*3.4. Culture-Independent Communities*

A total of 4116 OTUs containing 745708 high-quality reads were detected in the soybean root microbiome. The raw sequencing reads for this project were submitted to the National Center for Biotechnology Information Short Read Archive under accession numbers SRR8056376–SRR8056381. The predominant bacterial phyla were *Proteobacteria*, *Bacteroidetes* and *Actinobacteria* in the soybean roots. To compare the microbial communities obtained in healthy and diseased root samples from each group, the relative abundance of order *Rhizobiales* that can improve rhizobial nodulation and nitrogen fixation was significantly greater in the healthy samples, whereas the order *Actinobacteria* were more abundant in the diseased samples (Figure 5).

**Figure 5.** Analysis of culture-independent endophytic communities at order level in the soybean roots. DE, diseased sample; RE, healthy sample.

#### **4. Discussion**

The multifaceted approach adopted in this study, linking culture-independent and culture-dependent analysis, showed that actinobacteria were more abundant or diverse in the diseased soybean roots. This finding was in agreement with the previous study that the phylum *Actinobacteria* was higher in '*Candidatus* Liberibacter asiaticus'-infected citrus samples compared with that in healthy samples [31]. Another similar study also showed that potato plants infected with *Erwinia carotovora* subsp. *atroseptica* increased bacterial diversity [59]. The higher diversity of endophytic actinobacteria in diseased but healthy plants suggests that they may be involved in pathogen defense [60]. Indeed, extensive research has shown that endophytic actinobacteria has the capacity to control plant pathogens [22]. The in vitro antagonism assays demonstrated that four strains showed strong antifungal activity against *S. sclerotiorum*. Among the four antagonistic strains, all colonies of three strains were absolutely from diseased soybean roots, and another strain was also significantly enriched in diseased soybean roots compared to healthy soybean roots. A similar study has also shown that the rhizosphere soil of diseased tomato plant harbored a high percentage of antagonists [61]. Studies over the past few years have provided important information that plants possess a sophisticated defense mechanism by actively recruiting root-associated microbes from soil upon pathogen attack [18,62]. By adjusting the quantity and composition of its root secretion, plants can determine the composition of the root microbiome by affecting microbial diversity, density, and activity [63,64]. Our results seem to be consistent with previous observations. However, those strains with antagonistic activity in vitro may not be simply translated into biocontrol bacteria. Their biocontrol effects are influenced by various factors. For example, the antagonistic strains should reach a certain amount inside the plants to demonstrate a significant biocontrol effect [65,66]. Moreover, their secondary metabolite producing ability inside the plants may be influenced by plant physiological environment. Therefore, further work is required to assess the biocontrol efficiency in vivo and root-colonizing capacity of antagonistic strains by pathogen infection.

To learn more about the chemical nature of the antifungal activity, nine active compounds including six macrolides, two diketopiperazines and one 2-oxonanonoids, were finally obtained. Out of which, bafilomycin B1 (**3**) showed strongest inhibitory activity against *S. sclerotiorum*. Bafilomycin B1 has been reported to be produced by several *Streptomyces* strains and to show inhibitory activity against various fungi in vitro, such as *Rhizoctonia solani*, *Aspergillus fumigatus*, *Botrytis cinerea*, *Penicillium roqueforti*, and so on [67,68]. The antifungal activity of this compound against *S. sclerotiorum* was first reported in this paper. Azalomycins F4a (**1**) and F5a (**2**) were first isolated from the broth of *Streptomyces hygroscopicus* var. *azalomyceticus* [69]. Azalomycins F complex, including azalomycins F3a, F4a and F5a showed remarkable antifungal activity against asparagus (*Asparagus o*ffi*cinalis*) pathogens *Fusarium moliniforme* and *Fusarium oxysporumas* as well as powdery mildew pathogen *Botrytis* spp. [70]. The antifungal activity of the pure compound was first demonstrated in our research. Azalomycins possess broad-spectrum antibacterial and antifungal activities, and almost all of them were produced by *Streptomyces*, which were isolated repeatedly from soil and plant roots in the field by our laboratory (data no shown). This emphasizes the possible importance of *Streptomyces* producing azalomycins to protect plants against phytopathogens. A mixture of dinactin (**7**), trinactin and the major component tetranactin (**6**) is known as commercial pesticides polynactin (liuyangmycin in China), which can effectively control spider mites under wet conditions [71]. In addition, tetranactin (**6**) also exhibited significant antifungal activity against plant pathogen *Botrytis cinerea* with a minimum inhibitory concentration (MIC) of 24 <sup>μ</sup>g·mL–1 [72]. Besides dinactin (**7**) and tetranactin (**6**), the monomer (**4**) and dimer (**5**) of polynactin were also isolated in this study, all of which were active against *S. sclerotiorum*. The antifungal activities of actinolactomycin (**4**), dimeric dinactin (**5**) and dinactin (**7**) have not been reported as yet. The findings reported here shed new light on the application of polynactin. Natural indole diketopiperazines exhibited a wide range of biological activities including antitumor [73], antibacterial [74], antifungal [75] and antiviral activities [76]. FR900452 is sulfur-containing indole diketopiperazines that showed specific and potent inhibitory activity against the platelet aggregation induced by platelet-activating factor [77]. Maremycin G (**8**) and compound **9**, structurally related to FR900452, showed significant antifungal activity against *S. sclerotiorum*. To our knowledge, this is the first report of the antifungal property of maremycins. Further research is needed to confirm the efficacy of in vivo disease control provided by the nine active compounds under laboratory and field conditions.
