The Potency of Plant Growth Promoting Rhizobacteria (PGP

) of

Coastal Poaceae (Phragmites karka) to Stimulating of Paddy (Oryza

sativa L.) Growth

Yurnaliza

, Artha Joseva Hutapea

and Nunuk Priyani

Departement of Biology, Faculty Mathematics and Natural Sciences, Jl. Bioteknologi No. 1, Kampus USU, Universitas

Sumatera Utara, Medan, 2015, Indonesia.

Keywords: Coastal area, Paddy, PGPR, Phragmites karka.

Abstract: The aim of this research was to obtain and to observe the activity of plant growth promoting rhizobacteria

(PGPR) isolated from coastal Poaceae, Phragmites karka, on stimulating paddy’s growth. The bacteria were

qualitatively on three different selective media: Pikovskaya, JNFb, and tryptophan containing LB media to

observe the ability of bacteria to solubilize phosphate, to fix nitrogen, and to produce indol acetic acid (IAA).

The selected potential bacteria from each test were studied further as PGPR candidates on stimulating paddy

growth in sterilized soil medium. The results showed that there were 6 isolates able to solubilizing phosphate,

6 isolates capable to fixing nitrogen and 8 isolates able to producing IAA. The synergy test resulted in three

selected isolates (PP03, RG08 and PI05) were synergistic each other. The application of three isolates as

single culture and their consortium stimulated of paddy growth significantly comparing to control (without

bacteria) especially in the plant height and root length parameter. The best performance of paddy growth was

achieved from consortium of 3 bacterial isolates treatment.

1 INTRODUCTION

Indonesia is one of the most populous countries in

the world. Rapid population growth leads to increase

in food demand. Society and government use various

measures to maintain food availability. Food

production is needed to improve of food demand

fulfillment. Efforts that have been made to increase

food production still need to be improved. One effort

that can be done is extended farmlands by utilized

marginal land such as coastal area. Marginal land is

a nutrient-poor land with environmental conditions

do not support the plant growth. Coastal area have

sandy soils and high salinity content. Only tolerance

organism can grow in this area. Indonesian Centre for

Rice Research have breeding program to improve and

find a salinity tolerance of rice (Hairmansis et al., 2017).

Beside the plant tolerant to salinity, other efforts to

make marginal land become usable are attempted the

organism plant growth promoting tolerance to

salinity.

Plant Growth Promoting Rhizobacteria (PGPR)

was a numerous soil bacteria which occupy roots or

rhizosphere area, stimulating plant growth in various

mechanism and also induced plant resistance

(

Vessey,2003). Investigation of PGPR microorganism

from many sources is the purpose to making it as

biofertilizers. In association of plant and PGPR,

microorganism given some benefit action to

stimulating plant growth specifically by fixing

dinitrogen from atmosphere, solubilizing unsoluble

phosphate, producing growth hormone of Indole-3-

acetic acid (IAA), antimicrobial compound to

reduced plant pathogen and induced plant resistance

(Vessey, 2003), (Sandilya et al., 2016). Bacteria

Burkholderia cepaciai is one of species PGPR

bacteria that potential as stimulated growth of maize

and also have biocontrol activity to Fusarium spp.

(Bevivino et al., 1998).

Many PGPRs are diazotrophs (symbiosis or non

symbiosis nitrogen fixing bacteria). Many

diazotrophs bacteria are phosphate solubilizing and

IAA producer (Vessey, 2003). In lower conditions

of N or P level in soil, diazotroph microorganism

adapt in the rhizosphere but harmless to their host.

Population and activity of microbial in rhizosphere of

sandy soil land is more higher than in rhizosphere of

humus soil, and it’s call as rhizosphere effect

Yurnaliza, ., Hutapea, A. and Priyani, N.

The Potency of Plant Growth Promoting Rhizobacteria (PGPR) of Coastal Poaceae (Phragmites karka) to Stimulating of Paddy (Oryza sativa L.) Growth.

DOI: 10.5220/0010088600670072

In Proceedings of the International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches (ICOSTEERR 2018) - Research in Industry 4.0, pages

67-72

ISBN: 978-989-758-449-7

(Dotaniya and Meena, 2015). Low land moisture such as

sandy soil affect microorganism to move toward the

plant root zone. Availability nutrient in rhizosfer

influence the rhizosphere population microorganism

(

Dotaniya and Meena, 2015).

In this research will be found a new candidate the

plant growth promoting rhizobacteria of Phragmites

karka which collected from coastal area.

2 MATERIAL AND METHODS

2.1 Material Research

The soil from Phragmites karka rhizosphere were

collected as much as 100 g from Coastal location in

Percut Sei Tuan area. The soil was collected from 3

samples point and stored on sterile of sealed plastics

sample before transfer to laboratory. Seed of paddy

was obtained from commercial seed seller. Several

medium Pikovkayas, JNFb (medium free nitrogen

source) and Luria Bertani (LB)+ Tryptophan medium

were used for cultivation and selection of PGPR

activity.

2.2 Isolation of PGPR Bacteria

The amount of 1 g of soil sample was diluted into 9

ml of sterile distilled water and than homogenized.

Serial dilution was conducted to obtain a 1x 10

-5

dilution. A total of 0.1 ml of soil suspension from the

last dilution was spread to the surface of Pikovskaya,

JNFb and LB + tryptophan medium aseptically. The

culture medium was incubated for 2-3 days at room

temperature. Activity of phosphate solubilizing

bacteria were detected qualitatively by clear zone

formation around colony of bacterial on pikovskaya

medium. The positive test of nitrogen-fixing bacteria

were characterized by the presence of colonies

bacteria on JNFb medium. The medium of JNFb was

free nitrogen source. Presence of bacteria in JNFb

medium indicated the bacteria can fix bacteria from

atmosphere. Bacteria produced IAA in medium LB +

tryptophan broth medium were checked by added

Salkowski’s reagent (2% 0.5M FeCl

in 35% HClO

solution). The color medium become purple if there

was indol compound in the medium. The positive sign

bacteria growth in selective medium were purified

and further characterized.

2.3 Selection of Phosphate Solubilizing

Bacteria

The purified bacteria isolates were further tested

for their ability to dissolve the phosphate to

determine its solubility index, fixing nitrogen and

produce IAA. Phosphate solubility index (PSI) score

was obtained from the comparison of the diameter of

clear zone around the bacterial colony and diameter

of colony bacteria (Dotaniya and Meena, 2015).

2.4 Selection of Nitrogen Fixing

Bacteria

Nitrogen fixation from purified bacteria were

obtained from pellicle size formed of bacteria when

cultured on semi solid JNFb medium. The white

pellicle formed will be visible in the surface medium

on tube and measured after 10 days incubation at

room temperature.

2.5 Selection of IAA Producing

Bacteria

The IAA produced by each bacteria was measured

with colorimetric technique using the Salkowski’s

method (Ehmann, 1977). Bacterial suspension (3 ml)

with cell density in Optical Density (OD) 600 was

0.5 (≈ 10

Colony Forming Unit, CFU / ml) was

introduced into 27 ml of liquid LB + tryptophan broth

and incubated at 28°C for 6 days and shaken at 100

rpm. Every 2 days as much as 10 ml of culture fluid

is taken and then centrifuged at 5500 rpm for 10

minutes. The supernatant was transferred to a new

sterile tube and then added Salkowski reagent with 4

: 1 ratio (supernatant : salkowski) and incubated for

20 minutes at room temperature. Colorimetrically the

color change formed was measured by a

spectrophotometer at a wavelength of 535 nm. The

concentration of IAA from the sample was calibrated

from linear regression equation of pure IAA.

2.6 Application of Selected Bacteria to

Improved Paddy Growth

Paddy seeds were cultivated on 1 kg of sterile humus-

sandy soil with composition humus: sandy soil (3:1)

for 1 week. The potential bacteria were checked their

synergistic each other by cross inoculated onto agar

medium. When two bacteria was in synergism

condition, they can live together with no inhibited

each other. The selected bacteria was cultivated on

Nutrient Broth for 24 hours. Ten milliliter of bacterial

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

culture broth OD 600 (0,5) or in consortium form

were poured into rhizosphere of paddy seedling on

soil medium. Each treatment was 3 replications.

Parameters of paddy growth were analyzed as plant

height, root length, biomass of plant and how much

the dead plant. Observation of rice plant growth was

done at 30 days after planting.

3 RESULTS AND DISCUSSION

3.1 Isolation Results

A total of 20 bacteria were isolated from Phragmites

karka rhizosphere. Six isolates were each growth on

Pikovkaya and JNFB medium and also 8 isolates on

LB+Tryptophan medium. bacteria and 8 isolates are

IAA producing bacteria. Based on the colony

morphology observations showed that the 20 isolates

had various characteristics in shape, edge, elevation

and color. The colony shapes were circular, irregular

and rhizoid. Almost all isolates were had white color

and several cream. Only one isolate had orange color

(Table 1).

3.2 Phosphate Solubilizing, Nitrogen

Fixing and IAA Producing Bacteria

From 20 isolates bacterial tested were only 7

isolates potential to give positive results to all tree

test. Results for phosphate solubilizing activity

showed that 14 isolates bacterial were able to dissolve

phosphate and one isolate with the highest ability was

PP03. In the nitrogen fixing test was indicated by

pellicle size showed that only 9 isolates produced the

pellicle when growth on semisolid medium of JNFb

and seven isolates produced thick pellicle with the

size 5-9 mm. All isolates tested were IAA producer,

and only two isolates produced IAA in concentration

rage 28-40 ppm i.e. PP03 and RG08. Seven bacterial

isolates were only dissolved phosphate and produced

IAA and two isolates were only fixed nitrogen and

produced IAA. From all tested bacteria, four isolates

had one ability that only as IAA producer (Table 2).

Three isolates bacteria which have best perform to all

selected criteria were collected i.e. PP03, RG05 and

PI05) and then used in paddy growth application. The

synergistic test results showed that tree bacteria

synergistically (Table 1).

Three selected bacteria in this research were potential

as biofertilizer candidate. Existence of

microorganisms as biofertilizer increases the growth

of plants by enrichment of soil nutrients (nitrogen

fixation) or making nutrient like phosphate more

available to plant or increasing number of roots to be

increasing nutrient absorption from soil or producing

growth hormone to stimulated plant growth (

Vessey,

2003). PGPR may

Table 1: Morphological character of colony bacteria from Phragmites karka, cultivated on 3 different agar medium

No Code Agar medium Shape Edge Elevation Color

1 PP01 Pikovskaya Agar Irregular Entire Flat White

2 PP02 Pikovskaya Agar Irregular Undulate Flat White

3 PP03 Pikovskaya Agar Circular Filamentous Raised White

4 PP04 Pikovskaya Agar Circular Undulate Raised White

5 PP05 Pikovskaya Agar Circular Entire Flat White

6 PP06 Pikovskaya Agar Rhizoid Filamentous Flat White

7 RG01 JNFB Circular Entire Flat White

8 RG02 JNFB Circular Undulate Flat White

9 RG05 JNFB Rhizoid Filamentous Flat White

10 RG06 JNFB Circular Undulate Raised Orange

11 RG07 JNFB Circular Undulate Raised White

12 RG08 JNFB Rhizoid Filamentous Flat White

13 PI01 LB Irregular Filamentous Flat White

14 PI02 LB Irregular Entire Raised Cream

15 PI03 LB Circular Entire Flat White

16 PI04 LB Circular Entire Raised Cream

17 PI05 LB Irregular Filamentous Raised Cream

18 PI06 LB Irregular Entire Flat White

19 PI07 LB Irregular Filamentous Flat White

20 PI08 LB Rhizoid Filamentous Flat White

The Potency of Plant Growth Promoting Rhizobacteria (PGPR) of Coastal Poaceae (Phragmites karka) to Stimulating of Paddy (Oryza

sativa L.) Growth

colonize plant on rhizosphere or endophytes. Not any

soil bacteria can colonize in that area. Association

between rhizobacteria and plant were symbiotically

give benefit to plant. Non-symbiotic diazotroph

bacteria like Azospirillum, Azotobacter,

Gluconacetobacter diazotrophicus, Beijerinckia sp.

can live in rhizospher and promoting plant growth

(

Vessey,2003).

3.3 Analyzed of Paddy Growth

Application of 3 isolates bacteria to paddy seed were

observed to several plant growth parameter .

Inoculated of 3 bacteria and their consortium

treatment statistically were not give significant results

for plant biomass measured but had significantly

results than control (P0/no bacterial treatment) to

stimulated plant height and root length.

Table 2: Combination ability of phosphate solubilizing bacteria,nitrogen fixing bacteria and IAA producing bacteria

Bacterial

Isolate

Phosphate

solubilizing index

Pellicle size

Consentration of

IAA

1 PP01 + - +

2 PP02 + - ++

3 PP03 +++ +++ +++

4 PP04 + - ++

5 PP05 ++ - +

6 PP06 + - +

7 RG01 + +++ +

8 RG02 + +++ +

9 RG05 + +++ +

10 RG06 + - ++

11 RG07 + +++ +

12 RG08 + - +++

13 PI01 - + +

14 PI02 - - +

15 PI03 - - +

16 PI04 - - +

17 PI05 ++ +++ ++

18 PI06 + ++ +

19 PI07 - - ++

20 PI08 - +++ +

Noted. Phosphate solubilizing index: - (0), + (1-2), + + (2-3), + + + (3-4); Pellicle Size (mm): - (0), + (1-3), + + (3-5), + +

+ (5-9); IAA Consentration (ppm): - : 0, + (1-14), ++ (15-27), + + + (28-40)

Table 3: Average paddy seedling growth parameter after 30 days planting

Treatment

Average seedling Growth Parameters

Plant

height

(cm)

Root

length (cm)

Wet

weight

canopy

(g)

Weight of

wet root

(g)

Dry weight

of canopies

Dry

weight of

root(g)

P0 (without bacteria) 35,04

4,71

0,75

0,20

0,13

0,07

P1 (PP03) 39,99

8,67

0,71

0,36

0,11

0,09

P2 (RG08) 39,10

10,50

0,61

0,35

0,08

0,09

P3 (PI05) 42,09

5,08

0,73

0,42

0,10

P4 (PP03+RG08) 41,90

9,43

0,74

0,37

0,11

0,10

P5 (PP03+PI05) 41,31

8,10

0,48

0,19

0,08

0,06

P6 (RG08+PI05) 42,55

8,44

0,67

0,43

0,09

P7 (PP03+RG08+PI05) 44,84

7,81

0,80

0,47

0,13

0,14

Description: The average value that has the same letter in the same column shows no significant difference according to the DMRT test

at the real level of 5%

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

The best results for plant height was from P7

treatment and for length of roots was P2 treatment

(isolate of RG08) (Table 3). All treatment of PGPR

bacterial and their consortium provided best

performance to paddy growth than control, even

though from growth parameter P7 treatment was the

best results (Figure 1). The research about application

of biofertilizer to Sugarcane soil showed that

biofertilizer will substitute 25%-50% of NPK

fertilizer in soil (

Mulyani

et al., 2017). Inoculation of

PGPR bacteria to maize plant increased plant

performance in chlorophyll content, root and shoot

length (

Ullah et al., 2017)

Figure 1: Performance of paddy seedlings after treated with rhizobacteria of Phragmites karka and observed on 30 days

planting. Noted: P0 (without bacteria), P1-P7 were treated by P1 (PP03 isolate), P2 (RG08 isolate), P3 (PI05 isolate), P4

(PP03 and RG08 isolates), P5 (PP03 and PI05 isolates), P6 (RG08 and PI05 isolates) and P7 (PP03, RG08 and PI07 isolates).

4 CONCLUSION

Selective isolation of rhizobacteria from Phragmites

karka were obtain 20 isolates and were selected

three isolates as PGPR candidate. Only seven isolates

were able to fixing nitrogen, solubilizing phosphate

and producing IAA. Three selected isolates had the

best activity for all test. Application of three selected

isolates and their consortium to paddy growth

resulted best performance to paddy growth especially

for plant height and length of root than control

(without bacterial treatment).

ACKNOWLEDGEMENTS

We would to thank to head of Microbiological

laboratory, Department of Biology, Universitas

Sumatera Utara for supporting this research.

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ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches