**3. Results**

#### *3.1. Isolation and Distribution of Endophytic Actinobacteria*

A total of 1574 endophytic actinobacterial colonies were successfully isolated. Based on their phenotypic characteristics, the colonies were preliminary classified into 70 species. Among the 70 species, 15 species were from healthy soybean roots, 27 species were from diseased soybean roots, and 28 species were shared by healthy and diseased soybean roots. The diversity of actinobacteria from diseased soybean root were greater than those from healthy soybean root. 16S rRNA gene sequence analysis of the 70 isolates revealed that they were distributed under 14 genera: *Streptomyces*, *Micromonospora*, *Actinomadura*, *Nonomuraea*, *Microbacterium*, *Rhodococcus*, *Promicromonospora*, *Microbispora*, *Kribbella*, *Mycolicibacterium*, *Glycomyces*, *Saccharothrix*, *Streptosporangium* and *Cellulosimicrobium* within the class *Actinobacteria*. *Streptomyces* was the most frequently isolated genus (58%, 41 isolates), followed by *Micromonospora* (5 isolates) and *Nonomuraea* (4 isolates). Some rare genera, including *Rhodococcus*, *Kribbella*, *Glycomyces*, *Saccharothrix*, *Streptosporangium* and *Cellulosimicrobium*, were endemic to the diseased samples (Figure 1). The 16S rRNA gene sequences were deposited in GenBank with accession numbers: MH919371–MH919374 and MN058215–MH058280.

#### *3.2. In Vitro Antagonism of S. sclerotiorum and Identification of Bioactive Strains*

Strains DAAG3-11, DGS1-1, DDPA2-14 and DGS3-15 exhibited strong antagonistic activity against *S. sclerotiorum*, with inhibition activity rates ranging from 54.1% to 87.6% (Figure 2). Strains DGS1-1, DDPA2-14 and DGS3-15 were from diseased soybean roots. For strain DAAG3-11, 176 colonies were from diseased soybean roots, whereas 11 colonies were from healthy soybean roots. Based on the 16S rRNA gene sequences, strains DAAG3-11, DGS1-1, DDPA2-14 and DGS3-15 were closely related to *Streptomyces sporoclivatus*, *Streptomyces cavourensis*, *Streptomyces capitiformicae* and *Streptomyces pratensis*, respectively (Table 1).

**Figure 1.** Neighbor-joining phylogenetic tree of 16S rRNA gene sequences from 70 endophytic actinobacteria in this study and their phylogenetic neighbors. Numbers at nodes are bootstrap values (percentages of 1000 replications); only values > 50% are shown. GenBank accession numbers of 16S rRNA gene sequences are shown next to isolate names. A branch indicated by the same color belongs to the same genus. Isolates indicated by green and purple are endemic to the healthy and diseased samples, respectively. Isolates shared by healthy and diseased samples are indicated with red.

**Figure 2.** Dual culture plate assay between four endophytic actinobacteria against *S. sclerotiorum*.


**Table 1.** Antagonistic potential endophytic actinobacteria isolated from healthy and diseased soybean root, and similarity values for 16S rRNA gene sequences.

\* Values are the means±SE (*n*=4). Data within the same column followed by different letters are significantly different.
