Isolation and characterization of ACC Deaminase Producing Endophytic Bacillus mojavensis PRN2 from Pisum sativum

Endophytic bacteria reside inside healthy plant tissues and provide several benefits to their host, and help them to tolerate various stresses. Aminocyclopropane-1-carboxylate deaminase (ACCD) production is one of the mechanisms by which these bacteria help the plant to survive under ethylene stress.

The main focus of this study was to isolate endophytic bacteria and effectively screen them for ACCD production. The selected isolate was identified and assessed for plant growth-promoting potential under pot conditions.

Endophytic bacteria were isolated from root nodules of Pisum sativum plants, grown in northern India (Haryana state). ACCD activity was initially screened on DF minimal salt medium with ACC as a sole nitrogen source. To narrow down the number of the isolates, another screening method was adopted using a modified medium containing indicator dyes along with ACC. The strain producing ACCD as well as a significant amount of Indole 3 acetic acid (IAA) was identified using 16S rDNA gene sequencing and amplification of acdS gene. Its ability to promote plant growth was evaluated under pot culture conditions.

Twenty-six endophytic bacteria were isolated from nodules of P. sativum plants. Sixteen isolates showed growth on DF minimal salts medium supplemented with ACC along with negative control. On the modified medium containing indicator dyes, two isolates, PJN13 and PJN17, showed zones of the color gradient. The ACC deaminase activity was further confirmed by enzymatic assay. The strains PJN13 and PJN17 produced 160 and 130 µM of α-ketobutyrate m.g^-1 protein h^-1, respectively. The IAA production in the strain PJN13 (79.04 ± 0.78 µg.mL ^-1) was significantly more than that in the strain PJN17 (38.36 ± 1.89 µg.mL^-1). It could enhance pea plant growth parameters, including root and shoot length and fresh and dry weight from 1 to 4 times compare to the control (untreated pea plants) under pot conditions. The results of 16S rDNA amplification and sequencing showed that PJN13 has maximum similarity to Bacillus mojavensis, and the sequence submitted to GenBank under accession number MH298523. Also, a band about 800 bp was amplified for the acdS gene.

Though Bacillus is known as a predominant non-rhizobial endophytic genus, however in the present study, a B. mojavensis Bacillus mojavensis PRN2 (MH298523) was reported for the first time as an endophyte from the nodules of pea plants. The isolated strain possesses ACC deaminase activity along with IAA production capability, and high potentials as PGPE (Plant growth-promoting endophyte) for plant growth, so it has potential to be used as biofertilizers in pea fields.