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2011 International Conference on Food Engineering and Biotechnology
IPCBEE vol.9 (2011) © (2011)IACSIT Press, Singapoore
Effect of Phosphatic Fertilizer on Root Colonization of Aerobic rice by
Phosphate-Solubilizing Bacteria
Qurban Ali Panhwar 1, Radziah Othman 1, Zaharah Abdul Rahman 1, Sariah Meon 3 and Mohd Razi
Ismail 2
1
Department of Land Management. 2Institute of Tropical Agriculture, 3Department of Plant Protection.
Universiti Putra Malaysia, Selangor, Malaysia
Abstract. Phosphate-soubilizing bacteria (PSB) have the ability to colonize and associate with
plant roots. An experiment was conducted to study the colonization and association of Bacillus spp.
(PSB9 and PSB16 strains) on aerobic rice. Root colonization was examined under scanning and
transmission electron microscope (SEM and TEM). Bacterial association with different rates of P
fertilizer was determined during 30 days of growth. The population of PSB16 was affected by the
application of phosphatic fertilizer. The highest population was found in rhizosphere while lowest
was in non-rhizosphere. Bacterial inoculation significantly increased plant biomass and root growth
of aerobic rice. The PSB strains were able to colonize on the surface and interior of rice roots.
Key words: aerobic rice, phosphate-solubilizing bacteria, colonization, rhizosphere.
1. Introduction
Phosphorus is important for the plant growth and promotes root development, tillering, early flowering,
and performs other functions like metabolic activities, particularly in synthesis of protein (Tanwar and
Shaktawat, 2003). The rhizosphere microorganisms play a significant role for P solubilization in many crops
especially under P deficiencies (Khan et al., 2009). Among the soil microorganisms, PSB play an important
role in solubilizing P for the plants and allowing more efficient use of P fertilizers (Gyaneshwar et al., 1998).
These plant growth-promoting rhizobactera can colonize the root surface and some have been shown to
colonize endophytically (Naher et al., 2009). The association and colonization of PSB on surface of roots
involve direct competition with other rhizosphere microorganisms, while the endophytic population of PSB
may give more beneficial effects to the plants due to less competition.
The efficacy of PSB in solubilizing P can be decreased with increasing rates of chemical fertilizers
applied to soil. The strains can be utilized in combination with suitable doses of P fertilizers for better plant
growth with lower usage of fertilizers (Shaharoona et al., 2008). For a more sustainable and environmental
friendly crop production, it is necessary to enhance P solubilization by PSB in cereal crops like rice. Hence,
the present study was undertaken with the aim of assessing the potential of PSB colonization in aerobic rice
at different rates of P fertilizer.
2. Materials and Methods
Two PSB strains (Bacillus spp. PSB9 and PSB16) were used. The aerobic rice line MR 219-9 mutant
(M-9) obtained from Malaysian Agricultural Research and Development Institute (MARDI) and seeds were
surface sterilized by method modified from Amin et al. (2004). The phosphatic fertilizer, triple
superphosphate (TSP) was applied at 0, 30 and 60 kg P2O5 ha-1. The study was laid out as a factorial
experiment arranged in a completely randomized design (CRD) with 4 replications. Seedlings were grown
for 30 days in growth chamber condition and plant sampling was done at intervals of 10 days. Total bacterial
population from rhizosphere, non-rhizosphere and endosphere were determined. The bacterial colonization
on rice roots was observed by using electron microscopy. Five days old seedlings were inoculated with
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approximately 109 cfu mL-1 of bacterial strains and 5 days after inoculation root sample were washed with
sterile water. Samples were pre-fixed with 4% glutaraldehyde overnight, washed with 0.1 M sodium
cacodylate buffer and osmium tetraoxide buffer (1%) was used for post fixation. After a series of
dehydration in acetone (35, 50, 75, 95 & 100%) the samples were dried in a critical point dryer and mounted
on aluminum stubs, sputter coated in gold and viewed under scanning electron microscope (SEM) (JEOL
JSM-6400 attached with OXFORD INCA ENERGY 200 EDX). For transmission electron microscope (TEM)
observation the samples were infiltrated with acetone and resin mixture. Polymerized steps samples were cut,
coated with gold and observed under TEM (PHILIPS HMG 400).
Data Analysis
All data were statistically analyzed using the SAS Software Program (Version 9.1) and treatment means
were compared using Tukey’s test (P < 0.05).
3. Results
3.1.
Population of PSB at different P fertilizer rates
In general the highest population of PSB inoculated was found in the rhizosphere while lowest was in
non-rhizosphere of aerobic rice. Application of P fertilizer significantly affected the population of PSB16 but
not the PSB9 strain. The PSB16 at the rate of 30 kg P2O5 ha-1 produced significantly highest population in
non-rhizosphere, rhizosphere and endosphere.
10 days
9
7
5
3
1
-1
0
30
60
9
7
5
3
1
-1
0
10 days
20 days
c) Rhizosphere (PSB9)
Pop u latio n (lo1 0 cfu -1 )
-1
Population (log10 cfu g )
7
6
5
4
3
2
1
0
30
9
8
7
6
5
4
3
2
1
0
30 days
0
60
kg P2 O5 ha
e) Endosphere (PSB9)
10 days
20 days
30 days
60
8
7
6
5
4
3
2
1
10 days
f) Endosphere (PSB16)
20 days
Population (log10 cfu g-1 )
9
-1
30
kg P2O 5 ha-1
-1
Population (log10 cfu g )
10 days
20 days
8
0
30
60
d) Rhizosphere (PSB16)
30 days
9
0
30
kg P 2 O5 ha-1
kg P 2 O5 ha-1
0
10 days
20 days
30 days
b) Non-rhizosphere (PSB16)
20 days
30 days
P o p u la tio n ( lo g 1 0 cf u g-1 )
P o p u la tio n ( lo g 1 0 c f u g-1 )
a) Non-rhizosphere (PSB9)
10
30 days
8
6
4
2
0
0
60
30
60
kg P2O5 ha-1
-1
kg P2 O5 ha
Fig. 1: Effect of PSB9 and PSB16 at different P fertilizer rates on population of non-rhizosphere, rhizosphere and
endosphere of aerobic rice.
3.2.
Effect of PSB on plant biomass
Plant biomass of aerobic rice was significantly increased with PSB inoculation (Fig. 2). The PSB16
strain produced highest biomass at 30 kg P2O5 ha-1 but decreased at 60 kg P2O5 ha-1.
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Non-inoculated
90
PSB9
-1
Plant biomass (mg plant )
80
PSB16
70
60
50
40
30
20
10
0
0
30
60
Kg P2 O5 ha
-1
Fig. 2: Effect of PSB inoculation on plant biomass of aerobic rice
Bars indicate standard error, n = 5
3.3.
Root colonization
The micrographs of SEM and TEM showed that at 5 days after inoculation both bacterial strains
were able to colonize on the surfaces of primary and lateral roots, root hair zone, lateral root junction, in
crevices, root tips at the elongation and discrimination zones (Fig. 3. b–d). The transverse sections of the root
viewed under TEM showed both of the PSB strains were able to enter the intracellular and intercellular
spaces of cell wall and within the epidermis (Fig. 3. e-h).
a
b
e
f
c
d
g
h
Fig. 3: SEM micrograph pictures showing the location of PSB colonization inoculated on aerobic rice roots: (a) noninoculated, (b) on the lateral roots and in disrupted zones of the mucigel, (c) in the crevices and junction of lateral root
meristem, (d) on the root hair zone. TEM micrographs showing the PSB colonization of interior spaces: (e & f)
bacterial cells intracellular cell wall, (g & h) bacterial cells in inter cellular spaces and within epidermis.
4. Discussion
The study showed that there was a close association between PSB strains and rice plants. However,
bacterial population in aerobic rice was affected by the application of different P rates of PSB16 but not the
PSB9 in non-rhizosphere, rhizosphere and endosphere of the plants. Higher bacterial population was found
in both the rhizosphere and endosphere as compared to non-rhizosphere. Rhizosphere plays a major role in
PSB activity due to the root exudates. The release of root exudates and decaying plant materials supply
carbon compounds as major sources of energy to the bacteria (Werner, 1998).
The addition of different P rates with bacterial inoculation showed positive effect on rice seedlings. The
highest plant biomass was observed with PSB16 inoculation at 30 kg P2O5 ha-1 and was decreased with
higher P rate. Previous inoculation studies in maize crop showed that PSB strains (Serratia marcescens EB
67 and Pseudomonas sp. CDB 35) survived in rhizosphere, solubilized P and improved plant growth
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(Hameeda et al., 2008). Earlier studies also showed that such kind of bacteria can be moved from seeds to
seedlings, rendering benefits to the host plant (Ferreira et al., 2008).
Scanning electron microscopy showed evidence of bacterial colonization on the root surface, elongation
zones, root hair, lateral root junctions, root tips and in crevices. In addition micrographs of TEM illustrated
widespread colonization of the PSB from intra and intercellular spaces which extended into the root cortex.
Similar findings were observed by Naher et al. (2009) where the inoculated bacteria manifested intercellular
growth and formed aggregated cells and mucilaginous materials that were involved in the root attachment.
Endophytic bacteria mostly colonize the intercellular space and vascular systems of the host plant. Plant root
surface attachment with bacterial flagella has been shown to play a significant role in colonization (Burdman
et al., 2001). Previous findings by Islam et al. (2007), showed that inoculation of PSB on rice seedlings
resulted in intense colonization on surface of roots. The involvement of colonization depends on different
rice varieties and the rhizosphere environment with high microbial competition. The production of
extracellular polysaccharide and cell aggregation might help in colonization process.
5. Conclusion
Results of this study proved that locally isolated PSB strains were able to colonize rice the surface and
interior roots at different P fertilizer rates. The highest bacterial population was found in the rhizosphere.
Beneficial association of the inoculated strains produced highest plant biomass. Bacillus sp. PSB16 formed a
natural association with aerobic rice leading to improved plant growth.
6. Acknowledgements
The author is grateful to Universiti Putra Malaysia, and the Ministry of Science, Technology &
Innovation for providing the financial support for the project.
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