Research Article Isolation and characterization of ... 8 No 3/Sagar488-494.pdfSagar A. Desai Department of Chemistry, Bhagwan Mahavir College of Science & Technology, Nr. VIP Road,

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Bioscience Discovery, 8(3): 488-494, July - 2017

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Research Article

Isolation and characterization of gibberellic acid (GA3)

producing rhizobacteria from sugarcane roots

Sagar A. Desai

Department of Chemistry, Bhagwan Mahavir College of Science & Technology,

Nr. VIP Road, Bharthana-Vesu, Surat

[email protected]

Article Info

Abstract

Received: 23-05-2017,

Revised: 19-06-2017,

Accepted: 24-06-2017

Gibberellic acid (GA3) is an important plant hormone controlling plant’s growth

and development. It is being widely used in agriculture, horticulture, garden and

other area. Apart from plants, several fungus and bacterial stains can also

produce GA3 as their secondary metabolite. A total of 60 bacterial isolates were

isolated from rhizospheric soil collected from sugarcane farm in the vicinity of

Surat. These isolates were screened for gibberellic acid (GA3) production in a

nutrient medium and checked by spectrophotometric method. Out of all of these

two isolates namely K8 and K37 produced higher amount of gibberellic acid as

compared to others and thus selected for further investigation. The effect of pH,

incubation temperature, carbon and nitrogen supplementation on gibberellic acid

(GA3) production by both isolates were studied. The cell free supernatant of

these bacterial isolates was bioassayed on mung and sweet sorghum seeds and

found that it significantly promoted the growth of both plants. The isolates were

identified as Pseudomonas Spp. for K8 and Azotobacter Spp. for K37 on the

basis of cultural and biochemical conditions.

Keywords:

Azotobacter Spp.,

Gibberellins, plant growth

promoting rhizobacteria,

phytohormone,

Pseudomonas Spp.

INTRODUCTION

Plant growth regulators like gibberellins and

cytokinins are one of the important biotechnological

products commonly used in agriculture,

horticulture, gardens and other areas involving

plants (Bandelier and Renaud, 1997). Gibberellins

are biologically active, endogenous hormones of

higher plants that are involved in a range of

developmental processes in higher plants including

stem elongation, germination, seed dormancy, fruit

senescence and sex expression (Elezar and

Escamilla, 2000; Gelmi and Perez, 2000). Different

gibberellins (GAs) selectively affect different parts

of the plants. In addition to higher plants, certain

fungi and a few bacteria (Gutierrez et al., 2001) also

produce gibberellins. At present, species belonging

to fungal genera like Fusarium, Gibberella,

Sphaceloma, Neurospora and Phaeosphaeria have

been reported to produce gibberellins. These fungi

produce GA3 and/or GA4 as final metabolite

(MacMillan, 2002). Bacteria interacts with plant

roots in the rhizosphere and enhance plant growth

and development to some extent. Gibberellins

produced by plant growth promoting rhizobacteria

(PGPR) promotes the plant growth and increases

yields of many crop plants (Piccoli et al., 1997).

Among all, 136 GAs have been identified from

higher plants (128 species), 28GAs from fungi (7

species), and only 4GAs (GA1, GA3, GA4, and

GA20) from bacteria (7 species) (MacMillan,

2002). Gibberellic acid (GA3), the main product of

gibberellins in fungi and bacteria, is a terpeanoid

hormone that is an important phytohormone

regulating plant growth and development.

http://biosciencediscovery.com 489 ISSN: 2231-024X (Online)

Sagar A. Desai

It’s a high value industrially important biochemical

selling at 27-36 USD/g in international market

(Avinash et al., 2003). Currently, GA3 is largely

being produced by submerge fermentation of the

fungus Gibberella fujikuroi at an industrial scale

(Santos et al.,2003). It is also being synthesized

from several bacteria such as Azotobacter and

Azospirillium (Rademacher, 1994), Bacillus

siamensis BE76 (Ambawade and Pathade, 2013),

Pseudomonas spp. (Damam et. al., 2016).

The present study was aimed to isolate and

characterize indigenous rhizobacteria, capable of

producing gibberellic acid and to investigate the

effect of various physico-chemical parameters on

the GA3 production.

MATERIALS AND METHODS

Chemicals

All the chemicals used in this study were of

analytical grade and procured from Hi-media

laboratories, Mumbai, India.

Sample Collection

Soil samples were collected from sugarcane farm in

the vicinity of Surat, for isolation of

microorganisms. Two soil samples (Soil and Root

adherent soil) and one root sample were collected.

Samples were collected in sterile plastic bags and

stored at 4 °C until further use.

Isolation and Screening of Gibberellic

acidproducing bacteria

For isolation of bacterial stains, 0.1 ml of samples

(0.1 g of soil in sterile d/w) were spreaded on

Nutrient Agar, Actinomycetes Isolation Agar and

Ashby’s mannitol agar plates and incubated at 37

°C for 48 hr. After incubation, plates were observed

for morphologically distinct isolated colonies and

screened for gibberellic acid production.

For screening of GA3 producing bacterial isolates,

50 ml of sterile nutrient broth was inoculated with

loop full of isolated bacteria and incubated for 10

days at 30 °C at 100 rpm. After 10 days, cell free

supernatant was used to measure amount of GA3

produced by bacterial isolates by method described

below.

GA3 estimation by DNPH (2, 4 – Dinitrophenyl

hydrazine) method (Graham and Thomas, 1961)

For GA3 estimation equal volume of cell free

extract and ethyl acetate was taken in a test tube and

shook vigorously for 10 min. Ethyl acetate layer

was taken in separate tube, this was repeated thrice.

Ethyl acetate was allowed to evaporate at room

temperature. The remaining contents was dissolved

in absolute alcohol (Zeigler et al., 1980) .2 ml of

thissuspension was mixed with 1 ml of DNPH and

incubated at 100 °C for 5 min and cooled in

water bath. To this, 5 ml of 10% potassium

hydroxide was added and allowed to stand till red

wine color developed. 15 ml of sterile distilled

water was added and finally the content was diluted

to 1:2 using sterile distilled water. Color intensity

was measured at 430 nm in UV-VIS

Spectrophotometer (Shimadzu-UV-3600 Plus). For

standard curve, different aliquots of standard

gibberellic acid (0.8 mg/ml) was prepared using

absolute alcohol and estimated similarly.

Selected bacterial isolates were characterized at

Advanced Diagnostic Laboratory, Surat, Gujarat by

biochemical characterization on Phoenix

Instrument, version: 6.01 A and EpiCenter, version:

V6.20A.

Gibberellic acid production under Submerged

Condition

Gibberellic acid production was carried out in

submerged fermentation using 100 ml sterile

nutrient broth (Pandya and Desai, 2014). Selected

bacterial isolates were inoculated in separate flasks

and incubated for 10 days at 30 °C at 100 rpm.

Samples were removed aseptically at regular

interval and subjected to gibberellic acid estimation.

Optimization of process parameters

Effect of pH and incubation temperature on

Gibberellic acid production

For pH optimization, initial pH of the nutrient broth

was adjusted to 6, 7, 8 and 9. 1.5 ml of bacterial

suspension was inoculated incubated at 30°C at 100

rpm. Samples were removed at regular interval and

checked for amount of gibberellic acid produced.

For temperature optimization, 1.5 ml of bacterial

suspension was inoculated in nutrient broth and

incubated at various temperatures like room

temperature (30°C), 37°C and 40°C at 100 rpm.

Samples were removed at regular interval and

checked for amount of gibberellic acid produced.

Effect of carbon and nitrogen supplementation

on gibberellic acid production.

Effect of additional carbon supplementation

(glucose, lactose and sucrose) and nitrogen

supplementation (ammonium chloride and urea)

was tested for gibberellic acid production at various

concentration i.e. 0.2%, 0.5% and 1% (w/v). For

this, 100 ml of nutrient medium was supplemented

with different carbon and nitrogen sources at

different concentration. 1.5 ml of bacterial

suspension was inoculated and flasks were

incubated at 37°C at 100 rpm. Samples were



removed and checked for amount of gibberellic acid

produced.

Plant growth promoting capacity of microbial

isolate

Seeds of Phaseolus mungo (mung) and Sorghum

bicolor (sweet sorghum) were surface sterilized

with 0.1% aqueous solution of mercuric chloride

(Mineo, 1990). Seeds were germinated in sterilized

petri dishes lined with moistened cotton. Samples

were extracted with equal volume of ethyl acetate.

Upper aqueous layer was taken and allowed to

evaporate at room temperature and extracted

metabolites were dissolved in distilled water. 15 ml

of this was added to petri dishes containing 10 seeds

of both mung and sorghum. Germination (%),

length of root and length of shoot parameters were

observed after 7 days of test and controls (15 ml of

sterile distilled water) (Tam and Tiquia, 1994).

RESULTS AND DISCUSSION

Isolation and Screening of Gibberellic acid

producing bacteria

Total 60 visibly different, with distinct morphology,

designated as K 1-59 were isolated after 2 days of

incubation on different medium. Isolated bacteria

were preserved by streaking them on respective

agar slant from which they were isolated. Only two

isolates namely K8 and K37 showed gibberellic

acid production above 20 µg/ml which were 24

µg/ml and 22 µg/ml, respectively. Thus, they were

selected for further investigation. On the basis of

cultural and biochemical conditions, selected

isolates were identified as Pseudomonas Spp. for

K8 and Azotobacter Spp. for isolate K37 (Data not

shown).

Figure1. Gibberellic acid production under submerged condition.

Figure2. pH optimization for Gibberellic acid production.


Sagar A. Desai

Figure3. Temperature optimization for Gibberellic acid production.

Figure4. Effect of glucose supplementation on Gibberellic acid production.

Figure5. Effect of ammonium chloride supplementation on Gibberellic acid production.



Figure6. Effect of urea supplementation on Gibberellic acid production.

Gibberellic acid production under Submerged

Condition

Both K8 and K37 isolates were subjected to

gibberellic acid production under submerged

fermentation. Maximum gibberellic acid production

of 33 µg/ml was observed with isolate K8 after 9

days of incubation as shown in figure1.

Optimization of process parameters

Maximum gibberellic acid production of 40.8 µg/ml

was observed at pH 8 with isolate K8 suggesting

that higher pH is favorable for gibberellic acid

production by bacteria (Figure2). Similar results

were also obtained by Patil and Patil (2014) as they

reported that pH of the medium was favored near

about 8 by strains of Actinomycetes during the

course of gibberellic acid production.

For temperature optimization flasks with

fermentation medium were incubated at different

temperature. Maximum gibberellic acid production

of 42.5 µg/ml was observed at 30°C with isolate K8

and the level of gibberellic acid produced started

decreasing at higher temperatures (Figure3). Out

findings were supported by the work of Karakoc

and Aksoz (2006). They observed maximum

gibberellic acid production at 30°C by

Pseudomonas sp. This may be due to the effect of

elevated temperature on the enzyme activity

involved in biosynthesis of gibberellic acid.

Two different carbon sources glucose and lactose

were used at the concentrations of 0.2%, 0.5% and

1% (w/v) to study the effect of carbon

supplementation on gibberellic acid production. The

maximum gibberellic acid production of 87.6 µg/ml

was observed in flask containing 1% glucose

inoculated with isolate K8 (Figure: 4). Whereas,

maximum gibberellic acid production was observed

when 1% lactose was added in the flask inoculated

with isolate K8. Lale and Gadre (2010), also used

glucose as additional carbon source and reported

considerably higher production of gibberellins.

Similar results were also obtained by other

researchers (Hollmann et al., 1995; Escamilla et al.,

2000; Shukla et al., 2005).

Effect of additional nitrogen supplementation on

gibberellic acid production was studied. The

maximum gibberellic acid production of 76.8 µg/ml

was observed in flask supplemented with 0.5%

ammonium chloride inoculated with isolate K37

(Figure: 5). In case of urea as an additional nitrogen

supplementation, maximum gibberellic acid

production of 70.0 µg/ml was observed in flasks

containing 0.5% urea inoculated with isolate K8

(Figure: 6). Lale and Gadre (2010) also used

inorganic nitrogen sources like ammonium chloride

for gibberellic acid production.

Plant growth promoting capacity of microbial

isolate

The microbial isolates were bioassayed on

Phaseolus mungo (Mung) and Sorghum bicolor

(sweet sorghum) seeds for its growth promoting

capacity in terms of root and shoot length of crop

plants. Length of shoot and root was measured after

6 days of incubation. Results of shoot and root

development is shown in Table: 1. The results

confirm the previous reports of shoot length

promotion through microbial culture supernatant

treatment (Dobert et al., 1992; Fulchieri et al.,

1993).


Sagar A. Desai

Table 1 Plant growth promoting effect of gibberellic acid produced by bacterial isolates.

Shoot/Root length Control (D/W) K8 K37

Phaseolus mungo (Mung)

Shoot length (cm) 23.0 23.7 21.5

Root length (cm) 5.68 6.19 12.2

Sorghum bicolor (Sweet sorghum)

Shoot length (cm) 10.6 11.5 11.0

Root length (cm) 3.0 3.5 3.6

Current findings are supported by the studies

carried out by Choi et. al., 2005. They also found

significant increase in rice leaf sprouting when

treated with the cell free broth of Panicillium

commune KNU5379. Many bacterial isolates were

reported earlier showing plant growth promoting

activities and supports our findings. Ambawade and

Pathade (2013) obtained 0.24 mg/ml of GA

production by Bacillus siamensis BE76 isolated

from banana. Similarly, many other plant growth

promoting bacteria were isolated showing potential

of their use in agriculture and other areas of plant

research (Damam et al., 2016; Pawar et al., 2016).

CONCLUSION

From this study it could be concluded,

bacterial isolates were identified as Pseudomonas

Spp. and Azotobacter Spp. had potential of

gibberellic acid production and can be further

explored for its utilization for plant growth

promoting capacity. The effect of culture filtrate

showed on seed germination as well as shoot and

root induction. Thus, microbial sources of GA could

be explored and applied for large scale production

of GA which is found to be economically important

in many fields of plant generation like horticulture,

ornamental developmental of plants etc.

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How to Cite this Article:

Sagar A. Desai, 2017. Isolation and characterization of gibberellic acid (GA3) producing rhizobacteria from

sugarcane roots. Bioscience Discovery, 8(3):488-494.

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Research Article Isolation and characterization of ... 8 No 3/Sagar488-494.pdfSagar A. Desai Department of Chemistry, Bhagwan Mahavir College of Science & Technology, Nr. VIP Road,