PHENOTYPIC CHARACTERIZATION OF ACTINOBACTERIA ISOLATED FROM APHIDS ADELGES (APHRASTASIA) PECTINATAE (HEMIPTERA: ADELGIDAE)

Actinobacteria are Gram-positive bacteria with high guanine and cytosine content in their genetic structure [1]. Their metabolic versatility enables them to form diverse symbiotic relationships, including defensive associations with arthropods such as ants, beetles, and wasps, where they produce secondary metabolites that protect against pathogenic microorganisms [2]. Beyond their role in arthropod defense, they also contribute to plant health by colonizing surfaces or forming close symbiotic associations as rhizosphere or endophytic bacteria. Through the production of bioactive compounds like herbicides, fungicides, and biofertilizers, they promote plant growth and enhance resistance to diseases. This study investigates the phenotypic characterization of Actinobacteria isolated from aphids of the genus Adelges Vallot, 1836, sap-feeding pests that infect conifers of the family Pinaceae. Aphids maintain close associations with microorganisms through various interactions, including symbiotic relationships, the transmission of pathogens, and the dispersal of bacteria. The isolation of Actinobacteria began with the preparation of an aphid suspension, where aphids were crushed and plated on a microbiological medium. Based on their distinctive colony morphologies, five strains were subsequently isolated. These strains were designated C1, D8, D11, D15, and LB2. The 16S rRNA genes of the isolates were PCR-amplified and sequenced. Subsequent BLAST analysis confirmed their classification within the genus Frigoribacterium. Moreover, further examination of the 16S rRNA region revealed that these isolates share the greatest sequence similarity with Frigoribacterium faeni, indicating a close evolutionary relationship. Currently, delineation of prokaryotic taxa based on 16S rRNA gene sequence similarity stands at 98.7% for species and 94.5% for genera. The ultimate finding suggests that LB2 may belong to a particular species (sequence similarity 98.35%), whereas other strains (sequence similarity 95.95-96.43%) might potentially represent a novel species, however, additional genome analysis is needed. To further characterize these isolates, we conducted a phenotypic analysis, which included assessments of optimal growth temperature, pH tolerance, and salt tolerance. Additionally, motility tests as well as enzymatic activity assays, were performed, including catalase, gelatin hydrolysis, starch degradation, lipase, chitinase, and cellulase activity tests. Furthermore, glucose and ornithine fermentation, ability to reduce thiosulphate to sulphide tests, along with Biolog GEN III MicroPlate and API assays, were conducted to gain deeper insights into the metabolic properties of these bacterial strains. These assays assess the utilization of various carbon and nitrogen sources, enzymatic activities, and overall metabolic capabilities under standardized conditions.