Biosurfactant producing multifarious Streptomyces puniceus RHPR9 of Coscinium fenestratum rhizosphere promotes plant growth in chilli.

The present study involves isolation of Streptomyces spp. from rhizosphere of Coscinium fenestratum Gaertn, an endangered medicinal plant from Western Ghats of Karnataka, India. Four potential isolates were identified by 16S rRNA sequencing as Streptomyces sp. RHPR3, Streptomyces puniceus RHPR9, Streptomyces sp. RHPR14 and Streptomyces mediolani RHPR25. An enrichment culture method was used for the isolation of Streptomyces spp. for biosurfactant activity. Among four potential Streptomyces spp., S. puniceus RHPR9 showed highest Emulsification index (EI) (78±0.2%) and Emulsification assay (EA) (223±0.2 EU mL-1). Thin layer chromatography, Fourier transform infrared spectroscopy (FTIR) and mass spectrometric analysis revealed that as glycolipid. Further confirmed by presence of fatty acids like hexanoic acid methyl ester, decanoic acid by Gas chromatography mass spectroscopy (GC-MS) analysis. S. puniceus RHPR9 showed a significant IAA production (41µg mL-1), solubilized P (749.1 µg mL-1), growth promotion of chilli (Capsicum annuum L.) was evaluated using paper towel method and greenhouse conditions. S. puniceus RHPR9 showed a significant increase in seed vigor index (2047) and increase in plant biomass (65%) when compared to uninoculated control. To our knowledge, this is the first report on epiphytic S. puniceus RHPR9 isolated from an endangered medicinal plant C. fenestratum Gaertn, for biosurfactant production and plant growth promotion activities.

Statistical optimization of antifungal iturin A production from Bacillus amyloliquefaciens RHNK22 using agro-industrial wastes.

Biosurfactants are secondary metabolites with surface active properties and have wide application in agriculture, industrial and therapeutic products. The present study was aimed to screen bacteria for the production of biosurfactant, its characterization and development of a cost effective media formulation for iturin A production. A total of 100 bacterial isolates were isolated from different rhizosphere soil samples by enrichment culture method and screened for biosurfactant activity. Twenty isolates were selected for further studies based on their biosurfactant activity [emulsification index (EI%), emulsification assay (EA), surface tension (ST) reduction] and antagonistic activity. Among them one potential isolate Bacillus sp. RHNK22 showed good EI% and EA with different hydrocarbons tested in this study. Using biochemical methods and 16S rRNA gene sequence, it was identified as Bacillus amyloliquefaciens. Presence of iturin A in RHNK22 was identified by gene specific primers and confirmed as iturin A by FTIR and HPLC. B. amyloliquefaciens RHNK22 exhibited good surface active properties and antifungal activity against Sclerotium rolfsii and Macrophomina phaseolina. For cost-effective production of iturin A, 16 different agro-industrial wastes were screened as substrates, and Sunflower oil cake (SOC) was favouring high iturin A production. Further, using response surface methodology (RSM) model, there was a 3-fold increase in iturin A production (using SOC 4%, inoculum size 1%, at pH 6.0 and 37 °C temperature in 48 h). This is the first report on using SOC as a substrate for iturin A production.

Molecular dynamics and protein interaction studies of lipopeptide (Iturin A) on α- amylase of Spodoptera litura.

The small mottled willow moth (Spodoptera litura) is one of the best-known agricultural pest insects. To understand the insecticidal activity, we have selected iturin A compound produced by Bacillus amyloliquefaciens RHNK22 which showed the strongest and most common inhibitory effect on the Spodoptera litura protein. In this work we have identified the action of iturin A on α- amylase is a major digestive enzyme of Spodoptera litura using docking studies. A 3D model of α- amylase from Spodoptera litura was generated using 2HPH as a template with the help of Modeller7v7. With the aid of the molecular mechanics and molecular dynamics methods, the final model is obtained and is further checked by Procheck and Verify 3D graph programs, which showed that the final refined model is reliable. With this model, a adjustable docking study was performed with iturin A using GOLD software. The results indicated that ARG 18, THR15, LEU42 in α- amylase are important determinant residues in binding as they have strong hydrogen bonding interactions with iturin A. These hydrogen binding interactions play an important role for the stability of the complex.

Draft Genome Sequence of Bacillus amyloliquefaciens Strain RHNK22, Isolated from Rhizosphere with Biosurfactant (Surfactin, Iturin, and Fengycin) and Antifungal Activity.

Bacillus amyloliquefaciens strain RHNK22 isolated from groundnut rhizosphere showed direct and indirect plant growth-promoting traits along with biosurfactant activity and reduction in surface tension of water. Biosurfactants were identified as lipopeptides (surfactin, iturin, and fengycin) by molecular and biochemical analysis in our studies.