Abstract

This study was carried out to characterize and identify bacterial isolates on molecular basis from indigenous microorganisms (IMO) collected from major agroecosystems in Nueva Ecija, Philippines, including bamboo (Bambusa spinosa), forest, tomato (Solanum lycopersicum) , mango (Mangifera indica), rice (Oryza sativa), corn (Zea mays) and sweet potato (Ipomoea batatas) cropping systems. IMO samples were collected during September 2023 from three randomly selected sampling sites per agroecosystem. One ml of each IMO sample was aseptically obtained and serially diluted up to 1 × 10-8. Dilutions were plated on yeast mannitol extract agar supplemented with 0.15% Congo red to isolate potential nitrogen-fixing bacteria. A total of 695 isolates were collected, distributed as follows: mango (161), corn (145), forest (100), sweet potato (99), rice (94), bamboo (72) and tomato (24). Based on colony morphology, one representative isolate per agroecosystem was purified and subjected to morphological characterization, DNA extraction, PCR amplification, and 16S rRNA gene sequencing. Consensus sequences were generated using BioEdit and compared with reference sequences in the NCBI database using BLAST. Sequence alignment was performed using ClustalW. Thereafter, a phylogenetic tree was inferred through the Neighbor-Joining method with 1,000 bootstrap replications in MEGA 11 software. Pseudomonas putida was designated as the outgroup. Phylogenetic analysis revealed two major clades corresponding to the families Bacillaceae and Lactobacillaceae. Isolates from mango (Mg1), forest (Fr1), tomato (Tm1) and bamboo (Bm1) clustered with Bacillus subtilis (KP743119), supported by bootstrap values ranging from 91-100%. The rice isolate (Rc1) clustered with Lacticaseibacillus paracasei (KR265313) with 80% bootstrap support. Notably, isolates from corn and sweet potato did not cluster strongly with known reference sequences, suggesting the presence of potentially unclassified indigenous bacterial taxa within these agroecosystems.