9
2

3
.
8
1
5

3
.
8
8
5

3
.
9
3
9

3
.
9
6
6

4
.
0
3
8

Figure 3.3.11.1 Chromatogram of fatty acid profile study using FAME analysis of isolate Brevibacillus brevis MS1


Screening and characterization of PGPR





Screening and characterization of PGPR

95





















Table 3.3.11.1 Qualitative study of fatty acid profile for Brevibacillus brevis MS1



C
17:0 iso
3.0774 3.92
C
17:0 anteiso
3.1088 1.58
C
12:0 iso
1.5844 0.17
C
12:0
1.6736 0.13
C
13:0 iso
1.8416 2.80
C
13:0 anteiso
1.8665 1.36
C
14:0 iso
2.1293 2.25
C
15:1 iso 9c
2.3720 0.28
C
15:0 iso
2.4374 28.97
C
15:0 anteiso
2.4671 34.11
C
16:1 7c OH
2.6856 4.93
C
16:0 iso
2.7558 2.63
C
16:1 11c
2.8032 1.78
C
16:0
2.8735 1.57
C
17:1 iso 10c
3.0070 6.35
Summed Feature 4 3.0378
Fatty acid peak Retention Time Percent
3.52



96
min 0.5 1 1.5 2 2.5 3 3.5 4
pA
15
20
25
30
35
40
45
50
F I D 1 A , (E 08826.588\A 0051227.D )

0
.
7
3
4

1
.
6
7
3

1
.
8
4
1

2
.
1
2
9

2
.
2
3
9

2
.
4
3
7

2
.
4
6
7

2
.
5
5
2

2
.
6
8
5

2
.
7
1
6

2
.
7
5
6

2
.
7
8
1

2
.
8
0
3

2
.
8
7
3

2
.
9
0
0

2
.
9
2
5

2
.
9
5
7

3
.
0
0
6

3
.
0
3
7

3
.
0
7
7

3
.
1
0
9

3
.
3
1
5

3
.
4
2
2

3
.
4
4
7

3
.
4
6
2

3
.
5
1
3

3
.
5
7
3

3
.
6
0
5

3
.
7
3
4

3
.
8
8
7

3
.
9
0
1

4
.
0
3
7

4
.
2
7
1

Figure 3.3.11.2 Chromatogram of fatty acid profile study using FAME analysis of isolate Bacillus licheniformis MS3


Screening and characterization of PGPR





Screening and characterization of PGPR

97










Table 3.3.11.2 Qualitative study of fatty acid profile for Bacillus licheniformis MS3







C
17:0 iso
3.0372 8.10
C
17:0 anteiso
3.0773 10.29
C
18:0
3.4615 0.26
C
12:0
0.7343 0.10
C
13:0 iso
1.6727 0.18
C
14:0 iso
1.8411 1.28
C
14:0
2.1291 0.44
C
15:0 iso
2.2395 29.67
C
15:0 anteiso
2.4370 36.96
C
16:1 7c OH
2.5522 0.53
C
16:0 iso
2.7158 4.61
C
16:1 11c
2.7810 0.74
C
16:0
2.8032 3.51
C
15:0 iso 3 OH
2.9002 0.52
C
17:1 iso 10c
2.9567 0.88
Summed Feature 4 3.0060
Fatty acid peak Retention Time Percent
0.67



98
min 0.5 1 1.5 2 2.5 3 3.5 4
pA
15
20
25
30
35
40
45
50
F I D 1 A , (E 08826.588\A 0031225.D )

0
.
7
3
4

1
.
0
6
5

1
.
2
4
4

1
.
4
2
8

1
.
6
2
9

1
.
6
7
3

1
.
9
4
4

2
.
0
0
4

2
.
0
6
6

2
.
0
8
6

2
.
1
0
4

2
.
1
9
0

2
.
2
2
6

2
.
2
3
9

2
.
4
0
1

2
.
5
5
2

2
.
6
0
8

2
.
6
8
8

2
.
7
1
8

2
.
7
3
6

2
.
7
8
1

2
.
8
2
2

2
.
8
5
0

2
.
8
7
3

3
.
1
6
8

3
.
1
9
5

3
.
3
2
5

3
.
3
3
8

3
.
3
8
6

3
.
4
1
2

3
.
4
2
2

3
.
4
4
7

3
.
4
6
4

3
.
5
1
2

3
.
6
1
2

3
.
6
5
9

3
.
7
3
4

3
.
8
0
3

4
.
0
3
8

Figure 3.3.11.3 Chromatogram of fatty acid profile study using FAME analysis of isolate Acinetobacter calcoaceticus MS5


Screening and characterization of PGPR






Screening and characterization of PGPR





Fatty acid peak Retention Time Percent
C
10:0
1.2440 0.32
C
12:0
1.6730 9.00
C
13:0
1.9442 0.16

C
12:0 2 OH
2.0037 0.95

C
12:0 3 OH
2.0863 1.68

C
14:0
2.2392 8.32

C
16:1 7c OH
2.6881 1.70
Summed Feature 2 2.7185 12.37
C
16:0 N OH
2.7360 1.69
Summed Feature 3 2.8216 16.18
C
16:0
2.8732 19.66

C
17:0 cyclo
3.1676 7.98

C
17:0 10-CH3
3.3253 1.05

C
18:1 9c
3.4474 4.94
Summed Feature 8 3.4641 11.64
C
18:0
3.5123 0.71
C
19:0 cyclo 8c
3.8027 0.50

Table 3.3.11.3 Qualitative study of fatty acid profile for Acinetobacter calcoaceticus
MS5









99


Screening and characterization of PGPR

100
[DQ444285] Brevibacillus brevis YJ011

Figure 3.9 Phylogenetic analysis based on 16S rRNA gene sequences of MS1

Figure 3.10 Phylogenetic analysis based on 16S rRNA gene sequences of MS2


[EU605978] Brevibacillus brevis B12
[DQ431897] Uncultured low G+C bacterium clone 3G02-02
[AY591911] Brevibacillus brevis B15
Brevibacillus brevis MS1
[EF690427] Brevibacillus formosus isolate M13-7
[AF378234] Brevibacillus formosus LMG 16101
[AB112712] Brevibacillus formosus DSM 9885T
[EF368355] Brevibacillus agri 681-1
[AY897210] Brevibacillus brevis ZJY-1
[D78460] Brevibacillus formosus
[D78459] Brevibacillus choshinensis 100
38
38
22
EF179826.1
EU571123.1
EU047701.1
EF198245.1
AY946283.1
DQ068819.1
MS-2
EF179835.1
AY335554.1
FJ560465.1
EF179834.1
100
50
49
36
39
53
80
96
0.000237
-0.000007
-0.000004
0.002950
-0.000004
0.000542
0.000739
0.002485
0.002737
0.001473
0.001473
0.001935
0.001788
0.000383
0.000058
0.000004
0.000092
0.000440
0.001323
0.001668
93
49
55
100
69





Screening and characterization of PGPR
Alignment
Result
Alignment View ID
Description

Consensus
0.95
Sample MS-2 16S rDNA

EF179826.1
0.95 Uncultured bacterium clone 4.3 16S ribosomal RNA gene

EU047701.1 0.95 Enterobacter aerogenes strain HC050612-1 16S ribosomal RNA gene

EU571123.1 0.95 Enterobacter sp. 1-13 16S ribosomal RNA gene

FJ560465.1 0.96 Pantoea sp. M1R3 16S ribosomal RNA gene

EF198245.1 0.96 Enterobacter sp. MACL08B 16S ribosomal RNA gene

F179834.1 0.93 Uncultured bacterium clone N4.3 16S ribosomal RNA gene

AY335554.1 0.95 Enterobacter aerogenes strain HK 20-1 16S ribosomal RNA gene

EF179835.1 0.94 Uncultured bacterium clone N4.5 16S ribosomal RNA gene

AY946283.1 0.96 Enterobacter sp. 22-2005 16S ribosomal RNA gene

DQ068819.1 0.95 Uncultured bacterium clone f6s5 16S ribosomal RNA gene

Table 3.10 Alignment view using combination of NCBI GenBank databases for MS2
101


102


Distance Matrix
1 2 3
4
5 6 7 8 9 10 11
EU571123.1 1 --- 0.993 0.996 0.990 0.993 0.992 0.996 0.991 0.991 0.993 0.995
EF179834.1 2 0.007 --- 0.995 0.995 0.999 0.991 0.995 0.990 0.990 0.993 0.996
EU047701.1 3 0.004 0.005 --- 0.994 0.996 0.996 1 0.996 0.996 0.998 0.999
AY335554.1 4 0.010 0.005 0.006 --- 0.996 0.996 0.994 0.990 0.990 0.993 0.995
FJ560465.1 5 0.007 0.001 0.004 0.004 --- 0.992 0.996 0.991 0.991 0.993 0.996
EF179835.1 6 0.008 0.009 0.004 0.004 0.008 --- 0.996 0.992 0.992 0.996 0.996
EF179826.1 7 0.004 0.005 0.000 0.006 0.004 0.004 --- 0.996 0.996 0.998 0.999
AY946283.1 8 0.009 0.010 0.004 0.010 0.009 0.008 0.004 --- 0.997 0.996 0.995
DQ068819.1 9 0.009 0.010 0.004 0.010 0.009 0.008 0.004 0.003 --- 0.996 0.995
EF198245.1 10 0.007 0.007 0.002 0.007 0.007 0.004 0.002 0.004 0.004 --- 0.997
Contig1 11 0.005 0.004 0.001 0.005 0.004 0.004 0.001 0.005 0.005 --- 0.003


Screening and characterization of PGPR

Table 3.11 Distance Matrix Table generated using Sample MS2




Screening and characterization of PGPR

103
[EU158367] Bacillus licheniformis Am-2


[FJ190075] Bacillus licheniformis Am2
[DQ071568] Bacillus licheniformis MKU 8
[AY479984] Bacillus licheniformis
[GU191917] Bacillus licheniformis SB 3131
[HQ179577] Bacillus licheniformis MS3
[AY728013] Bacillus subtilis JM4
[EU672846] Bacillus licheniformis TS-01
[EU231634] Bacillus licheniformis TCCC11029
[AB188216] Bacillus sp. TUT1217
[EF059752] Bacillus licheniformis
[DQ993676] Bacillus licheniformis BCRC 15413
76
71
100
99
100
70
100
87
44


Figure 3.11 Phylogenetic analysis based on 16S rRNA gene sequences of MS3





Figure 3.12 Phylogenetic analysis based on 16S rRNA gene sequences of MS4
GQ856255.1
FJ380958.1
EU379292.1|
FJ357606.1
FJ357601.1
EU379288.1
MS4
EU855211.1
EU005372.1
FJ217189.1
FJ217190.1
45
53
50
67
47
99
33
75
0.005382
0.000550
-0.000003
-0.000005
0.001260
0.000736
0.000735
0.000690
0.000000
0.000631
0.000000
0.000002
0.000104
0.000502
0.000187
0.004519
0.000316
0.000353
0.000285
0.000781




Screening and characterization of PGPR
Alignment
Result
Alignment View ID
Description

Consensus
0.89
Sample MS4 16s rDNA
FJ357601.1
0.94 Micrococcaceae bacterium BBN3B-01d 16S ribosomal RNA gene
EU379292.1 0.93 Micrococcus luteus strain 5N-5 16S ribosomal RNA gene
EU379288.1 0.94 Micrococcus luteus strain 4RS-9d 16S ribosomal RNA gene
EU855211.1 0.93 Micrococcus sp. CTSP34 16S ribosomal RNA gene
GQ856255.1 0.93 Micrococcus luteus 16S ribosomal RNA gene
FJ380958.1 0.94 Micrococcus luteus strain BQN1B-04d 16S ribosomal RNA gene
EU005372.1 0.92 Micrococcus endophyticus strain YIM 56238 16S ribosomal RNA gene
FJ217189.1 0.92 Micrococcus luteus strain BQAB-01d 16S ribosomal RNA gene
FJ357606.1 0.94 Micrococcus sp. BBN3N-02d 16S ribosomal RNA gene
FJ217190.1 Micrococcus luteus strain BQAB-02d 16S ribosomal RNA gene 0.92

Table 3.12 Alignment view using combination of NCBI GenBank databases


104


105

Distance Matrix
1 2 3
4
5 6 7 8 9 10 11
EU379288.1 1 --- 0.997 0.996 0.993 0.991 0.992 0.997 0.996 0.992 0.992 0.981
FJ380958.1 2 0.003 --- 0.997 0.994 0.993 0.993 0.999 0.999 0.993 0.995 0.981
EU379292.1 3 0.004 0.003 --- 0.991 0.990 0.990 0.996 0.996 0.990 0.992 0.984
EU005372.1 4 0.007 0.006 0.009 --- 0.999 0.999 0.994 0.993 0.999 0.989 0.986
EU855211.1 5 0.009 0.007 0.010 0.002 --- 0.998 0.993 0.992 0.998 0.987 0.986
FJ217189.1 6 0.008 0.007 0.010 0.001 0.002 --- 0.993 0.993 1 0.988 0.985
FJ357601.1 7 0.003 0.002 0.004 0.006 0.007 0.007 --- 0.999 0.993 0.993 0.981
FJ357606.1 8 0.004 0.001 0.004 0.007 0.008 0.007 0.001 --- 0.993 0.994 0.980
FJ217190.1 9 0.008 0.007 0.010 0.001 0.002 0.000 0.007 0.007 --- 0.988 0.985
GQ856255.1 10 0.008 0.005 0.008 0.011 0.013 0.012 0.007 0.006 0.012 --- 0.980
Consensus 11 0.019 0.019 0.016 0.014 0.014 0.015 0.019 0.020 0.015 --- 0.020


Screening and characterization of PGPR

Table 3.13 Distance Matrix Table generated using Sample MS4




Screening and characterization of PGPR




[EU921468] Acinetobacter calcoaceticus HIRFA36
[EU921466] Acinetobacter calcoaceticus HIRFA33
[EU921469] Acinetobacter calcoaceticus HIRFA37
[EU919797] Uncultured bacterium clone b89
[HQ179580] Acinetobacter calcoaceticus MS5
[FJ887883] Acinetobacter baumannii N1
[FJ457253] Acinetobacter sp. S275
[EU921465] Acinetobacter sp. HIRFA32
[EU921462] Acinetobacter calcoaceticus HIRVA26
[EU921461] Acinetobacter calcoaceticus GWRVA25
[EU921460] Acinetobacter calcoaceticus GWRVA22
[EU921459] Acinetobacter calcoaceticus GWRVA21
100
100
25
15
100
85
62
52
28


Figure 3.13 Phylogenetic analysis based on 16S rRNA gene sequences of MS5

3.3.11.1 Nucleotide sequence deposited
Sequence data were aligned and analyzed for finding the closest homology. Sequence
data reported in present study has been deposited in the GenBank nucleotide sequence
database under the accession numbers HQ179578 for MS2, HQ179577 for MS3,
HQ179579 for MS4 and HQ179580 for MS5. while accession number for MS1 is in the
process.
3.3.12 Seed bacterization study
Germination parameters was observed to know the extent of completeness of
germination, rapidity of germination and peak of germination which reflects the quality
of seeds, seedling produced using bacterial treatments. Seed germination is the process
where the radical and plumule of the seed emerge out from seed coat when favourable
environment is acquainted. Daily record of seed (Table 3.14) that had emerged out of the
surface of soil was kept.
106


Screening and characterization of PGPR
107

Recording of germination was carried up to 22 days (pic. 3.3) and at the end of 22 days
all the seeds that had not germinated were taken out. These ungerminated seeds were
counted and they were cut open to find whether they were still viable or not. The highest
germination percentage (Table 3.15) was observed in MS5 (63.33%) and the germination
capacity (80%) followed by MS3 (60%) with germination capacity (73.33%). The
germination capacity of one seed, based on a binary answer (germinated/non
germinated), is one qualitative attribute of the germination process, generally converted
in a quantitative attribute, commonly percentage. The lowest germination percentage was
recorded from uninoculated control (40%).
Germination energy is the percent by number of seed in a given sample which germinate
up to the time of peak germination. Where peak germination is the highest number of
germination in a particular day (William 1985). Germination value is a measure
combining speed and completeness of seed germination with a single figure where
germination speed was calculated as sum of the number of newly germinated seed at time
t divided by number of days since sowing (Czabator 1962). The highest germination
speed (5.33) was shown on the 6
th
day of the seed sown which is very fast in comparision
to the uninoculated control and the highest germination energy was shown by MS5
(29.77). Seedling vigor Index of the seedlings was calculated according to Abdul-Baki
and Anderson (1973) as germination percent (X) Seedling total length.
In our study the maximum seedling vigor index (929.05) was reported in MS5 followed
by MS3 (895.8) and minimum (542.8) was in the control test. Vigor index reflects the
health of the seedlings produced and so it takes into account the germination percent and
radical length. Higher the value of vigor index betters the seedling health. Generally
mechanical scarification and chemical treatments turn out to be an excellent treatment to
overcome seed dormancy as reported earlier in the case of hard coated seeds in different
studies.



Screening and characterization of PGPR
108

This agrees with results from several authors (Carvalho et al. 1980) for Erythrina
speciosa; Bianchetti and Ramos (1981) for Peltophorum dubium; Candido et al. (1982)
for Enterolobium contortisiliquum; Nassif and Perez (1997) for Pterogyne nitens; Jeller
and Perez (1999) for Cassia excelsa; and Lopes et al. (1998) for Caesalpinea ferrea,
Cassia grandis and Samanea saman). Seed treatments involving water soaking and
sulfuric acid for 5 or 15 minutes were inefficient to break dormancy of E.
contortisiliquum seeds.
The best recorded results of total germination, first count of germination test and speed of
germination index were obtained with mechanical scarification, chemical scarification
(30, 60, 120 or 180 minutes) and mechanical scarification followed by water soaking at
room temperature. Mechanical scarification should be considered as the best treatment to
overcome "timburi" seed dormancy if practical aspects are important as in forest
nurseries of tropical countries. In our case treatment of Jatropha curcas seeds with
bacterial culture shows excellent results for different germination parameter and seeds
were found more viable after cutting than the mechanical and chemical scarification.
Vivas et al. (2005) reported that B. brevis increased the presymbiotic growth
(germination rate growth and mycelial development) of Glomus mosseae. Spore
germination and mycelial development of both G. mosseae isolate were reduced as much
as the amount of Cd or Zn increased in the growth medium. In medium supplemented
with 20 g Cd/ml, the spore germination was only 12% after 20 days of incubation, but
the coinoculation with B. brevis increased this value to 40% after only 15 days. The
corresponding bacterial effect increasing mycelial growth ranged from 125% (without
Zn) to 232% (200 g Zn/ml) in the case of G. mosseae isolated from Zn-polluted soil.
Mycelial growth under 5 g Cd/ml (without bacterium) was similarly reduced from that
produced at 15 g Cd/ml in the presence of the bacteria. As well, 50 g Zn/ml (without
bacterium) reduced hyphal growth as much as 200 g Zn/ml did in the presence of B.
brevis.



Screening and characterization of PGPR



Picture 3.4 Effect of selected isolates of PGPR after 30 DAS on the growth of Jatropha
curcas
109




Picture 3.3 Germination of Jatropha treated seeds after 22 day after sowing






Screening and characterization of PGPR
Treatments (T)/ Replicates (r)
C T
1
T
2
T
3
T
4
T
5
Day
r
1
r
2
r
3
r
1
r
2
r
3
r
1
r
2
r
3
r
1
r
2
r
3
r
1
r
2
r
3
r
1
r
2
r
3
DT C T CG% Peak
Value
Germi.
Speed
1st
2nd
3rd
4th 1 1 1 1 4 4 2.22 0.55 1
5th 2 1 2 2 2 2 2 1 2 1 1 2 1 2 23 27 15 3 4.6
6th 3 2 3 2 1 3 1 3 2 2 1 2 1 1 2 1 2 32 59 32.77 5.46 5.33
7th 1 1 1 2 2 1 2 2 1 1 2 1 1 1 2 21 80 44.44 6.34 3
8th 1 1 1 2 1 1 1 1 1 1 1 2 1 15 95 52.77 6.59 1.87
9th 1 1 1 1 1 5 100 55.55 6.17 0.55
10th 1 2 1 1 5 105 58.33 5.83 0.5
11th 2 1 1 1 5 110 61.11 5.55 0.45
12th 1 1 2 1 2 7 117 65 5.41 0.58
13th 1 1 1 3 120 66.66 5.12 0.23
14th
15th 1 1 1 1 4 124 68.88 4.59 0.26
16th 1 1 125 69.44 4.34 0.06
17th 1 2 3 128 71.11 4.18 0.17
18th
19th 1 1 129 71.66 3.77 0.05
20th 1 1 130 72.22 3.61 0.05
21st 1 1 131 72.77 3.46 0.04
22nd
Total 6 6 7 6 6 9 7 8 8 7 8 7 9 6 7 8 8 8 131

Table 3.14 Daily germination count of the jatropha seeds and calculation of germination parameters. DT (Daily total); CT
(Cumulative total); CG % (Cumulative germination percent); C (Control); T (Treatments); r (Replicates)
110


111

Treatments Percentage Germination Germination capacity % Germination energy Seedling vigor index
Control 40 % 63.33 23.25 542.87
MS1 50 %
Table 3.15 Germination parameter study shown by the selected isolatess in comparison with the control. These parameters were
calculated after the germination count up to 28
th
day after the seeds sown in the pot.
63.33 24.75 728.52
MS2 46.66 % 76.66 24.87 663.03
MS3 60 % 73.33 27.53 895.84
MS4 50 % 73.33 23.85 701
MS5 63.33 % 929.05


Screening and characterization of PGPR
29.77 80




Screening and characterization of PGPR
112

3.3.13 Influence of selected PGPR on the growth of Jatropha curcas
Study of Jatropha curcas plant growth under the influence of five selected isolates i.e
MS1, MS2, MS3, MS4 and MS5 showed increased growth of plants in terms of root
length, shoot length, number of leaves and fresh weight as well as dry weight. MS3 and
MS5 were found to be the most effective PGPR for Jatropha plant.
Brevibacillus brevis MS1 was found to increase maximum root length (fig. 3.14) ranges
between 7.36 % to 6.92 % from 30 (pic. 3.4)

to 120 DAS (days after sowing), increase
root dry weight (fig. 3.18) 223.07 % (30 DAS) and 18.08 % (120 DAS), root fresh
weight (fig. 3.16) 77.35 % (60 DAS), shoot dry weight (fig. 3.19) 38.80 % (30 DAS)
and 18.08 % (120 DAS), shoot fresh weight (fig. 3.17) 115.76 % (60 DAS) and 134.87
% (90 DAS) as well as increase shoot width (fig. 3.20) 41.40 % (60 DAS), 49.81 % (90
DAS), 43.79 % (120 DAS) compare to the uninoculated control. While the biomass (fig.
3.26) was found to increase 102.46 % (60 DAS) and 91.70 % (90 DAS) compare to
control. Desai et al. (2007) reported that Bacillus pumilus (IM-3) supplemented with
chitin showed over all growth promotion of Jatropha curcas effect resulting in enhanced
shoot length (113%), dry shoot mass (360%), dry root mass (467%), dry total plant mass
(346%), leaf area (256%), and chlorophyll content (74%) over control. Treating seeds
with strain IM-3 without chitin resulted in enhanced dry shoot mass (473%), dry total
plant mass (407%), and chlorophyll content (82%).
However, Bacillus polymyxa (KRU-22) with chitin supported maximum root length
(143%). Either strain IM-3 alone or in combination with other promising strains could be
promoted further for enhanced initial seedling growth of Jatropha. B. brevis is a plant
growth promoting rhizobacterium (PGPR) (Kloepper 1992) and its positive effect on root
biomass was greater than that observed on the shoot.




Screening and characterization of PGPR
113

Enterobacter cloacae MS2 was found to increase maximum root dry weight 27.53 % (90
DAS), root fresh weight 31.10 % (120 DAS), shoot dry weight 2.53 % (90 DAS), shoot
fresh weight 80.74 % (30 DAS) and 125.13 % (120 DAS) as well as increase leaf number
(fig. 3.21) 21.45 % (90 DAS), leaf length (fig. 3.22) 46.44 % (30 DAS) and leaf width
(fig. 3.23) 27.38 % (30 DAS) compare to the uninoculated control. While the biomass
was found to increase 113.96 % (60 DAS) and 97.51 % (90 DAS) compare to control.
Deepa et al. (2010) studied plant growth promotion potential of strains NII-0907 (E.
aerogenes), NII-0929 (E. aerogenes), NII-0931 (E. cloacea) and NII-0934 (E. asburiae)
members of the genus Enterobacter. All the four Enterobacter species were very good
phosphate solubilizers (60.1 to 79.5 g/ml/day after 10
th
day of incubation); IAA
producers (23.8 to 104.8 g /ml/day after 48h of incubation); HCN producers and
siderophore producers. They were also studied their considerable influence on cowpea
and recorded 153.8, 46, 50.7, 87.6 and 47.8, 39.2, 50.0, 72.8% higher root and shoot
lengths in isolates NII-0907, NII-0929, NII-0931 and NII-0934 respectively compared
with uninoculated control. E. cloacae suppress P. ultimum infections when applied as a
coating on to seeds of plants such as carrot, cotton, cucumber, lettuce. Radish, sunflower,
tomato and wheat (Windstam and Nelason 2008).
Bacillus licheniformis MS3 was found to increase maximum root length ranges between
22.23 % to 10.49 % from 30

to 120 DAS (days after sowing), increase root dry weight
276.92 % (30 DAS) and 78.84 % (90 DAS) (pic. 3.6), root fresh weight 77.98 % (60
DAS) and 51.41 % (120), shoot dry weight 44.77 % (30 DAS) and 103.09 % (60 DAS),
shoot fresh weight 80.74 % (30 DAS) and 129.75 % (90 DAS) as well as increase shoot
width 63.95 % (30 DAS), 57.53 % (90 DAS), 51.33 % (120 DAS), leaf number 28.52 %
(120 DAS), leaf length 49.06 % (30 DAS) and leaf width 30.04 % (120 DAS) compare to
the uninoculated control.





Screening and characterization of PGPR


0
5
10
15
20
25
30
Control MS1 MS2 MS3 MS4 MS5
Isolates
R
o
o
t

l
e
n
g
t
h

(
c
m
)
30D 60 D 90 D 120 D


Figure 3.14 Effect of selected strains of PGPR on the root length of Jatropha curcas plant


0
5
10
15
20
25
Control MS1 MS2 MS3 MS4 MS5
Isolates
S
h
o
o
t

l
e
n
g
t
h

(
c
m
)
30 D 60 D 90 D 120 D

Figure 3.15 Effect of selected strains of PGPR on the shoot length of Jatropha curcas
plant
114


Screening and characterization of PGPR

0
1
2
3
4
5
6
7
Control MS1 MS2 MS3 MS4 MS5
Isolates
R
o
o
t

f
r
e
s
h

w
e
i
g
h
t

(
g
m
s
)
30 D 60 D 90 D 120 D

Figure 3.16 Effect of selected strains of PGPR on the root fresh weight of Jatropha
curcas plant


0
2
4
6
8
10
12
14
16
18
20
Control MS1 MS2 MS3 MS4 MS5
Isolates
S
h
o
o
t

f
r
e
s
h

w
e
i
g
h
t

(
g
m
s
)
30 D 60 D 90 D 120 D

Figure 3.17 Effect of selected strains of PGPR on the shoot fresh weight of Jatropha
curcas plant
115


Screening and characterization of PGPR


0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Control MS1 MS2 MS3 MS4 MS5
Isolates
R
o
o
t

D
r
y

w
e
i
g
h
t

(
g
m
s
)
30 D 60 D 90 D 120 D

Figure 3.18 Effect of selected strains of PGPR on the root dry weight of Jatropha curcas
plant


0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Control MS1 MS2 MS3 MS4 MS5
Isolates
S
h
o
o
t

D
r
y

w
e
i
g
h
t

(
g
m
s
)
30 D 60 D 90 D 120 D

Figure 3.19 Effect of selected strains of PGPR on the shoot dry weight of Jatropha
curcas plant
116


Screening and characterization of PGPR


0
5
10
15
20
25
30
35
40
Control MS1 MS2 MS3 MS4 MS5
Isolates
S
h
o
o
t

w
i
d
t
h

(
m
m
)
30 D 60 D 90 D 120 D

Figure 3.20 Effect of selected strains of PGPR on the shoot width of Jatropha curcas
plant


0
1
2
3
4
5
6
7
8
9
N
u
m
b
e
r

o
f

l
e
a
f
Control MS1 MS2 MS3 MS4 MS5
Isolates
30 D 60 D 90 D 120 D

Figure 3.21 Effect of selected strains of PGPR on the number of leaf of Jatropha curcas
plant
117


Screening and characterization of PGPR


0
2
4
6
8
10
12
Control MS1 MS2 MS3 MS4 MS5
Isolates
L
e
a
f

l
e
n
g
t
h

(
c
m
)
30 D 60 D 90 D 120 D

Figure 3.22 Effect of selected strains of PGPR on the leaf length of Jatropha curcas plant



0
2
4
6
8
10
12
14
Control MS1 MS2 MS3 MS4 MS5
Isolates
L
e
a
f

w
i
d
t
h

(
c
m
)
30 D 60 D 90 D 120 D

Figure 3.23 Effect of selected strains of PGPR on the leaf width of Jatropha curcas plant
118


Screening and characterization of PGPR



Picture 3.5 Study of vegetative parameters of Jatropha curcas treated with the selected
isolates of PGPR 90 DAS.



30 DAS 60 DAS
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Chl a Chl b Chl a Chl b
C
h
l
o
r
o
p
h
y
l
l

(
m
g
/
g
r
a
m

w
t
.
)
Control MS1 MS2 MS3 MS4 MS5

Figure 3.24 Effect of selected strains of PGPR on the chlorophyll content of Jatropha
curcas leaf (30 DAS and 60 DAS)
119


Screening and characterization of PGPR


90 DAS 120 DAS
0
0.5
1
1.5
2
2.5
Chl a Chl b Chl a Chl b
C
h
l
o
r
o
p
h
y
l
l

(
m
g
/
g
r
a
m

w
t
.
)
Control MS1 MS2 MS3 MS4 MS5

Figure 3.25 Figure 3.26 Effect of selected strains of PGPR on the chlorophyll content of
Jatropha curcas leaf (90 DAS and 120 DAS)

0
2
4
6
8
10
12
14
16
18
B
i
o
m
a
s
s

(
g
m
s
)
Control MS1 MS2 MS3 MS4 MS5
Isolates
30 D 60 D 90 D 120 D

Figure 3.26 Effect of selected strains of PGPR on the biomass of Jatropha curcas plant

120


Screening and characterization of PGPR
121

While the biomass was found to increase maximum 95.63 % (30 DAS) and 105.53 % (90
DAS) compare to control. Cakmakci et al. (2007) reported that Bacillus OSU-142,
Bacillus M-13, and Bacillus licheniformis RC02 increased root length in comparison to
the control and P fertiliser. Of the bacterial inoculations, Bacillus M-13 produced the
highest root length, while Ps. putida RC06 produced the lowest root length. Statistically
significant differences in root and shoot weight, and bacterial count were observed
between all bacterial inoculates and the control.
Inoculation with N
2
-fixing and P-solubilising bacteria increased barley root weight by
17.9% -32.1%, depending on the species, while N fertiliser increased root weight by
28.6% compared to the control. Rapid establishment of roots, whether by elongation of
primary roots or by proliferation of lateral and adventitious roots, is beneficial to young
barley seedlings. PGPR inoculation may effectively increase the surface area of roots
(Richardson 2001) and root weight (Cakmakci et al. 2007b). Inoculation with P.
polymyxa increased the mass of root adhering soil in wheat (Bezzate et al. 2000), and
increased shoot and root growth in rice (Sudha et al. 1999).
Micrococcus sp. MS4 was found to increase maximum root length 7.85 % (120 DAS),
increase root dry weight 18.84 % (90 DAS), root fresh weight 34.47 % (90 DAS), shoot
length 36.82 % (90 DAS), shoot fresh weight 121.17 % (60 DAS) and 124.31 % (120
DAS) as well as increase leaf number 11.74 % (120 DAS), leaf length 5.24 % (90 DAS)
and leaf width 26.44 % (120 DAS) compare to the uninoculated control. To the best my
knowledge there is no any other report of Micrococcus sps with the growth promotion of
Jatropha curcas so this is the first report which shows significant results with the
Jatropha curcas plant. Kumar et al. (2009) has reported the development of vegetatively
propagated Jatropha on control soil, FYM (Farmyard manure) and vermicompost. Data
on survival percentage showed, 100% of Jatropha cuttings were fresh upto 25 DAS with
all treatments, but survival percentage was significantly reduced in the following order;
control soil (83%) < FYM (92%) < vermicompost (98%) at 45 DAS.


Screening and characterization of PGPR
122

Plant height showed insignificant increased in all treatments and further it was increased
significantly with FYM (13.14% and 8.29%) and vermicompost (25.13 & 17.53%) over
control.
For chlorophyll content (fig. 3.24 and fig. 3.25) interaction of strains and dates were
significant. The maximum chlorophyll content (Chla and Chlb) in MS1 was found (1.03
mg/g and 0.65 mg/g) at 120 DAS, in MS2 (1.21 mg/g and 1.07 mg/g) at 120 DAS, in
MS3 (1.79 mg/g) at 120 DAS and (0.95 mg/g) at 30 DAS, in MS4 (1.98 mg/g and 1.56
mg/g) at 120 DAS and in MS5 was found (1.99 mg/g) at 120 DAS and (1.34 mg/g) at 30
DAS. While in control maximum chlorophyll content (Chla and Chlb) was found (0.78
mg/g and 0.89 mg/g) at 120 DAS, which was much lesser than all the five treatments.
Acienetobacter calcoaceticus MS5 was found to increase maximum root length 32.79 %
(60 DAS), increase root dry weight 307.69 % (30 DAS) and 67.08 % (120 DAS), root
fresh weight 86.79 % (60 DAS), shoot length (fig. 3.15) 42.26 % (90 DAS), shoot dry
weight 47.76 % (30 DAS) and 96 % (60 DAS), shoot fresh weight 124.54 % (60 DAS)
and 133.50 % (90 DAS) as well as increase shoot width 52.35 % (60 DAS), 48.05 % (90
DAS), 49.10 % (120 DAS), leaf length 44 % (30 DAS), leaf width 27.94 % (30 DAS)
compare to the uninoculated control. While the biomass was found to increase 84.42 %
(30 DAS) and 94.08 % (120 DAS) compare to control. Sarode et al (2009) reported
growth promotion as well as phytopathogen suppression activities of A. calcoaceticus
with wheat plant. Influence of this strain on wheat growth showed 25.2% increase in the
rate of germination, 45.08% and 12.76% in the root length and dry weight, respectively.
Subsequently, 2.71% and 24.29% increase in the shoot length and dry weight
respectively were observed over control. Kumar et al. (2009) reported the effect of
bioinoculants on percentage seed germination of Jatropha curcas and survival at 0.4% of
Na
2
CO
3
was found to be in order of; Azotobacter + AMF > AMF > Azotobacter +
Microfoss > Microfoss > Azotobacter > control (no germination) while at 0.5 % Na
2
CO
3

germination was almost nil with all treatments. The survival percentages with respect to
all treatments were found to be significant at 0.4%, Na
2
CO
3
level over control.


Screening and characterization of PGPR
123

The combination of AM fungi and Azotobacter increased plant height, shoot diameter,
shoot dry weight, leaf relative water content and soluble sugar content and decreased
level of soluble protein at 0.4 % of Na
2
CO
3
over other treatments.
Conclusion
The overall improvement in seedling vigour through a significant increase in various
physiological parameters suggests that these strains have a plant-growth promoting
ability on Jatropha seedlings and hence could be used for seed inoculation for better
establishment of seedlings. The plants with enhanced seedling vigour can help in better
establishment of plantations. All the five isolates Brevibacillus brevis MS1, Enterobacter
cloacae MS2, Bacillus licheniformis MS3, Micrococcus sps MS4 and Acinetobacter
calcoaceticus MS5 were suitable PGPR for the growth promotion of Jatropha curcas.
Considering the plant growth promoting abilities of these five isolates for bioinoculant
preparation is possible. This study show that these isolates having best characteristics of
plant growth promoting potential that help in the seed germination, root and shoot length
promotion and also increase the biomass of the plant Jatropha curcas. It is evident that
the increases in plant height, leaf number and leaf area have contributed to increased
yield. The nutrients enrichment of rhizosphere soil inoculated with microbial inoculant
attributed to the increased soil microbiologist process that contributes towards fertility
status of the soil. Moreover, these isolates have positive impacts on soil characteristics
and health necessary for better growth of planted biomass. This study showed the
practical benefits of employing PGPR for a sustainable farming system and especially the
best three cultures MS1, MS3 and MS5.