Physiological and molecular characterization of endophytic and epiphytic pink-pigmented facultative methylotrophs (PPFMS) isolated from paddy (Oryza Sativa) and palm oil (Elaeis Guineensis) leaves

Biofertilizer is a formulation of living cells of beneficial bacteria to stimulate plant development via microbially-driven processes without negatively affecting the ecology. The pink-pigmented facultative methylotrophs (PPFMs) can promote the growth of plants and are ubiquitous in the environment. PPFMs can utilize methanol as their energy source. Due to their plant growth-promoting traits, PPFMs demonstrated a significant potential as biofertilizer candidates. This study isolated PPFMs from two types of samples: paddy leaves and oil palm leaves. Isolation was conducted on ammonium mineral salt supplemented with 0.5% methanol. Nine isolates designated as ENPM1, ENPM2, ENPM3, EPPM1, ENPD1, ENPD2, ENPD3, EPPD1, and EPPD4 were selected for physiological and molecular characterization based on their colony morphologies. All isolates showed the same cellular morphology: pink-pigmented, strictly aerobic, Gram-negative, and bacilli-like shape. In this study, the PPFMs isolates underwent molecular identification by 16S rRNA sequence analysis and the mxaF gene. BLAST analyses of 16S rRNA gene sequences indicated the isolated bacteria were identified to have close identity with Methylorubrum sp. bacteria. In addition, for the mxaF gene sequences based on BLAST analysis, the most isolated bacteria were identified as lanthanide-dependent methanol dehydrogenase at a range of 94% to 99% of the percentage of identity, except ENPD1, ENPD2, and EPPD1 were identified as methanol/ethanol family PQQ-dependent dehydrogenase at a range of 98% to 99% of the percentage of identity. The plant growth-promoting activity of the PPFMs isolates was determined using the phosphate solubilization index (PSI) test, vanadate molybdate method, and phytase assay, and the production of IAA was measured using the Salkowski reagent test. The results showed that only ENPM1, ENPM2, ENPM3, ENPD1, EPPD1, and EPPD4 are able to solubilize phosphate in Pikovkaya’s plate, with ENPM3 having the highest activity, showing a solubilization index (SI) of 1.483 ± 0.448. However, phosphate solubilization activity, ENPD3, was recorded at the highest concentration with 182.48 ± 0.1975 ug/ml. Besides, the ENPM1 recorded was the highest for phytase enzyme activity with 90.12 U± 0.1305. Furthermore, the isolates that could produce IAA with ENPD1 as the highest IAA producer (8.634 ± 0.0793 μg/ml). When PPFMs bacteria priming by PPFMs was examined on mung beans, it was shown to have a significant positive impact on the germination and growth of mung bean seeds. Every PPFM isolate germinated more quickly than the controls. The ENPD3 treatment produced the most significant mung bean on seed vigour index (1763 ± 160.72) and 100% germination rate.