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).
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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,
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
Çakmakçi 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
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 ± 10C for three days. An optical density of
0.5 recorded at λ 535 nm was achieved by dilution to maintain uniform cell density (108-
109 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 70°C.
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 Pikovskyaya’s medium after 3 days of incubation at 30 ± 2 oC (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 µg/ml) followed by MS1 (47 µg/ml), MS2 (37 µg/ml), MS4 (18.2 µg/ml) and MS5 (18
µg/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 pikovskyaya’s medium with a decline in pH (Tank and Saraf 2003)
Screening and characterization of PGPR
0123456789
10
4 8 12 16 20 24
Duration (h)
Log1
0 C
FU/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)
Pho
spha
te s
olub
iliza
tion μg
/ml
21
MS1 MS2 MS3 MS4 MS5
Figure 3.2 Phosphate solubilization by selected PGPR strains
74
Screening and characterization of PGPR
Isolate 0 day 7th day 14th day 21st 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 21st day after inoculation
0
5
10
15
20
25
30
MS1 MS2 MS3 MS4 MS5
Isolates
Zone
of P
sol
ubili
zatio
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)
IAA
pro
duct
ion μg
/ml
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 µg/ml) after 96 h of incubation in dark followed by MS3 (47
µg/ml), MS4 (39 µg/ml), MS5 (32 µg/ml) and MS2 (27 µg/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 µg/ml of IAA by A. vinelandii where as
Chandra et al. (2007) reported a production of 24 µg/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 0.01µg/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
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 28th 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