*2.4.2 Primers and PCR conditions*

To identify the antagonistic bacterial isolates, the primer sets 27F (50 -AGA GTT TGATCM TGG CTC AG-30 ) and 1518R (50 -AAG GAG GTG ATC CAN CCR CA-30 ) specific to 16SrDNA were used for amplification of 16SrDNA from the prepared genomic DNA template [61]. The PCR conditions were as follows: initial denaturation at 95°C for 5 min, 35 cycles denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 2 min and finally a 7 min extension at 72°C. PCR products were visualized by electrophoresis on 1.0% agarose gel containing 0.5% of ethidium bromide using a Gel documentation System after separating the PCR products in the agarose gel for 50 min at 80 volt.

### *2.4.3 Sequencing of PCR products*

A partial nucleotide sequencing of 16SrDNA was performed from amplified PCR products using primers 27F (5<sup>0</sup> -AGA GTT TGATCM TGG CTC AG-3<sup>0</sup> ) in the Macrogen Lab, South Korea via Biotech Concern Bangladesh. The sequencing was done directly from PCR products according to the standard protocols for the ABI 3730xl DNA genetic analyzer (Applied Biosystems, Foster City, CA, USA) with BigDye® Terminator v1.1 and 3.1 Cycle Sequencing Kits.

#### *2.4.4 Processing of sequence data*

The sequencing data were processed and nucleotide sequence data was exported using Chromas software version 2.6.4.The quality of nucleic acid sequences was evaluated using Chromas (Version 2.6) software to avoid the use of low quality bases.

### *2.4.5 Analyses of nucleotide sequences*

The nucleotide sequences were analyzed using online bioinformatics tools. The DNA sequences of 16Sr DNA of the bacterial isolates were compared with 16Sr DNA of the bacterial spp. and the sequences of ITS region of the fungal isolates were compared with ITS region of the fungal spp. that were available in the NCBI database using Basic Local Alignment Search Tool (BLAST) algorithm to identify closely related sequences (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

#### **2.5 Formulation of plant growth promoting antagonistic bacterial species**

The pure cultures of thirty two selected potential bacterial antagonists were grown on LB agar medium for 24 hrs. Then the bacterial isolates were transferred in LB broth for about six hours by taking a loopful of bacteria from the LB agar plate. After that the liquid culture was then centrifuged and resuspended the pellet in previously prepared 200 ml peptone broth aimed to fortify the carrier materials. This culture broth was then grown for another two hours with shaking. After that 5 ml of sterile 100% glycerol was added to this 200 ml culture. Then the cultures of the bacterial antagonists (200 ml fortified with 1% peptone and 1% glycerol) were added to the mixture of 500 g talcum powder amended with 5 g carboxy methyl cellulose (CMC) and 7.5 g Calcium carbonate which were autoclaved for two consecutive days at 121°C under 15PSI pressure for 30 min each. The formulations were then being dried overnight in the tray. After that the formulations were powdered with hand, the formulated bacterial antagonists were packed in plastic bags. The formulated bacterial antagonists were then kept at both room and 4-8°C temperature in the refrigerator.

*Potential Role of Rice Plant Growth Promoting Phylloplane and Rhizospheric Bacteria… DOI: http://dx.doi.org/10.5772/intechopen.99854*
