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Procedia Engineering 32 (2012) 177 183

I-SEEC2011

Screening of Endophytic Bacteria from Organic Rice Tissue

for Indole Acetic Acid Production
P. Phetcharat a* and A. Duangpaeng b
a

Department of Biotechnology, Faculty of Technology, Udon Thani Rajabhat University, Udon Thani 41000 Thailand
b
Department of Plant Production Technology, Faculty of Technology,Udon Thani Rajabhat University,
Udon Thani 41000 Thailand
Elsevier use only: Received 30 September 2011; Revised 10 November 2011; Accepted 25 November 2011.

Abstract
The objectives of this study were to isolate and characterize endophytic bacteria from various rice tissues focusing on
their ability to produce indole acetic acid (IAA). Rice tissue samples were collected from different three types of rice
farm; 1 year, 3 years organic rice, and conventional rice farms in Udon Thani, Thailand. Seventy-one isolates of
endophytic bacteria were screened using PDA and TSA medium. The majority of strains isolated from root tissues
were totally 26 isolates, exclusively collected from 3 years organic rice farm. Phenotypic characteristics of all isolates
illustrated that 34 isolates were identified as Pesudomonas sp., while other isolates were also identified as Bacillus,
Azotobacter, and Enterobacter species. The study of IAA production indicated that 4 isolates efficiently produced
IAA over than 10 Pg/ml. The O-1-R-4 (2) isolate produced the highest IAA (14.58 Pg/ml), and it was identified as
Bacillus sp. This effective result will be used for further investigation on the feasibility of commercial production of
IAA up-scale fermentation.

2010 Published by Elsevier Ltd. Selection and/or peer-review under responsibility of I-SEEC2011
Keywords: Organic rice; Endophytic bacteria; Indole acetic acid; Root; Stem

1. Introduction
Organic rice is typically grown and processed without the use of any synthetic chemicals as found, for
example, in fertilizers, insecticides, pesticides, herbicides, fungicides, preservatives, seed treatments and
hormones growth regulators, etc. It mainly relies on organic sources to maintain soil health, supply plant
* Corresponding author. Tel.: +6-681-574-6434; fax: +66 4221 1978
E-mail address: parinyapan@gmail.com.

1877-7058 2012 Published by Elsevier Ltd.

doi:10.1016/j.proeng.2012.01.1254

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nutrients and minimize insects, weeds and other pests>1@. Efficiency of organic rice expansion could be
done by living microorganisms in ecosystem. Their metabolic activities will help to circulate many
nutrients in soil.
Endophytic bacteria generally live in plant tissues without doing substantive harm or gaining benefit
other than securing residency. They have been found in numerous plant species with most being members
of common soil bacteria genera such as Pseudomonas, Bacillus and Azospirillum >2@. Endophytic bacteria
can be isolated from surface-disinfected plant tissue or extracted from internal plant tissue >3@. Several
reports indicated that both gram-positive and gram-negative bacterial endophytes have been isolated from
difference plants such as soybean, wheat, corn, sorghum, cucumber fruits, sugar beet roots and rice >2, 3,
4, 5@. However some endophytic bacteria exert several beneficial effects on host plants, such as
stimulation of plant growth, nitrogen fixation and induction of resistance to plants pathogens >6@.
Endophytes also promote the growth of plants, especially through secretion of plant growth regulators;
e.g. indole-acetic acid, via phosphate-solubilizing activity, by supplying biologically fixed nitrogen. In
addition, endophytic bacteria supply essential vitamins to plants >7@. The production of auxin-like
compounds increased shoot growth and tillering. Other effects of endophytes infection on the host plant
include osmotic adjustment, stomatal regulation, modification of root morphology, enhanced uptake of
minerals and alteration of nitrogen accumulation and metabolism >4, 8@. The aims of this study were to
isolate and characterize endophytic bacteria from various rice tissues focusing on their ability to produce
indole acetic acid (IAA). This information will be used for further investigation on the feasibility of
commercial production of IAA up-scale fermentation.
2. Materials and methods
2.1 Isolation of endophytic bacteria.
The rice plants were collected from different three types of rice farm; 1 year, 3 years organic rice
farms, and conventional rice farm in Udon Thani, Thailand. The healthy rice plants were carefully
removed, washed under tap water to remove soil and separated to three parts; root, lower stem (5-10 cm.
from root) and higher stem (10-15 cm. form root). All rice tissues were put in beaker, soaked in distilled
water and drained. After that rice tissues were rinsed in 70% ethyl alcohol for 30 seconds and then
sterilized with 0.2% HgCl2 for 3 minutes for root and lower stem, 5 minutes for higher stem. The tissues
were then washed ten times with sterile water, cut into small pieces and homogenized in a blender
containing 90 ml sterile distilled water. Macerated tissues were diluted into 10-1 dilution by adding 9
volumes of sterile distilled water. Serial dilution was made up to 10-5 dilution by taking 1 ml of wellshaken suspension and adding into 9 ml water blank tubes. 100 Pl from 10-4 and 10-5 dilutions were
spread plated on two different media; PDA and TSA and then incubated at 35 qC for 24 hrs >6@.
2.2 Morphological and physiological characterization
Gram staining and capsule staining were carried out according to standard staining protocols.
Cellulase activity: the test isolates were spot-inoculation on cellulose agar plates and incubated for 1
week at 30 qC. Bacterial cellulose activity was observed. The medium was flooded with Congo red dye
for 15 minutes, drained, rinsed with 1M NaOH. The Congo red reacts with cellulose to form a red-colored
complex. Any clear area around the growth of the culture after the addition of the Congo red dye indicates
the breakdown of cellulose by the organism due to its production of cellulase, an extracellular enzyme
>7@.

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Pectinase activity test: the test isolates were spot-inoculated on the pectin agar plates and incubated
for one week at 30 qC. The plates then were flooded with 1% aqueous Red ruthenium solution for one
hour, drained, rinsed with water and observed. Red ruthenium is bound to unhydrolysed pectin and give
the red color. Halo zone around isolates colony was observed >9@.
Motility test: each isolate was spot-inoculated on the center of semi-solid nutrient agar plates (0.2%
agar) and incubated at 30 qC. The diffusion of colony was observed and recorded at 24 hours.
Antibiotic resistance test: the test isolates were spot-inoculated on the nutrient agar plates
incorporated with filter sterilized streptomycin at the rate of 100 Pg/ml and incubated for 48 hrs at 30 qC.
The antibiotics resistance was recorded as positive if the test colony appeared on the plates, as compared
to the control plate in which no antibiotic was added.
Fluorescence pigment production test: the test isolates were spot-inoculated on the Kings B
medium agar plates and incubated at 30 qC. The plates were exposed to UV light to examine the
fluorescence ability after 24-48 hrs of incubation.
IAA production test: 5 Pl of LB (Luria Bertani) broth alone and LB broth medium added with Ltryptophan at the rate of 100 Pg/ml. The test tubes were covered with brown paper and incubated at 28 qC
for 24 hours on a rotary shaker. The broth was centrifuged at 10000 rpm for 15 minutes. 2 ml of
supernatant was collected and 2-3 drops of o-phosphoric acid were added. The aliquots were shaken,
added 4 ml of reagent (1 ml of 0.5M FeCl3 in 49 ml of 35% perchloric acid (HClO4)) and vortexed
thoroughly. The samples were incubated at room temperature for 25 minutes and their absorbance was
read at 530 nm. Auxin quantification value was recorded by extrapolating calibration curve made by
using IAA standard (10-100 Pg/ml) >10@.
3. Results
3.1 Isolation of endophytic bacteria
Seventy-one isolates of endophytic bacteria were isolated from three parts of rice tissue (root, lower
stem and higher stem) using PDA and TSA medium. Rice tissue samples were collected from different
three types of rice farm; 1 year, 3 years organic rice farms, and conventional rice farm in Udon Thani,
Thailand. The serial dilutions of macerated rice samples were spreaded on TSA and PDA medium. The
colony forming units (CFU) were determined after appropriate inoculation at 30 qC. A majority of the
microorganisms were screened and isolated (26 strains) from root tissue of 3 years organic rice farm. As
shown in Table 1. Bacterial isolates were identified based on Grams staining and physiological tests.
3.2 Characterization of bacterial colony
Figure 1 showed the colony characteristics of some isolates on TSA medium. They were classified into
three group of colony morphology depending on color, form, elevation, and margin. The members of
group one were white circular, convex and entire; those in group two were pale orange, circular and
undulate; and those in group three were light yellow circular, undulate with creamy colonies.

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P. Phetcharat and A. Duangpaeng / Procedia Engineering 32 (2012) 177 183

Table 1. Isolation frequency and total population of endophytic bacteria in rice tissues
Number of bacterial isolates
Rice farm type

Type of tissue

C1/

2/

O-1

O-33/

st

1 time of isolation

2nd time of
isolation

Total numbers of
both isolation

R4/

11

N5/

S6/

11

11

Total number of endophytic bacteria

1/

C : Conventional rice farm

2/
O1 : 1 year organic rice farm
3/
O3 : 3 year organic rice farm

(a)

71
4/

R : Root
5/
N : Lower stem
6/
S : higher stem

(b)

(c)

Fig. 1. (a) The colony characteristics of O-3-R-2 isolate on TSA medium (b) The colony characteristics of O-3-R-3 isolate on TSA
medium (c) The colony characteristics of O-1-S-3 isolate on TSA medium

3.3 Morphological and physiological characterization

The result from Grams staining clearly exhibited 40 gram-positive and 31 gram-negative isolates.
However, the result fairly differenced from that of recently researched by Hung et al. (2004) and found that
gram-positive and gram-negative isolates were equally distributed between two species of soybean. The
result also found that 6 isolates formed capsule (Table 2). When grown on 0.2% agar, 30 isolates were
found to be motile. 71 isolates of endophytic bacteria were screened for growth on NA amened with
Streptomycin at the rate of 100 Pg/ml. 43 isolates were able to grow in the presence of the antibiotic.
When grown on Kings B medium (specific for fluorescent Pseudomonads), 34 isolates were found to be

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putative fluorescent Pseudomonads. Twenty-five isolates gave a clear zone of hydrolysis on pectin agar
plate. When grown on cellulose agar medium, there were 13 isolates able to grow.
Table 2. Isolation frequency and total population of endophytic bacteria in rice tissues
Rice farm
type

Grams staining
Positive

Capsule
staining

Motility

Antibiotic
resistance

Fluorescence

Pectinase
activity

Cellulase
activity

Negative

IAA
Production

Conventional

15

10

10

13

21

1 year organic

10

10

19

10

10

18

3 year organic

15

11

12

24

11

10

27

Total

40

31

30

43

34

25

13

66

The IAA production ability of endophytic bacteria was detected. 66 isolates produced IAA in both of
presence and absence of the precursor tryptophan. 4 isolates efficiently produced IAA over than 10 Pg/ml
(Table 3). The O-1-R-4 (2) isolate produced the highest IAA (14.58 Pg/ml).
Table 3. The ability of IAA production of the first 4 isolates of endophytic bacteria
The Endophytic bacterial isolate

Ability of IAA production ( Pg/ml).

O-1-R-4 (2)
O-1-N-2 (2)
C-R-8 (2)
C-R-5 (2)

14.58
13.54
13.17
12.68

The characteristics of O-1-R-4 (2) isolate were determined. The colony was white and circular, rodshaped, motile, gram positive, endospore forming, not showed the ability of pectin hydrolysis, cellulose
hydrolysis and fluorescence ability. The O-1-R-4 (2) isolate was identified as Bacillus sp.
4. Discussion
This investigation is to describe the indigenous endophytic bacteria isolated from three types of rice
tissue, which collected from conventional, 1 year organic rice, and 3 year organic rice farms. Seventy-one
isolates of endophytic bacteria were screened from those samples, using PDA and TSA medium. There
was significant phenotypic variation in the types of indigenous bacteria. Several factors may explain these
differences, including rice tissue types and the type of rice farm. Previous researches have reported that
there were both phenotypic and genotypic variations in the types of endophytes isolated from two species;
one cultivated (Glycine max) and another wild (G. soja) of soybean >6@. Moreover, Stoltzfus et al. (1997)
selected 133 isolates of endophytic bacteria from root and stem of rice tissues of diverse varieties grown
in different soil types >4@.
The diversity of a collection of seventy-one putative endophytic bacteria was assessed using
phenotypic characterization methods. Colony morphology gave an indication of the variation among the
endophytes. The isolates studied were chosen for their dominance as well as uniqueness or difference
with other in colony morphology. The studying of all isolates morphological and physiological
characteristics indicated that 34 isolates to be Pseudomonas because they grown on KingB medium, the
selective medium for Pesudomonas. The other isolates to be Bacillus, Azotobacter and Enterobacter
species. Result of the previous study evidenced that most of the endophytic bacteria isolated from

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medicinal plants belonged to Bacillus and Pseudomonas species >2@. Bai et al. (2002) also isolated
Bacillus sp. from nodules of soybean >11@. In addition, the recent study of endophytic bacteria isolated
from rice tissue found the species of Pseudomonas, Bacillus, Azospirillum and others >4@.
In addition to this study, 25 isolates secreted pectinase and 13 isolates secreated cellulase. It can be
summarized that hydrolytic enzymes, pectinase and cellulose may play an importance role in the
mechanisms by which endophytic bacteria penetrate into and persist in the host plant. Also, these enzyme
might be involved in the invasion of host plants by endophytes, as reported for Azoarcus sp. >12@.
The motility of endophytic bacteria was also studied. 26 isolates were motile. Due to the motility and
pectinolytic activity may confer an advantage for intercellular ingress and spreading of endophytic
bacteria into the host plants, the cell wall of the host plants contain cellulose, whereas the middle lamella
between cell walls contain mainly pectin >13@.
The ability of IAA production was investigated. There were 66 isolates could produced IAA, but only
4 isolates efficiently produced IAA over than 10 Pg/ml. The O-1-R-4 (2) isolate was produced the highest
IAA (14.58 Pg/ml). Bandara et al. (2006) found that endophytic bacteria and fungi isolated from rice also
produced IAA with variable quantity >8@. In addition, the previous research reported that there were 15
isolates of endophytic bacteria produced IAA over than 25 Pg/ml >6@. IAA has many different effects, as
all auxins do, such as inducing cell elongation and cell division with all subsequent results for plant
growth and development.
This study significantly demonstrated the occurrence and diversity of culturable endophytic bacteria in
various rice tissues. This can be utilized in future application, such as effect of rice seed treatment with
endophytic bacteria, delivery of degradative enzymes for controlling certain plant diseases, and
production of IAA in large scale including other useful products.
5. Conclusion
Seventy-one isolates of endophytic bacteria were screened from three parts of rice tissue using PDA
and TSA medium. Rice tissue samples were collected from different three types of rice farm; 1 year, 3
years organic rice, and conventional rice farms in Udon Thani, Thailand. The isolates were examined for
colony forming, Grams staining, physiological characteristics, and indole acetic acid productivity. The
majority of strains isolated from root tissues were totally 26 isolates, exclusively collected from 3 years
organic rice farm. Phenotypic characteristics of all isolates illustrated that 34 isolates were identified as
Pesudomonas sp., while other isolates were also identified as Bacillus, Azotobacter, and Enterobacter
species. The study of IAA production indicated that 4 isolates efficiently produced IAA over than 10
Pg/ml. The O-1-R-4 (2) isolate produced the highest IAA (14.58 Pg/ml), and it was identified as Bacillus
sp.
Acknowledgements
The authors acknowledged the help from Office of the National Research Council of Thailand
scholarship, the support from Department of Biotechnology and Department of Plant Production
Technology, and the facility provided from Center of Science and Technology for research and
community development, Udon Thani Rajabhat University.

P. Phetcharat and A. Duangpaeng / Procedia Engineering 32 (2012) 177 183

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