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Colonization of Oryza sativa Roots by Plant Growth-promoting rhizobacterium, Bacillus sphaericus UPMB10 and Subsequent Inoculant Formulation
Abstract:
The Bacillus sphaericus UPMB10 plant growth-promoting strain was selected for colonization and inoculant formulation studies. The UPMB10 strain is a Grampositive, endospore-forming bacteria. The biochemical assay results showed that this strain was able to hydrolyze complex carbon source (potato starch) but did not utilize simple sugars. The UPMB10 strain was able to grow in the presence of 10 μg mL-1 of the antibiotic streptomycin and 10U of bacitracin which may be an important inherent characteristic in the competitiveness of this plant growth-promoting rhizobacterium. The results of the in vitro colonization study showed that B. sphaericus strain UPMB10 could successfully proliferate on and colonize the roots of Oryza sativa L. MR220 for up to 28 days after inoculation. Enumeration of bacteria colonizing the rice root indicated that the UPMB10 strain colonized both the rhizoplane and the root interiors at up to 108 and 104 cfu g-1 root tissue, respectively. The production of root cell wall degrading enzymes, cellulase, and pectinase, by this strain may have facilitated cellular entry. Scanning electron microscopy of colonization showed dense colonization at areas rich in root exudates specifically the elongation zone, junction of lateral roots, and junctions of epidermal cells for up to 21 days. Clusters of microcolonies formed a mucigel within the mucilaginous sheath of the root. Colonies were also seen colonizing beneath what could possibly be the epidermal cuticle of the roots, especially in the relatively older primary roots. The colonization of the rice roots by this strain displayed adhesion structures which attach the cells with the root surface of rice and between each other. The process of root colonization also elicited a varying pattern of bacterial morphology during temporal colonization of rice roots. Normal rods of this strain were sized 0.5-0.6 μm × 1.5-1.9 μm. Some colonies of cells appeared rounder and fatter (0.9-1.1 μm × 1.3-1.8 μm) with widths twice that of the normal rods. Results also showed that colonies elongating (lengths 5.3-6.2 μm) in readiness for cell replication possibly as a result of receiving sufficient nutrients from the colonized sites on the roots. The UPMB10 strain was successfully tagged with the green fluorescent protein (GFP) marker in the plasmid specifically constructed for this study (pSV101gfp3) by electroporation. The presence of GFP was confirmed by polymerase chain reaction (PCR), SDS-PAGE, and Western blotting. The fluorescence signal of the transformants, however was too weak to be used to follow colonization on roots in non-sterile environments.
The effects of different bacterial liquid media, carrier materials (coirdust, ground oil palm fronds [GOPF], and GOPF amended with Kusokom® compost), temperature (20, 30, and 40°C), moisture potentials (pF 2.19 and 2.54), and storage period (six months) on viable cell growth were evaluated. Tryptic soy broth was found to be suitable for the cultivation of a high number of viable bacterial cells (108 cfu mL-1) for long term inoculant production. Ground oil palm fronds (GOPF) + 25% Kusokom® compost was found to be suitable carrier for maintaining and increasing the number of B. sphaericus UPMB10 viable cells (log10 9.28 cfu g-1) at near room temperature (30°C) for up to six months. The results indicated that there were interaction between the factors studied for the inoculant formulation. Consequently, the growth and survival of UPMB10 in the carriers depended on the storage temperature and time (p = .05). Highest viable growth in GOPF, coir dust, GOPF + 50% Kusokom® compost and GOPF + 25% Kusokom® compost was recorded at temperatures 20, 30, 40, and 30°C, respectively, and at storage time D21, D7, D28, and D14, respectively. Results of the interactions between carrier and temperature indicated that growth and viability was more favorable at 30°C in GOPF and GOPF + 25% Kusokom® compost, while GOPF + 50% Kusokom® compost treatment was able to tolerate storage temperature of 40°C. In all treatments the viable population was significantly higher at D161 (log10 8.61 cfu g-1) and D21 compared with D1 (log10 8.00 cfu g-1). The outcome of this study showed the potential of producing a commercial inoculant from GOPF + 25% Kusokom® compost for Malaysia and other tropical countries with high numbers of B. sphaericus UPMB10 that showed high colonizing ability.