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 1Department 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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2011 International Conference on Food Engineering and Biotechnology IPCBEE vol.9 (2011) © (2011)IACSIT Press, Singapoore

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.

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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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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).

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Fig. 3: SEM micrograph pictures showing the location of PSB colonization inoculated on aerobic rice roots: (a) non-inoculated, (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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