The rhizosphere soil of Machilus pingii was collected from the primitive ecological nature reserve of ‘Yingge’ mountain (1109°43′05′′E, 19°02′03′′N) in Hainan province, China. The subtropical region has a hot climate in summer (23–36°C) and cold in winter (16–24°C) with an average humidity of 60–80% and annual rainfall of 1,500–2,600 mm. The samples were transported to the laboratory in sterile polythene bags and stored in an insulated container at 4°C.

Actinomycetes were isolated by a gradient dilution separation method (Sadeghian et al., 2016). The air-dried soil was ground to powder and passed through a 60-mesh sieve. Five grams of soil samples were dissolved in 45 ml of sterile water. After being incubated at 55°C for 20 min in a water bath, the mixture was cultured at 28°C, 180 rpm, for 1 h. The homogenate was diluted with sterile water into 10^–1, 10^–2, and 10^–3 using a serial dilution method (Li et al., 2021a). One hundred fifty microliters of each dilution were spread on the four-separation agar media, namely, humic acid–vitamin (HV), glucose–aspartic acid (GA), starch casein agar (SCA), and Gause’s No. 1. These media were supplemented with potassium dichromate (50 mg/L) and nystatin (50 mg/L) to inhibit fungal and bacterial contamination. The plates were cultured at 28°C for 5–7 days. The colonies were streaked repeatedly on the ISP2 agar medium for purification until a single colony was obtained. The purified isolates were kept on the Gause’s No. 1 agar medium at 4°C and 20% (v/v) of glycerol at −80°C.

The antagonistic activity of actinomycetes against F. oxysporum TR4 was initially assayed using an agar diffusion method (Wei et al., 2020). A 5-mm hyphal disk of F. oxysporum TR4 was manufactured using a hole puncher and placed in the middle of a potato dextrose agar (PDA, 200 g/L of potato, 20 g/L of dextrose, and 16 g/L of agar) plate. The selected isolate was inoculated in four symmetrical points of about 2.5-cm distance from the pathogen. The F. oxysporum TR4 plate without the inoculation of the isolate was used as a control. All plates were cultured at 28°C for 5–7 days under dark conditions. The inhibition activity was measured until the mycelia of F. oxysporum TR4 covered the entire surface of the plate. The inhibition percentage was calculated using the following formula:

where C and T were growth diameters of tested pathogens in the control and treated plates, respectively.

Selected actinomycetes with antifungal activity in a preliminary screening were subjected to small-scale fermentation. Briefly, the actinomycete was cultured in a broth medium (15 g of corn flour, 3 g of beef extract, 10 g of yeast extract, 10 g of soluble starch, 10 g of glucose, 0.5 g of K₂HPO₄, 0.5 g of NaCl, 2 g of CaCO₃, 0.5 g of MgSO₄, pH 7.2–7.4) at 28°C, 180 rpm, for 7 days. The fermentation broth was centrifuged at 13,000 rpm, and the supernatant was mixed with an equal volume of ethanol (95%, v/v). The extract was concentrated using a separating funnel and evaporated with a rotary evaporator (EYELA, N-1300, Japan). The dried extract was diluted with ethanol (50%, v/v) to a final concentration of 10 mg/ml. Antifungal activity was determined by the agar diffusion method as mentioned above after the plates were cultured at 28°C for 7 days.

To detect the growth characteristics of actinomycetes, the isolate was first cultured in six standard ISP media (yeast extract–malt agar, ISP2; oatmeal agar, ISP3; inorganic salts–starch agar, ISP4; glycerol–asparagine agar, ISP5; peptone yeast–iron agar, ISP6; tyrosine agar, ISP7), PDA, and Gause’s No. 1 (Shirling and Gottlieb, 1968). These plates were incubated at 28°C for 7 days under dark conditions. Colors of substrate and aerial mycelia as well as diffusible pigments were judged by comparing them with the ISCC-NBS color charts (Qi et al., 2019). Some biochemical profiles including nitrate reduction, gelation liquefaction, hydrolysis of cellulose, starch, Tween 20, Tween 40, Tween 80, and urease activities were also measured according to the previous description of Li et al. (2016). IAA production was qualitatively assayed as the development of pink color in the plate. Liquid chrome azurol S (CAS) assay was used to assess siderophore production on ISP2 (Milagres et al., 1999). Chitinase activity was quantitated by measuring the reducing end group of N-acetyl glucosamine (Lee et al., 2012). The β-1,3-glucanase activity was measured using laminarin (Sigma, MA, United States) as a substrate (Lee et al., 2012). Resistance evaluation to 20 standard antibiotics was tested by a disk diffusion method (Jing et al., 2020).

To detect the broad-spectrum antifungal activity of actinomycete, 18 phytopathogenic fungi were selected, namely, Fusarium graminearum Schwabe (ATCC MYA-4620), Botrytis cinerea (ATCC 11542), Cryphonectria parasitica (ATCC 9414), Colletotrichum acutatum (ATCC 56815), Curvularia fallax (ATCC 34598), Colletotrichum fragariae (ATCC 58689), Colletotrichum gloeosporioides (ATCC MYA-456), Colletotrichum gloeosporioides (Penz) Saec (ATCC 36351), Curvularia lunata (ATCC 42011), Colletotrichum musae (ATCC 44422), Fusarium oxysporum f. sp. cubense race 1 (ATCC 31271), Fusarium oxysporum f. sp. cubense tropical race 4 (ATCC 76255), Fusarium oxysporum (Schl.) f. sp. cucumerinum (ATCC 204378), Fusarium oxysporum f. sp. lycopersici (ATCC MYA-1199), Colletotrichum gloeosporioides Penz. (ATCC MYA-4130), Alternaria tenuissima (ATCC 96828), Pyricularia oryzae (ATCC 52083), and Colletotrichum fructicola (ATCC 16103). These phytopathogenic fungi were kept in the National Banana Industry Research and Development Center, China Academy of Tropical Agricultural Sciences, Haikou, China. Antifungal activities of the selected actinomycetes were evaluated based on the radial growth inhibition of phytopathogenic fungi in vitro (Wei et al., 2020). The control plates were poured with only phytopathogenic fungi. All experiments were performed in triplicate.

The extract stock was diluted to 80 μg/ml using sterile water. Seven temperature gradients, namely, 50, 60, 70, 80, 90, 100, and 121°C, were selected to treat the isolate extract for 1 h. Different pH values were prepared from 3 to 10 at an interval of one unit using 1 mol/L HCl or 1 mol/L NaOH. The extract was irradiated by an ultraviolet lamp with an illumination intensity of 100 μW cm^–2 for 1, 3, 5, 7, 9, 11, and 13 h. Antifungal activity against F. oxysporum TR4 was used to evaluate extract stability after treatment of different physical factors. Hemolytic assay of human red blood cells treated with the extract was carried out to test its safety as in our previous description (Li et al., 2021b).

The actinomycete strain with the highest antifungal activity was identified by using a molecular procedure. The isolate was cultured in the ISP2 liquid medium at 28°C for 4 days. Total genomic DNA was extracted using a Rapid Bacterial Genomic DNA Isolation Kit (Biotech Corporation, Beijing, China) according to the standard manufacturer protocol. The complete genome was sequenced using a paired-end sequencing method in the Illumina HiSeq × Ten platform (Illumina, San Diego, CA, United States) by the Shanghai Majorbio Bio-pharm Technology Co., Ltd. A multilocus sequence analysis (MLSA) was performed using 31 housekeeping genes, namely, dnaG, frr, infC, nusA, pgk, pyrG, rplA, rplB, rplC, rplD, rplE, rplF, rplK, rplL, rplM, rplN, rplP, rplS, rplT, rpmA, rpoB, rpsB, rpsC, rpsE, rpsI, rpsJ, rpsK, rpsM, rpsS, smpB, and tsf, from the selected actinomycete genome (Brady et al., 2008). The MLSA phylogenetic tree was constructed using the neighbor-joining method in MEGA 7.0 (Kumar et al., 2016). The confidence level was investigated using bootstrap analysis on 1,000 replicates. Average nucleotide identity (ANI) between genomes was calculated using the online OrthoANI (Yoon et al., 2017). The genome sequence of the standard strain (Streptomyces albospinus JCM3399) was downloaded from the public genome database of EzBioCloud¹.

Sequencing data of the genome were analyzed on an online platform of Majorbio Cloud². Clean sequences were assembled using SOAPdenovo v2.04. The open reading frames (ORFs) and genome annotation were predicted by the Rapid Annotation using Subsystem Technology (Brettin et al., 2015). These genes were annotated using the Clusters of Orthologous Groups (COGs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) (Ogata et al., 1999; Tatusov et al., 2000). Identification of potential biosynthetic gene clusters (BGCs) was proposed by the bioinformatics tool website antiSMASH v4.0.2 software (Weber et al., 2015)³. Gene function analysis was performed through manual BLAST on NCBI⁴. The genome sequence data (accession number: SRR15506182) was deposited in the GenBank database.

The biocontrol and growth-promoting effects of the selected actinomycete were evaluated in greenhouse experiments. The genotype (cv. Brazilian, AAA group) of banana susceptible to F. oxysporum TR4 was multiplied by micropropagation. Plantlets with four to five leaves were transferred to soil and grew at 28°C with 70–80% of relative humidity. The selected actinomycete was cultured in a sterilized soybean liquid medium at 180 rpm for 7 days at 28°C. F. oxysporum TR4 was inoculated into a PDA broth at 200 rpm for 10 days at 28°C. The conidial suspension was filtered through eight layers of sterile gauze. The spore suspension of F. oxysporum TR4 was adjusted to 10⁶ CFU ml^–1 with sterile distilled water. Roots of banana plantlets were wounded with a scalpel and then dipped into 30 ml of fungal inoculum suspension for 30 min. The fermentation broth of the selected actinomycete (10⁶ CFU g^–1 soil) was also added to the roots of each plant. Three experimental groups were set, namely, medium treatment (T1), selected actinomycete + F. oxysporum TR4 treatment (T2), and F. oxysporum TR4 treatment (T3). The banana plantlets were kept in a greenhouse and maintained at approximately 70% of relative humidity at 28°C. The expression levels of defense genes and antioxidant enzyme activity were determined at 1, 2, 3, 4, 5, and 6 days post inoculation (dpi). Plant growth indicators including chlorophyll content, leaf area, leaf thickness, stem diameter, plant height, dry weight, and fresh weight were also measured at 45 dpi (Wei et al., 2020). The disease index was evaluated according to the description of Jing et al. (2020). Data were acquired from three independent experiments, and 36 plantlets were selected for each experiment. All experiments were carried out in triplicates.

Five grams of frozen root samples were ground in 10 ml of precooled phosphate buffer (50 mM, pH 7.8) containing 2% of polyvinylpyrrolidone. The homogenate was transferred to a 15-ml tube and centrifuged at 15,000 rpm for 15 min at 4°C. The supernatant was used to determine the activity of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and polyphenol oxidase (PPO) activities as described by Sun et al. (2013). H₂O₂ levels were measured according to the method of Ferguson et al. (1983). The content of malondialdehyde (MDA) was assayed using the thiobarbituric acid reaction method (Zhang et al., 2009).

Total RNA of banana roots was extracted by the routine TRIzol method. The quality and quantity of RNA were measured by NanoDrop (Thermo Scientific, United States). cDNA was synthesized using the M-MLV reverse transcriptase (Takara, Kyoto, Japan) after treatment with the RNase-free DNase (Takara, Kyoto, Japan). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed in a LightCycler^® 480 System (Roche Diagnostics, Mannheim, Germany). A 10-μl reaction system contained 50 ng of first-strand cDNA, 10 μM of forward and reverse primers, and 5 μl of TB Green Advantage qPCR Premix (2×) (Takara, Kyoto, Japan). The reaction program was set as follows: 95°C for 1 min, followed by 40 cycles of denaturation at 92°C for 5 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s. The representative defense marker genes such as Maβ-1,3-Glu (accession number: AF001523) and mitogen-activated protein kinase 1 (MaMAPK1, accession number: XM018826311) were selected. Results were normalized using the expression levels of 18S rRNA gene (accession number: U42083). The prime sequences were shown in our previous study (Zhang et al., 2019). The data obtained from different PCR runs were analyzed using the mean of CT values from three biological replicates. The expression ratios were calculated using the 2^–ΔΔCt method (Zhang et al., 2019).

The chemical compounds in the extract were identified using gas chromatography–mass spectrometry (GC-MS) as in our previous description (Li et al., 2021b). The strain extract was first dissolved in the chromatographic-grade methanol and filtered through a 0.2-μm filter. The solution was injected into a gas capillary column (DB-FFAP, 30 m × 0.25 mm × 0.25 μm) of a gas chromatography (5973 Inert XL MSD, Agilent, United States). Helium was used as a carrier gas with a flow rate of 1 ml min^–1. The mass spectrometer (EI with replaceable horn) was operated in the electron ionization (EI) mode at 70 eV with a continuous scan from 50 to 800 m/z. The peaks were identified by matching the mass spectra with the National Institute of Standards and Technology (NIST, United States) library.

All experiments were performed in three biological triplicates. Data were expressed as the mean ± standard deviation. The greenhouse experiments were carried out in a randomized design. All data were analyzed using analysis of variance (ANOVA) by the SPSS statistical package (version 22, SPSS Inc., Chicago, IL, United States). The t-test compared the mean of an outcome variable for different subjects. The LSD’s multiple range test was applied to determine the significant difference at the level of p < 0.05.

The primitive nature reserve of ‘Yingge’ mountain possessing great biodiversity was selected for the isolation of antifungal actinomycetes. A total of 60 actinomycetes were isolated from the rhizosphere soil of M. pingii using four culture media of HV (18), GA (12), SCA (10), and Gause’s No. 1 (20). Isolated actinomycetes showed well-developed aerial and substrate mycelia in Figure 1. Most aerial mycelia appeared hairy, granular, or powdery. Some isolates produced colored pigments in the substrate and were hard to pick from the media. It was a representative trait of the genus Streptomyces. All the isolates were deposited at the National Banana Industry Research and Development Center, the Institute of Tropical Bioscience and Biotechnology, China Academy of Tropical Agricultural Sciences, Haikou, China.

Initially, all the isolates were subjected to a primary screening test against F. oxysporum TR4. We assessed the inhibition ability of the mycelial growth of F. oxysporum TR4. By antagonistic experiments in plates, 17 out of 60 isolates showed significant antifungal activities (Supplementary Figure 1), accounting for 28.3% of the total actinomycetes screened. The inhibitory activity of each isolate was further determined. Eleven actinomycetes owned more than 60% of antifungal activity against F. oxysporum TR4. Particularly, the strain numbered BITDG-11 had the best antifungal activity. The inhibition rate reached 80.48 ± 1.49%.

A small-scale fermentation of the 17 isolates with prominent zones of inhibition from the primary screening was carried out. The antagonistic ability of their extracts was further investigated. The results demonstrated that extracts of all the selected isolates showed antifungal activity against F. oxysporum TR4, ranging from 27 to 60% (Supplementary Figure 1). The strong inhibition zone sizes were observed in extracts of four isolates (BITDG-11, BITDH-4, BITDG-7, BITDG-19, and BITDA-7). The inhibition rates were 58.44, 45.10, 43.92, 42.89, and 42.62%, respectively. Combined with primary and rescreening results, strain BITDG-11 was selected as an excellent isolate for the subsequent study.

The cultural traits of strain BITDG-11 were recorded on eight media selected (Table 1). It had a good growth on ISP2 and a moderate growth on the rest of the media except for ISP4. The color of aerial mycelia varied from white to dark gray. Aerial hypha exhibited dark gray on ISP3, pale yellow on ISP5, and white on ISP2, ISP6, ISP7, PDA, and Gause’s No. 1 media. Light yellow colors of the hyphal substrate were observed on all the media except for light gray on ISP5. Soluble pigment was produced on ISP5, ISP7, and Gause’s No. 1. Production of melanin pigment on ISP7 could be taken as an identification criterion for the genus Streptomyces (Atta et al., 2011). Therefore, strain BITDG-11 exhibited typical morphological characteristics of Streptomyces.

The analysis of physiological and biochemical characteristics showed that strain BITDG-11 had the ability to hydrolyze starch and produce IAA. A positive response was observed to gelatin liquefaction and nitrate reduction. Enzyme activities of lipase and urease were also detected (Figure 2A). The level of chitinase was sharply increased during the exponential phase and reached the peak (3.50 U/mg protein) at 72 h (Figure 2B). The highest level of β-1,3-glucanase (0.49 U/mg protein) was found at 72 h of the incubation period (Figure 2C). Antibiotic susceptibility testing revealed that strain BITDG-11 manifested tolerance to 15 out of 22 kinds of antibiotics and sensitivity to erythromycin, kanamycin, gentamicin, amikacin, neomycin, midenomycin, and ofloxacin (Supplementary Table S1).

Based on the alignment of 16S rRNA sequence (1,523 bp, accession number: MW110901), the sequence shared 98.80% of similarity with that of S. albospinus NBRC 13846. It confirmed that the strain belonged to a member of the genus Streptomyces. However, 58% of the bootstrap value in the neighbor-joining tree indicated that strain BITDG-11 was not distinguished from closely related species using 16S rRNA genes.

In order to further identify the species of strain BITDG-11, its whole genome was sequenced. Sequences of 31 housekeeping genes were selected to perform the MLSA. The method was considered as an alternative strategy for defining the Streptomyces systematics due to the high efficiency of species resolution and reproducibility (Rong and Huang, 2010). The neighbor-joining tree based on MLSA showed that strain BITDG-11 formed a distinct clade with S. albospinus JCM3399 with 70% of bootstrap value (Figure 2D). We further compared the strain BITDG-11 genome (accession number: SRR15506182) with the standard strain genome (S. albospinus JCM3399) (Supplementary Table S2). The ANI of 86.55% was below the threshold value of 95–96% for distinguishing a novel species (Richter and Rosselló-Móra, 2009). Thus, strain BITDG-11 might be a novel species of the genus Streptomyces and named after Streptomyces sp. BITDG-11.

To assess whether Streptomyces BITDG-11 had a broad-spectrum antifungal activity, 18 fungal pathogens were selected in our study. Compared with the growth diameters of different phytopathogenic fungi in the control plate, the strain had strong antifungal activities against all pathogens selected (Figure 3). The inhibition rates ranged from 65 to 90%. The maximal and minimal inhibition rates were observed against C. parasitica (96.78% ± 1.83) and C. musae (65.01% ± 1.87), respectively.

To explore and apply Streptomyces BITDG-11 as biopesticides in the future, the stability of the extract expressed as antifungal activity was evaluated after treatment in different temperatures, pH values, and UV doses, respectively. The results showed that different temperatures reduced antifungal activity of the extract against F. oxysporum TR4, but 57.05% was kept after treatments at 121°C for 1 h (Figure 4A). For different pH treatments, the strongest antifungal activity was detected at pH 7.0–8.0 and decreased to 52% at pH 3.0 (Figure 4B). Additionally, the extract stability was negatively correlated with the increase of UV dose. A slight decrease was observed in the activity change of the extract treated with UV radiation after 7 h (Figure 4C). Although an obvious decrease (LSD’s multiple range test, p < 0.05) of antifungal activity was detected in Streptomyces BITDG-11 extract after treatment with different physical factors, more than 50% of residual antifungal activity was retained in all the groups. It suggested that the Streptomyces BITDG-11 extract was relatively stable to some extent. To investigate the extract toxicity on eukaryotic cells, hemolytic activity was analyzed. Human red blood cells were measured as the release of hemoglobin after treatment at 37°C for 1 h. Compared with the 100% hemolytic activity of Triton X-100 (0.1%, v/v), no obvious hemolytic activity was observed in the extract treatment group (Supplementary Figure 2).

Streptomyces BITDG-11 perfectly inhibited the growth of different phytopathogenic fungi in vitro, indicating that it was a promising biocontrol agent. With an objective to evaluate in vivo biocontrol efficiency, we analyzed the effects of Streptomyces BITDG-11 fermentation broth on the infection inhibition of F. oxysporum TR4. By contrast, the bottom leaves of banana plantlets exhibited a significant chlorotic symptom at 45 dpi in the T3 group (Figure 5A). No obvious disease symptom was detected in the group of Streptomyces BITDG-11 + F. oxysporum TR4. Therefore, the protective treatment with Streptomyces BITDG-11 effectively prevented the infection of F. oxysporum TR4 and reduced disease index of plants (Figure 5B). We also evaluated actinomycetes and fungal numbers in the rhizosphere of banana seedlings (Supplementary Figure 3). The colony-forming units of F. oxysporum TR4 were very low when compared to other treated soils. The results showed that Streptomyces BITDG-11 significantly increased competing bacterial and fungal counts along with the pathogen in the soil.

Moreover, various agronomic traits of plant health and condition were measured in Figures 5C–H. Streptomyces BITDG-11 significantly (LSD’s multiple range test, p < 0.05) increased the fresh and dry weight of banana plantlets at 45 dpi (Figure 5C). Compared to those of control (T1, 45.5 ± 2.2 cm) or F. oxysporum TR4 treatment (T3, 34.0 ± 1.8 cm), the height of banana plantlets treated with Streptomyces BITDG-11 + F. oxysporum TR4 reached 50.9 ± 2.0 cm (Figure 5D). F. oxysporum TR4 significantly inhibited the increase of plant agronomic indicators (Figures 5E–H). Similar plant-growth-promoting effects were observed in stem diameters. Although no obvious difference of leaf thickness was detected between dual disposal and controls, a significant increase was found in leaf area and chlorophyll content of plants treated with Streptomyces BITDG-11.

As is well-known, the stress condition could induce the production of reactive oxygen species, thereby resulting in oxidative damage of plant cells. This effect can be assayed by quantifying H₂O₂ content and CAT activity. In our study, H₂O₂ content in banana roots was increased after F. oxysporum TR4 treatment (Figure 6A). Although a similar increase was detected in banana roots treated with F. oxysporum TR4 + Streptomyces BITDG-11, H₂O₂ content was reduced significantly (LSD’s multiple range test, p < 0.05) by inducing high CAT activity (Figure 6B). Hence, Streptomyces BITDG-11 effectively alleviated the cell membrane damage. It was supported by the fact that MDA content was lower in roots treated with Streptomyces BITDG-11 (Figure 6C). Moreover, the strain also increased the higher activities of defense-related enzymes (PPO, POD, and SOD) in comparison with F. oxysporum TR4 or control treatment alone (Figures 6D–F). The maximal values of POD, PPO, and SOD were 83.20, 70.67, and 351.31 U/g in the banana roots after inoculation with Streptomyces BITDG-11 combined with the challenge with F. oxysporum TR4 at 3, 6, and 2 dpi, respectively.

To further determine the defense mechanism of Streptomyces BITDG-11-induced resistance to Fusarium wilt of banana, the transcription levels of MaMAPK1 (early defense response marker gene) and Maβ-1,3-Glu (downstream defense marker gene) were analyzed by qRT-PCR (Figure 7). In comparison with control and F. oxysporum TR4-only treatment, the transcripts of two tested genes were much stronger in the banana roots treated with Streptomyces BITDG-11 + F. oxysporum TR4 and reached their maximum values at 4 and 3 dpi, respectively. In addition, high transcription levels of defense genes were kept by Streptomyces BITDG-11 treatment until 6 dpi. These results indicated that Streptomyces BITDG-11 could prime the banana plants to enhance resistance to F. oxysporum TR4 by simultaneously activating the MAPK-mediated signaling pathway of defense response.

The Streptomyces BITDG-11 genome was sequenced and contained 10,590,458 bp. The genome with 71.78% of GC content included seven rRNA genes, 78 tRNA genes, and 10,366 coding sequences (CDSs) (Figure 8A). By annotation, 68.45, 28.51, and 57.77% of CDSs were assigned to COG, KEGG, and GO, respectively. For 7,096 genes in COG, the top five categories contained transcription (749), amino acid transport and metabolism (502), energy production and conversion (398), and carbohydrate transport and metabolism (393). Notably, 2,402 of COG genes were clustered into an unknown function category (Figure 8B). The GO annotation showed that these genes were classified mainly into the biological process (2063), cellular component (2113), and molecular function (5023) (Supplementary Figure 4). Interestingly, 78% of genes participated in the regulation of metabolism in KEGG (Supplementary Figure 5). By analysis of the antiSMASH software, 43 BGCs related to biosynthesis of the secondary metabolites were found in genome sequences of Streptomyces BITDG-11 (Supplementary Table S3). BGCs included one terpene-type I PKS-nucleoside-NRPS, one type III PKS, one thiopeptide-transatpks-type I PKS-NRPS, five terpene, three NRPS, four type I PKS, one type II PKS, one type I PKS-NRPS, one NRPS-lantipeptide, one lantipeptide-terpene, two lantipeptide, three bacteriocin, one ectoine, two butyrolactone, one terpene-NRPS, one type II PKS-oligosaccharide-type I PKS, one transatpks-NRPS, two siderophore, one indole, etc. Genome analysis further revealed that seven BGCs showed more than 70% of similarity with toyocamycin (100%), naringenin (100%), lantipeptide B (100%), ectoine (100%), mannopeptimycin (81%), desferrioxamine (80%), and rimocidin (72%) (Figure 8C).

Gas chromatography–mass spectrometry was performed to identify the chemical composition in the Streptomyces BITDG-11 extract. In a comparison of their mass spectra with the NIST library, 15 compounds were identified based on their retention time and molecular weight (Table 2). The peak area represented the proportion of the given compound in the total extract. According to the available library data, chemical compounds were identified as tetradecanoic acid, tricosene, cis-1-chloro-9-octadecene, pentadecanoic acid, n-hexadecanoic acid, linoleic acid ethyl ester, dibutyl phthalate, 1,2-bis(p-(trans-styryl)phenyl)-cis-ethylene, 3,3′-dihydroxy-4,4′-dinitrohexafluorobiphenyl, 1,2- bis(p-(cis-styryl)phenyl)-trans-ethylene, 4′-(3-(6-methyl-3-pyri dyl)-1-(p-tolyl)-2-pyrazolin-5-yl)acetanilide, colchiceinamide, 3,3′-dihydroxy-4,4′-dinitrohexafluorobiphenyl, 2-imidazoline, 3,5-di(4-chlorophenyl)-1-(4-fluorophenyl), naphtho[2,3-c]fura n-1(3H)-one, 4-(3,4-dimethoxyphenyl)-3a,4,9,9a-tetrahydro-6,7-dimethoxy, and bis(2-ethylhexyl) phthalate.