Perlite-Immobilized Streptomyces Biofilms Establish Persistent Rhizosphere Populations and Promote Mung Bean (Vigna radiata) Growth Under Greenhouse Conditions

Streptomyces spp. are abundant actinobacteria in the rhizosphere, recognized for their biocontrol and plant growth-promoting (PGP) abilities. However, their potential as biofilm-based biofertilizers and the use of inert carriers for formulation remain largely unexplored. This study aimed to select PGP actinobacterial strains with strong biofilm-forming ability, induce their biofilm formation on perlite, an agricultural inert carrier, and evaluate their effects on mung bean (Vigna radiata) growth under greenhouse conditions. Forty-one actinobacterial strains isolated from the rhizosphere of organic Persea americana orchards were screened for PGP traits and biofilm formation using crystal violet (CV) microtiter assay, sliding test (ST), and Congo Red agar. The CV assay revealed 30 °C as optimal for biofilm development, and the combined CV-ST-CRA results identified several high-biofilm producers. Three Streptomyces strains (ARI_A20-3, ARI_A20-5, ARI_A30-32) with 16s rRNA sequences identified as S. aff. flaveolus, S. aff. aureus, and S. aff. griseorubens were selected based on consistent biofilm performance and PGP profiles. Their biofilms were induced on perlite and characterized using bright-field microscopy and scanning electron microscopy (SEM). The results confirmed the formation of mature, compact biofilms that covered and penetrated the mineral surface. Perlite-based biofilm inoculants were applied to V. radiata in sterilized soil, with biochar under greenhouse conditions. The single-strain treatment containing ARI_A20-5 (T2) and the two-strain treatment containing ARI_A20-3 and ARI_A30-32 (T5) produced the largest increases in plant biomass, reaching approximately 10.6-fold and 7.6-fold of the non-inoculated control in total dry biomass, and 4.9-fold and 4.1-fold in root dry biomass, respectively. Rhizosphere viable counts showed 1–2 orders of magnitude increases in Streptomyces CFU over 12 weeks, and post-harvest SEM confirmed persistent colonization, with spores on perlite and rhizospheric/endophytic hyphae on roots. These results demonstrate that perlite-immobilized Streptomyces biofilms can establish persistent populations in the mung bean rhizosphere and significantly improve plant biomass relative to non-inoculated controls under greenhouse conditions, supporting their further development as carrier-based inoculants and motivating shelf-life and field evaluation.