chapter 3 screening and characterization of pgpr on their plant...

Screening and characterization of PGPR

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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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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.


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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.

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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)


Table 3.1 plant growth promoting rhizobacteria and their crop responses to the respective plants


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_________________________________________________________

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).


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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 oC. Growth of all isolates was then

measured spectrometrically. The fast growers were then selected for further studies.


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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 Pikovaskay’s medium amended

with bromophenyl blue. These plates were then incubated at 37o 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 (SnCl2. 2H2O)

method (Gaur 1990). A simultaneous change in the pH was also recorded in the

supernatant on systronics digital pH meter (µ 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

Salkowsky’s reagent method (Sarwer and Kremer 1995). One ml of culture supernatant

was mixed with 1 ml of Salkowsky’s 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.


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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 0C. 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 oC 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 oC 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 Arnow’s method (Arnows 1937) and for

Hydroxymate type of siderophores Csaky’s 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.


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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 µl inoculum with approximately 3 x 108 c.f.u. ml-1) were inoculated in 10

ml peptone water and incubated at 30oC for 48–72 h. Nessler’s 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 oC. Bacterial cell mass was then harvested by centrifugation at

8000 g for 10 min at 4 oC. 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 oC, 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 µl 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.


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The supernatant of the tube was then discarded and pellet was suspended in 600 µl 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 µL of the toluenized cell suspension was then

placed in 1.5 ml micro-centrifuge tubes. 20 µl of 0.5 M ACC was then added to the

suspension, briefly vortexed and then incubated at 30 oC 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 µl of 0.56 M HCl.

Thereupon, 300 µl of 2, 4- dinitrophenylhydrazine reagent was added to the glass tube,

the content vortexed and then incubated at 30 oC 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 α-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 oC 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.


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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 Durham’s vials. Five ml of medium was filled in tubes with

inverted Durham’s 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 µl 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.


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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.


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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).


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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)


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

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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


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.

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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.


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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 μg/ml) grown in nitrogen free medium

followed by S. mutans and B. subtilis (220 and 206 μg/ml respectively) in nitrogen free

medium after 7 days of incubation at 37°C. 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


79

(Table 3.4 and Pic. 3.2). The isolate MS5 showed siderophore production (32 µg/ml)

followed by MS2 (28 µg/ml), MS1 (25 µg/ml), whereas MS3 and MS4 produced (22

µg/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 Csaky’s 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, Csaky’s and Arnow’s 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 µg/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, β-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


pyoverdins are labile in presence of acids or O2. 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

EPS

prod

uctio

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)

Sid

erop

hore

pro

duct

ion

μg/m

l

MS1 MS2 MS3 MS4 MS5

Figure 3.6 Siderophore productions (Quantitative estimation) by selected isolates

80


Picture 3.2 Siderophore productions (Qualitative test) by selected isolates

-20

0

20

40

60

80

100

0 10 11 12 1

Days

Amm

onia

pro

duct

ion

(µg/

ml)

3

MS1 MS2 MS3 MS4 MS5

Figure 3.7 Ammonia productions by the selected PGPR

81


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 10th to 13th days of incubation as per method

given by Dye (1968). Maximum concentration of ammonia production was observed in

isolates MS5 and MS3 was 42 µg/ml (10th d) and 42 µg/ml (11th d) followed by MS1 41

µg/ml (12th d), MS4 39 µg/ml (11th d) and MS2 32 µg/ml (12th d) (fig. 3.7). Consecutive

reading after 11th 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 11th day after that there is decrease in ammonia production. Ammonia

released by diazotrophs is one of the most important traits of PGPR’s 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%).


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)


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 α-ketobutyrate/mg/h followed by MS1 (79 nm α-ketobutyrate/mg/h), MS5 (72 nm

α-ketobutyrate/mg/h), MS2 (52 nm α-ketobutyrate/mg/h) and MS4 (48 nm α-

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 µM/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 µM/mg/ml

of α-ketobutryic acid where as A. brasiliense Cd/pRKTACC mutant produced only 11

µM/mg/ml of α-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).


0

10

20

30

40

50

60

70

80

90

MS1 MS2 MS3 MS4 MS5

Isolates

AC

C d

eam

inas

e ac

tivity

(n

m α

-ket

obut

yrat

e m

g-1

h-1)

Figure 3.8 ACC deaminase productions by selected isolates

85


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.


87

Antibiotic Conc. (µg)

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.


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 µm 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

H2S 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 H2S production or

gas production in the butt region (Table 3.8).


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 H2S production -ve -ve -ve -ve -ve Gas production -ve -ve -ve -ve -ve

Table 3.8 Results of TSI test


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.


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 C15:0 anteiso

(34.11 %), C15:0 iso (28.97 %) and C17:1 iso ω10c (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.


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 C15:0 anteiso

(39.96 %), C15:0 iso (29.67 %), C17:1 iso (8.10 %) and C17:1 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 C12:0 (9.00 %),

C14:0 (8.32 %), C16:0 (19.66 %) and C17:0 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.


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

m in0 .5 1 1 .5 2 2 .5 3 3 .5 4

p A

1 5

2 0

2 5

3 0

3 5

4 0

F ID 1 A , (E 0 8 8 2 6 .5 8 8 \A 0 0 4 1 2 2 6 .D )

0.7

34

1.5

84 1

.611

1.6

29 1

.674

1.8

42 1

.867

1.9

22

2.1

29 2

.161 2

.239

2.3

43 2

.372

2.4

37 2

.467

2.4

85 2

.504 2

.552

2.6

08 2

.686

2.7

16 2

.756

2.7

82 2

.803

2.8

22 2

.873

2.9

31 3

.007

3.0

38 3.0

77 3

.109

3.1

25 3

.162

3.1

91 3

.226

3.2

66 3.3

15 3

.356

3.3

72 3

.398

3.4

23 3

.447 3

.461

3.4

98 3

.513

3.5

33 3.5

60 3

.577 3

.613

3.6

36 3

.653

3.7

34 3

.780

3.7

92 3

.815

3.8

85 3

.939

3.9

66 4

.038

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



95

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

C17:0 iso 3.0774 3.92 C17:0 anteiso 3.1088 1.58

C12:0 iso 1.5844 0.17 C12:0 1.6736 0.13 C13:0 iso 1.8416 2.80 C13:0 anteiso 1.8665 1.36 C14:0 iso 2.1293 2.25 C15:1 iso ω9c 2.3720 0.28 C15:0 iso 2.4374 28.97 C15:0 anteiso 2.4671 34.11 C16:1 ω7c OH 2.6856 4.93 C16:0 iso 2.7558 2.63 C16:1 ω11c 2.8032 1.78 C16:0 2.8735 1.57 C17:1 iso ω10c 3.0070 6.35 Summed Feature 4 3.0378

Fatty acid peak Retention Time Percent

3.52

96

m in0 .5 1 1 .5 2 2 .5 3 3 .5 4

p A

1 5

2 0

2 5

3 0

3 5

4 0

4 5

5 0

F ID 1 A , ( E 0 8 8 2 6 .5 8 8 \A 0 0 5 1 2 2 7 .D )

0.7

34

1.6

73

1.8

41

2.1

29

2.2

39

2.4

37 2

.467

2.5

52 2.6

85 2

.716

2.7

56 2

.781 2

.803

2.8

73 2

.900

2.9

25 2

.957 3

.006

3.0

37 3

.077

3.1

09

3.3

15

3.4

22 3

.447

3.4

62 3

.513

3.5

73 3

.605

3.7

34

3.8

87 3

.901 4.0

37

4.2

71

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



97

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

C17:0 iso 3.0372 8.10 C17:0 anteiso 3.0773 10.29 C18:0 3.4615 0.26

C12:0 0.7343 0.10 C13:0 iso 1.6727 0.18 C14:0 iso 1.8411 1.28 C14:0 2.1291 0.44 C15:0 iso 2.2395 29.67 C15:0 anteiso 2.4370 36.96 C16:1 ω7c OH 2.5522 0.53 C16:0 iso 2.7158 4.61 C16:1 ω11c 2.7810 0.74 C16:0 2.8032 3.51 C15:0 iso 3 OH 2.9002 0.52 C17:1 iso ω10c 2.9567 0.88 Summed Feature 4 3.0060


0.67

98

m in0 .5 1 1 .5 2 2 .5 3 3 .5 4

p A

1 5

2 0

2 5

3 0

3 5

4 0

4 5

5 0

F ID 1 A , (E 0 8 8 2 6 .5 8 8 \A 0 0 3 1 2 2 5 .D )

0.7

34

1.0

65

1.2

44

1.4

28

1.6

29 1

.673

1.9

44 2.0

04 2

.066

2.0

86 2

.104

2.1

90 2.2

26 2

.239

2.4

01

2.5

52 2

.608 2

.688

2.7

18 2

.736

2.7

81 2

.822

2.8

50 2

.873

3.1

68 3

.195 3.3

25 3

.338

3.3

86 3

.412

3.4

22 3

.447

3.4

64 3

.512

3.6

12 3

.659

3.7

34 3

.803

4.0

38

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




C10:0 1.2440 0.32 C12:0 1.6730 9.00 C13:0 1.9442 0.16 C12:0 2 OH 2.0037 0.95

C12:0 3 OH 2.0863 1.68 C14:0 2.2392 8.32 C16:1 ω7c OH 2.6881 1.70

Summed Feature 2 2.7185 12.37 C16:0 N OH 2.7360 1.69 Summed Feature 3 2.8216 16.18 C16:0 2.8732 19.66

C17:0 cyclo 3.1676 7.98

C17:0 10-CH3 3.3253 1.05 C18:1 ω9c 3.4474 4.94 Summed Feature 8 3.4641 11.64

C18:0 3.5123 0.71 C19:0 cyclo ω8c 3.8027 0.50

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

99


100

[DQ444285] Brevibacillus brevis YJ011

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


[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

EF179834.1100

50

49

3639

53

8096

0.000237

-0.000007

-0.000004

0.002950

-0.000004

0.000542

0.000739

0.002485

0.002737

0.001473

0.001473

0.0019350.001788

0.000383

0.000058

0.0000040.000092

0.000440

0.0013230.001668

93

4955

100

69


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


Table 3.11 Distance Matrix Table generated using Sample MS2


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 7671

100

99

100

70

100

87

44



GQ856255.1

FJ380958.1

EU379292.1|

FJ357606.1

FJ357601.1

EU379288.1

MS4

EU855211.1

EU005372.1

FJ217189.1

FJ217190.1

4553

50

67

47

99

3375

0.005382

0.000550

-0.000003

-0.000005

0.001260

0.000736

0.000735

0.000690

0.000000

0.000631

0.000000

0.0000020.000104

0.000502

0.000187

0.004519

0.000316

0.000353

0.0002850.000781


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


Table 3.13 Distance Matrix Table generated using Sample MS4


[EU921468] Acinetobacter calcoaceticus HIRFA36



[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



100100

25

15

100

85

62

52

28


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


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 6th 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.


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 µg 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 µg Zn/ml) in the case of G. mosseae isolated from Zn-polluted soil.

Mycelial growth under 5 µg Cd/ml (without bacterium) was similarly reduced from that

produced at 15 µg Cd/ml in the presence of the bacteria. As well, 50 µg Zn/ml (without

bacterium) reduced hyphal growth as much as 200 µg Zn/ml did in the presence of B.

brevis.


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


Treatments (T)/ Replicates (r) C T1 T2 T3 T4 T5

Day

r1 r2 r3 r1 r2 r3 r1 r2 r3 r1 r2 r3 r1 r2 r3 r1 r2 r3

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 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


29.77 80


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.


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 µg/ml/day after 10th day of incubation); IAA

producers (23.8 to 104.8 µg /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.


0

5

10

15

20

25

30

Control MS1 MS2 MS3 MS4 MS5

Isolates

Roo

t len

gth

(cm

)

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


Isolates

Shoo

t len

gth

(cm

)

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


0

1

2

3

4

5

6

7


Isolates

Roo

t fre

sh w

eigh

t (gm

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

02468

101214161820


Isolates

Sho

ot fr

esh

wei

ght (

gms)

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


0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6


Isolates

Root

Dry

wei

ght (

gms)

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

00.5

11.5

22.5

33.5

44.5


Isolates

Shoo

t Dry

wei

ght (

gms)

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


0

5

10

15

20

25

30

35

40


Isolates

Shoo

t wid

th (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

0123456789

Num

ber o

f lea

f


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


0

2

4

6

8

10

12


Isolates

Leaf

leng

th (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


Isolates

Leaf

wid

th (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


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

Chlo

roph

yll (

mg/

gram

wt.)


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

119


90 DAS 120 DAS

0

0.5

1

1.5

2

2.5

Chl a Chl b Chl a Chl b

Chlo

roph

yll (

mg/

gram

wt.)


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)

02468

1012141618

Bio

mas

s (g

ms)


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


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 N2-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.


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

Na2CO3 was found to be in order of; Azotobacter + AMF > AMF > Azotobacter +

Microfoss > Microfoss > Azotobacter > control (no germination) while at 0.5 % Na2CO3

germination was almost nil with all treatments. The survival percentages with respect to

all treatments were found to be significant at 0.4%, Na2CO3 level over control.


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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 Na2CO3 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.

chapter 3 screening and characterization of pgpr on their plant...

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