Asmiaty Sahur et al, International Journal of Advances in Agricultural Science and Technology,
Vol.4 Issue.1, January- 2017, pg. 07-15 ISSN: 2348-1358
Impact Factor: 6.057
Isolation and Characterization of Indigenous
Rhizosphere Bacteria Producing Gibberellic
Acid and Indole Acetic Acid from Local
Soybeans in South Sulawesi
Asmiaty Sahur1, Ambo Ala2, Baharuddin Patanjengi3, Elkawakib Syam'un4
1
Department of Agronomy, Faculty of Agriculture Hasanuddin University, Indonesia
[email protected] 2
Department of Agronomy, Faculty of Agriculture Hasanuddin University, Indonesia
[email protected] 3
Center for Biotechnology, Research Center (PKP) HasanuddinUniversity, Makassar 90245 Indonesia
[email protected] 4
Department of Agronomy, Faculty of Agriculture Hasanuddin University, Indonesia
[email protected]Abstract: This study aimed to isolate and characterize the indigenous rhizosphere bacteria producing
Gibberellic Acid and Indole Acetic Acid as plant growth regulators isolated from local soybean of South
Sulawesi, Indonesia. Several root samples and soil samples of soybean plants were collected from the
rhizosphere of local soybeans in three different areas of South Sulawesi such as Soppeng, Bone and Takalar.
There were fifty six isolates of bacteria taken from Soybean roots and soil and grouped into gram-positive
bacteria and gram negative bacteria. There were 35 isolates that produced a thick slime or felt slimy when
cultured on Natrium Broth media and the remaining 21 produced spores. The results showed considerable
potential for the bacterial isolated to produce Gibbrelic Acid in high concentration. The best isolates of thick
slime producing Gibberellin Acid was RK 30 (4.670 mg/ml), RK 17 (3.797 mg/ml), RK 15 (3.703) and RK 35
(3.222 mg/ml). There were 4 isolates that produced high concentrations of Indole Acetic Acid including RK
32 (2.794 mg / ml) followed by RK 8 (1.810 mg/ml), RK 23 (1.714 mg / ml), and RK 30 (1.678mg / ml)
respectively.
Keywords: Rhizosphere bacteria, Gibberellic Acid, Indole Acetic Acid, Soybeans
1. Introduction
Soybean is one of the legumes that contain vegetable protein that is high enough range around 34% to be
comparable with animal protein for human nutrition (Ditjentan, 2004). Soybeans are both a source of vegetable
protein, and serve as a functional food to prevent the onset of degenerative diseases, such as coronary heart
disease and hypertension. Substances called iso flavones contained in soy products function as an antioxidant.
Now soybeans are widely used as a source of alternative energy (biofuels). As a source of vegetable protein,
soybeans are generally consumed in the form of processed products, such as: tofu, tempeh, soy sauce, tauco, soy
milk and other forms of snacks (Sudaryanto and Swastika 2007).
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Asmiaty Sahur et al, International Journal of Advances in Agricultural Science and Technology,
Vol.4 Issue.1, January- 2017, pg. 07-15 ISSN: 2348-1358
Impact Factor: 6.057
However, traditional agricultural practices have relied on extensive cultivation of land and the use of pesticides
and chemical fertilizers to produce high soybean production. As a result, some of side effects on this practice
have emerged. They include erosion and loss of topsoil and soil structure damaged from cultivation of the soil,
the use of highly hazardous pesticides in the food chain, which can cause contamination and eutrophication of
fresh water and the marine environment. Therefore there is a need to find solution to solve those problems. One
solution in order to solve those problems is to use bacteria as bio-fertilizer.
According to Klooper et al., (1996), the existence of bacteria in the rhizosphere can lead to invasion of the root
system and result in the colonization process helping the plants to grow as well as supplying nutrients to the host
plants. However, those bacteria may not always be synergistic and become pathogens. In addition, Klooper
concluded that these bacteria have the potential to support and increase the availability of many nutrients
including Nitrogen Phosphorus as well as performing as a plant growth hormone.
One of the plant growth hormones that can be produced by rhizosphere bacteria is Gibberellic Acid. It is one of
the hormones promoting the growth of plants economically and industrially. As a hormone plant growth
promoter, Gibberellin Acid can be produced by higher plants around the rhizosphere area by bacteria (Pandya
and Desay, 2014). A common structural element of the Gibberelic is the tetracyclic diterpenoid acids which are
produced in the growth and metabolism process of plants, such as at the germination process of seed, promoting
seedling processes, and leaf as well as stem of plant growth (Bottini, Cassan, Piccoli, 2004).
The aim of the study is to isolate and characterize the physiological ability of rhizosphere bacteria
isolated from local soybeans in South Sulawesi. They might have potential to be used as bio-stimulants or as
bio-fertilizer which can be a p p l i e d t o th e plants and confer protection against soil borne pathogens.
2. Materials and Methods
2.1 Location of Bacteria Site.
Soil samples have been taken at a depth of 0-20 cm in the three areas of soybean rhizosphere in South
Sulawesi, Indonesia. In each area, one healthy soybean were taken and placed in the plastic bag for isolation
process. The samples of soil around the rhizosphere area were collected in one bag and labeled those with the
criteria of the site where the samples have been collected.
2.2 Isolation soil samples from rhizosphere soybean
Isolation of rhizosphere bacteria carried out by a serial dilution method. Ten grams of rhizosphere soil was
weighed and dissolved in 90 ml of sterile water, then shake for 30 minutes. One ml of rhizosphere soil
suspension was added to a reaction tube containing 9 ml of sterile water to get suspension with 10-2
concentration of the dilution. The same process were done for several level of concentration such as 10-3, 10-4,
and then 10-6 respectively.
The next step is took 0.1 ml of the suspension and it was grown on media specific such as Yeast Mannitol
Agar and Tap Water Yeast Extract in a petri dish B o t h medium which already contains rhizosphere
bacteria were incubated for 24 hours at room temperature. Every single colonies were grown to reisolate and
-8
made as pure culture and planted on the Nutrient Agar media 10 suspension. Subsequently 0.1 ml of the
suspension was grown on NA medium in a petri dish. NA medium which already contains rhizosphere
bacteria were incubated for 24 hours at room temperature. Every single colony were grown to reisolate and
made as pure culture.
2.3 Characterization of Rhizosphere Bacteria Isolated from Soybean Plants
2.3.1 Analyzing Gibberellic acid (GA3)
The production of Gibberellic acid was tested by culturing the bacteria on to nutrient broth media (Borrow et
al., 1995). Furthermore one ml of bacterial isolates were added to the media and incubated at 37˚C for seven
days. The cultures then were centrifuged at 8000 g for 10 min to remove the bacterial cells. Fifteen cultures
were added to 5 ml of zinc acetate. Account after 2 minutes was added 2 ml of potassium Ferro cyanide
solution and centrifuged at 8000 g for 10 min. Five ml of the supernatant was added to five ml of 30
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Asmiaty Sahur et al, International Journal of Advances in Agricultural Science and Technology,
Vol.4 Issue.1, January- 2017, pg. 07-15 ISSN: 2348-1358
Impact Factor: 6.057
per cent hydrocloric acid and the mixture was incubated at 20 0 C for 75 minutes. The blank cuvette
was prepared with five percent hydrocloric acid. Absorbance was measured at 254 nm in the UV-VIS
spectrophotometer. From a standard curve prepared by using gibberellic acid solution of known quantities,
produced of GA by the culture was calculated and expressed as mg/l.
2.3.2 Analyzing Indole Acetic Acid ( IAA )
Production of indole -3-acetic acid (IAA) by bacteria was tested using nutrient broth and Salkowski reagent
(Gutierrez et al., 2009). PGPR isolates cultured in NB is equipped with L-tryptophan (0,1g/l ) at room
temperature in the dark for five days, and the supernatant was taken after centrifugation. One ml of the
supernatant was added to one ml of Salkowski reagent (12 g l-1FeCl3 in 429 ml/l H2 SO4) (Glickman et al.,
1995) and incubated in dark for 24 hours at room temperature. The intensity of pink color developed was
read at 535 nm using a UV-VIS spectrophotometer. From a standard curve prepared with known
concentration of IAA, the quantity in the culture filtrate was determined and expressed as mg/l.
3. Results and Discussion
3.1 Isolated Rhizosphere Bacteria from Three Different Areas
Observation of several isolates indicated that between three different source of Soybean grown,
Rhizosphere bacteria from Soppeng is the highest amount of bacteria isolated, and some of them can
be isolated and grown from Bone and then Takalar. The result also gave indication that the bacteria
from rhizophere most commonly grown compare to bacteria isolated from root of soybean plants.
Our result showed that the rhizosphere bacteria isolated from three different areas of south Sulawesi is
rhizosphere bacteria producing plant growth and development is also regulated by phytohormones.
The production of phytohormone such as Gibberellin, Indole Acetic Acid which produced by
PGPR’s have been reported by many researchers, however not many evidence regarding production
of gibberellins by the plant growth promoting rhizobacteria has been reported (Amar et al.,2013) .
Yet, it has been reported to be produced by certain rhizosphere bacteria’s such as Bradyrhizobium
japonicum (Boiero et al, 2007). Gibberellins also can alter the plant morphology by the elongation of
stem tissues. (Amar et al., 2013).
The results showed in the figure one presented the source of isolates producing Gibberellin acid and
Indole Acetic Acid. The highest numbers of isolates were successfully isolated from rhizosphere from
Soil in Soppeng area followed by Bone and then Takalar. The isolated of root rhizosphere mainly from
Bone and then Soppeng. None of bacteria of the plant’s root was found in Takalar area.
(1a) (1b)
Figure 1: (1a) Isolates Producing Gibberellin and Indole Acetic Acid (IAA) which isolated from
Rhizosphere of Soybean Plants, (1b) Isolates Producing Gibberellin and Indole Acetic Acid (IAA)
which isolated from Root of Soybean Plants.
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Asmiaty Sahur et al, International Journal of Advances in Agricultural Science and Technology,
Vol.4 Issue.1, January- 2017, pg. 07-15 ISSN: 2348-1358
Impact Factor: 6.057
The observation of the colour of colonies (after 24 hours) incubation in selective media of rhizosphere
bacteria, it was shown that fifty-six isolates were generally dominated by yellow, white, cream, and
orange (Table 1). The colour of colonies was shown in Figure 2.
Table 1: Characteristic of Bacteria Isolated from Soybean Plants Which Has Gram Negative
Age Gram
Isolates Code Varities Location Colours
(Days) Reaction
Rk 1 Anjasmara 80 Soppeng Yellow -
Rk 2 Anjasmara 80 Soppeng White -
Rk 3 Anjasmara 80 Soppeng White -
Rk 4 Anjasmara 80 Soppeng Yellow -
Rk 5 Anjasmara 80 Soppeng Cream -
Rk 6 Anjasmara 80 Soppeng Yellow -
Rk 7 Anjasmara 80 Soppeng Cream -
Rk 8 Anjasmara 80 Soppeng White -
Rk 9 Anjasmara 80 Soppeng White -
Rk 10 Anjasmara 80 Soppeng Cream -
Rk 11 Anjasmara 80 Soppeng Cream -
Rk 12 Anjasmara 80 Soppeng White -
Rk 13 Anjasmara 80 Soppeng White -
Rk 14 Anjasmara 80 Soppeng White -
Rk 15 Anjasmara 80 Soppeng Cream -
Rk 16 Anjasmara 80 Soppeng White -
Rk 17 Anjasmara 80 Soppeng White -
Rk 18 Anjasmara 80 Soppeng Cream -
Rk 19 Anjasmara 80 Soppeng White -
Rk 20 Anjasmara 12 Soppeng Yellow -
Rk 21 Anjasmara 12 Soppeng Yellow -
Rk 22 Anjasmara 12 Soppeng Orange -
Rk 23 Anjasmara 12 Soppeng Yellow -
Rk 24 Anjasmara 12 Soppeng White -
Rk 25 Wilis 12 Takalar White -
Rk 26 Anjasmara 12 Takalar White -
Rk 27 Anjasmara 12 Takalar White -
Rk 28 Anjasmara 12 Takalar White -
Rk 29 Anjasmara 12 Takalar Cream -
Rk 30 Anjasmara 12 Takalar White -
Rk 31 Anjasmara 12 Takalar Yellow -
Rk 32 Anjasmara 12 Takalar White -
Rk 33 Anjasmara 12 Bone White -
Rk 34 Anjasmara 12 Bone White -
Rk 35 Anjasmara 1i Bone White -
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Asmiaty Sahur et al, International Journal of Advances in Agricultural Science and Technology,
Vol.4 Issue.1, January- 2017, pg. 07-15 ISSN: 2348-1358
Impact Factor: 6.057
Table 2: Characteristic of Bacteria Isolated from Soybean Plants Which Has Gram Positive
Age Gram
Isolates Code Varities Location Colour
(Days) Reaction
AK 1 Anjasmara 80 Soppeng Putih +
AK 2 Anjasmara 80 Soppeng Putih +
AK 3 Anjasmara 80 Soppeng Krem +
AK 4 Anjasmara 80 Soppeng Putih +
AK 5 Anjasmara 80 Soppeng Kuning +
AK 6 Anjasmara 80 Soppeng Krem +
AK 7 Anjasmara 80 Soppeng Krem +
AK 8 Anjasmara 80 Soppeng Putih +
AK 9 Anjasmara 14 Soppeng Putih +
AK 10 Anjasmara 14 Soppeng Putih +
AK 11 Anjasmara 14 Soppeng Krem +
AK 12 Anjasmara 14 Soppeng Krem +
AK 13 Anjasmara 14 Soppeng Krem +
AK 14 Anjasmara 14 Soppeng Putih +
AK 15 Anjasmara 14 Soppeng Putih +
AK 16 Anjasmara 14 Soppeng Putih +
AK 17 Anjasmara 14 Soppeng Putih +
AK 18 Anjasmara 14 Soppeng Putih +
AK 19 Anjasmara 14 Soppeng Putih +
AK 20 Anjasmara 14 Soppeng Putih +
AK 21 Anjasmara 14 Soppeng Putih +
Figure 2: Some of Isolates Producing GA and IAA
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Asmiaty Sahur et al, International Journal of Advances in Agricultural Science and Technology,
Vol.4 Issue.1, January- 2017, pg. 07-15 ISSN: 2348-1358
Impact Factor: 6.057
3.2 Production of Indole Acetic Acid (IAA)
The ability of the bacterial isolates to produce IAA was detected by the development of pink color after
the addition of salkowski reagent to the culture. Some species of bacteria have the ability to produce
IAA. Many evidence suggests that PGPR can affect plant growth and development as it can produce
phytohormones. Phytohormones such as auxin (IAA) is known to stimulate cell elongation and
cell division differentiation (Achmad et al., 2005), and gene regulation (Ryu et al., 2008). Indole
acetic acid is the common natural auxin that shows all auxin activity and extensively affects plants
physiology (Etesemi et al., 2009). Indole acetic acid is a phytohormone which is known to be involved
in root initiation, cell division and cell enlargement (Glickman et al., 1995). In our study, all 35 isolate
were able to produce IAA growing in medium addition of tryptophan. Maximum IAA
production was recorded in isolate RK 32 (2.794 mg l-1) as compared to other isolates. The minimum
amount of IAA production was recorded in RK 4 (0.190 mg l-1). Furthermore, the highest production
of IAA which identified as gram positive bacteria is AK 7 (3.208 mg l-1) and the lowest is AK 17
(0.034 mg l-1)
3.3 Production of GA3
A number of isolates was isolated from soybean crop and it is expected to have a potential in
producing gibberellin, because gibberellin can give positive influence on the germination of plants in
improving the cell elongation.
The analysis is using standard methods (Borrow et al., 1995) shows that the fifty six isolates can
produce GA3 at different concentrations (Table 3). The results of the analysis of the capability of
producing GA3 concentration is in the range of 0.034 mg l -1 sampai 3.222 mg/l. Isolates RK 35 (3.222
mg/l) showed high concentrations while the lowest concentration shown by isolates AK 21(0.084
mg/l).
According to Bottini et al., (2004), bacteria can increase the levels of GA in cultured isolates because
there is a process conjugation of root exudates of Gibberellin. Gibberellin plays an important role in
controlling the developmental processes of plants that includes germination, cell elongation, and the
development of flowers and seed (Lakitan, 1996) .It was also expressed by King and Evans (2003),
that gibberellin has a very useful processes associated with the induction of flowers and the growth of
flowers and fruits. The capability of gibberellin in promoting plants growth is stronger than the AIA
(Reinoso et al., 1993). Gibberellin is also involved in stimulating root growth, abundance of root hairs,
and inhibition of differentiation of flower buds on angiosperms, setting dormant vegetative buds and
generative phase, and inhibiting senescence process in many organs at various species of plants
(Bottini and Luna, 1993; Fulchieri et al., 1993; Tanimoto (1987) in Bottini et al., 2004).
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Asmiaty Sahur et al, International Journal of Advances in Agricultural Science and Technology,
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Impact Factor: 6.057
Table 3: (a) Level of Concentration Isolates Producing IAA and Gibbrellin Acid that has gram negative
(b) Level of Concentration Isolates Producing IAA and Gibbrellin Acid that has gram positive
3a 3b
IAA GA IAA GA
Isolates Isolates
(mg l-1) (mg l-1) (mg l-1) (mg l-1)
RK1 1.714 2.902 AK1 2.890 0.099
RK2 0.651 2.512 AK2 2.982 0.049
RK3 0.698 2.389 AK3 2.751 0.050
RK4 0.19 2.628 AK4 2.610 0.037
RK 5 1.54 2.573 AK5 2.664 0.061
RK 6 1.365 2.539 AK6 2.685 0.071
RK 7 0.921 2.687 AK7 3.208 0.059
RK 8 0.968 2.455 AK8 2.899 0.127
RK 9 0.571 2.521 AK9 2.786 0.077
RK 10 0.571 2.858 AK10 2.806 0.097
RK 11 0.714 2.949 AK11 3.066 0.072
RK 12 0.328 2.439 AK12 2.898 0.096
RK 13 0.413 2.535 AK13 2.714 0.057
RK 14 0.937 2.856 AK14 2.898 0.060
RK 15 1.333 2.645 AK15 3.001 0.068
RK 16 0.889 2.743 AK16 0.127 0.088
RK 17 1.095 3.797 AK17 0.034 0.104
RK 18 0.905 3.176 AK18 0.062 0.088
RK 19 0.683 2.628 AK19 0.028 0.250
RK 20 0.365 2.588 AK20 0.051 0.214
RK 21 0.889 2.538 AK21 0.072 0.080
RK 22 0.968 3.135
RK 23 1.698 2.622
RK 24 0.635 3.293
RK 25 1.079 3.703
RK 26 0.857 2.419
RK 27 0.302 2.502
RK 28 0.841 2.698
RK 29 0.73 2.658
RK 30 1.429 4.67
RK 31 1.222 2.654
RK 32 2.794 2.476
RK 33 0.429 2.571
RK 34 0.825 2.588
RK 35 0.638 3.222
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Asmiaty Sahur et al, International Journal of Advances in Agricultural Science and Technology,
Vol.4 Issue.1, January- 2017, pg. 07-15 ISSN: 2348-1358
Impact Factor: 6.057
Figure 3: (a) Level concentration of Indole Acetic Acid produced by Rhizosphere Bacteria that has negative
gram Isolated from Soybean plants.
(b) Level concentration of Gibrellic Acid produced by Rhizosphere bacteria that has negative gram
isolated from Soybean Plants.
(c) Level concentration of Indole Acetic Acid produced by Rhizosphere Bacteria that has positive
gram Isolated from Soybean plants.
(d) Level concentration of Gibrellic Acid produced by Rhizosphere bacteria that has positive gram
isolated from Soybean Plants
Our results indicated that the comparison between two different types of rhizosphere bacteria has different
pattern in the number of phytohormone they produced such as Indole Acetic Acid and Gibberellin Acid. As
shown in figure 3a and 3b rhizosphere bacteria with negative gram has produced Gibberellin Acid highest than
Indole Acetic Acid compare to bacteria with positive gram has produced Gibberellin Acid lower than Indole
Acetic Acid as indicated in figure 3c and 3d.
4. Conclusions
The vast majority of bacteria isolated from three different areas of South Sulawesi was successfully grown from
Takalar. In terms of characterization Bacteria isolated from rhizosphere of Indigenous soybean of South
Sulawesi had more than one physiological characters such they can produce Gibberellin acid and Indole Acetic
Acid. Isolates RK 32 (2.794 mg/l) produced the highest level of IAA concentration and RK 30 (4.670 mg/l)
secreted the highest number of GA concentration. Furthermore the isolates with positive gram that produced the
highest concentration of GA was AK 20 (0.214 mg/l) and produced IAA AK 11 (3.066 mg/l).
© 2017, IJAAST All Rights Reserved, www.ijaast.com 14
Asmiaty Sahur et al, International Journal of Advances in Agricultural Science and Technology,
Vol.4 Issue.1, January- 2017, pg. 07-15 ISSN: 2348-1358
Impact Factor: 6.057
5. Acknowledgements
The author thanks to those who already helped in doing the isolation process also the student who helps took
several plants samples from three different areas of South Sulawesi. Also the author acknowledged Dr.Malcolm
Wagener, The University of Queensland, Australia, for his valuable inputs and comments into this manuscript.
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