*2.3. Phenotypic and Molecular Characterization of Actinobacterial Isolates*

The purified colonies were cultivated on ISP 3 at 28 ◦C for two weeks, and then grouped according to their phenotypic characteristics, including the characteristics of colonies on plates, color of aerial and substrate mycelium, spore mass color, spore chain morphology, and production of diffusible pigment. Those colonies with the same characteristics were classified as one species. The number of species was counted to compare the diversity of root-associated endophytic actinobacteria from healthy and diseased soybean.

Different phenotypic isolates were further subjected to 16S rRNA gene sequence analysis for the genus and species identification. The total DNA was extracted using the lysozyme-sodium dodecyl sulfate-phenol/chloroform method [38]. The primers and procedure for PCR amplification were carried out as described by Kim et al. [39]. The PCR products were purified and ligated into the vector pMD19-T (Takara Biomedical Technology, Beijing, China) and sequenced by an Applied Biosystems DNA sequencer (model 3730XL). The almost full-length 16S rRNA gene sequences (~1500 bp) were obtained and aligned with multiple sequences obtained from the GenBank/EMBL/DDBJ databases using CLUSTAL X 1.83 software. Phylogenetic tree was constructed with neighbor-joining method [40] using Molecular Evolutionary Genetics Analysis (MEGA) software version 7.0 [41]. The bootstrap method with 1000 repetitions was using to assess the topology of the phylogenetic tree [42]. Phylogenetic distances were calculated according to the Kimura two-parameter model [43]. The 16S rRNA gene sequence similarities were determined using the EzBiocloud server [44]. The obtained gene sequences have been deposited in the GenBank database.
