A total of 144 endophytic actinomycetes were isolated previously from different tissues of 23 medicinal plants in “Wuzhishan” Nature Reserve, Hainan, China the related data will be published in Phytopathology, but the publication period is a little long. Antagonistic activity was evaluated against Foc TR4 (ATCC 76255) in vitro as previously described (Jing et al., 2020). Foc TR4 was cultured on the potato dextrose agar (PDA) medium at 28°C for 7 days. An agar disc (5 mm in diameter) with Foc TR4 was placed on the center of Petri dishes 6 cm away from an endophytic actinomycete. Plates without endophytic actinomycetes were served as a control. After inoculation at 28°C for 10 days, the inhibition percentage was calculated as described by Wei et al. (2020). All experiments were performed in triplicate. The endophytic strain 8ZJF-2 isolated from the roots of C. capitulata exhibited strong antifungal activity.

To further analyze whether strain 8ZJF-2 owned a broad-spectrum antifungal activity, the antagonistic activities were investigated against ten phytopathogenic fungi, including Curvularia lunata (ATCC 42011) from banana, Colletotrichum fragariae (ATCC 58718) from strawberry, Fusarium oxysporum f. sp. cucumerinum (ATCC 36332) from cucumber, Fusarium graminearum Sehw (ATCC 11696) from wheat, Fusarium oxysporum f. sp. cubense race 1 (ACCC 31271) from banana, Colletotrichum gloeosporioides (Penz) Penz and Sacc 1884 (ACCC 36351) from mango, Pyricularia oryzae (ATCC 52083) from rice, Alternaria tenuissima (ATCC 58124) from cotton, Colletotrichum acutatum (ATCC 56815) from loquat, and Colletotrichum gloeosporioides (ATCC 16330) from mango. Antifungal activity of strain 8ZJF-2 was detected as the above-mentioned method. The inhibition zones were measured in millimeters (Zhang et al., 2021).

The strain 8ZJF-21 was inoculated in various types of growth media including PDA and different International Streptomyces Project media (ISP2, ISP3, ISP4, ISP5, ISP6, and ISP7) for 7 days at 28°C under dark conditions (Yun et al., 2021; Zhang et al., 2021). Cultural characteristics such as colonial morphology and diffusible pigment production were detected in the different media according to Shirling and Gottlieb (1966). Based on Bergey’s manual of systematic bacteriology, strain 8ZJF-21 was classified by observing the color of aerial and substrate mycelia (Brinley-Morgan and McCullough, 1974). Phenotypic profile of strain 8ZJF-2 spore chain was observed by scanning electron microscopy (SEM, model S-4800, Hitachi Limited, Japan). Utilization of nitrogen and carbon sources was studied according to Qi et al. (2019). The capability of strain 8ZJF-21 to produce important enzymes (proteases, lipases, celluloses, nitrate reductases, pectinases, gelatinases, and ureases), indoleacetic acid (IAA), siderophores, and H₂S were determined (Jing et al., 2020; Wei et al., 2020; Zhang et al., 2021). Physiological tests were performed by inoculating strain 8ZJF-21 on the selected medium (ISP2) at different temperatures (20°C–50°C), pH (3.0–11.0), and NaCl (0–20% w/v).

Strain 8ZJF-21 was cultured in the ISP2 liquid medium at 200 rpm and 28°C for 4 days. Total genomic DNA was extracted using a Rapid Bacterial Genomic DNA Isolation Kit (Biotake corporation, Beijing, China). The sequencing libraries were generated using the Illumina TruSeq™ RNA Sample Preparation Kit (Illumina, San Diego, CA, United States). The complete genome was sequenced in the Illumina Hiseq × Ten platform (Illumina, San Diego, CA, United States) by the Shanghai Majorbio Bio-pharm Technology Co. Ltd. Sequencing data were analyzed using an online platform of the Majorbio Cloud¹ and was deposited in GenBank with accession number JAJQWY000000000. The open reading frames (ORFs) were predicted by the Rapid Annotation using Subsystem Technology (Brettin et al., 2015). Functional annotation was performed using the Clusters of Orthologous Group (COG), the Gene Ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Ogata et al., 1999; Tatusov et al., 2000). Biosynthetic gene clusters (BGCs) were identified by the online antiSMASH v4.0.2 software (Weber et al., 2015).

The 16S rDNA sequence was extracted from the sequenced genome of strain 8ZJF-21. Sequence alignment was performed against the EzTaxon-e database.² The phylogenetic trees were constructed using a neighbor-joining (NJ) method of MEGA 7.0 (Kumar et al., 2016). Evolutionary distance was calculated using the maximum-parsimony algorithm. The confidence level was calculated using the bootstrap analysis on 1,000 replicates. Average nucleotide identity (ANI) was obtained by comparing genomes of the type strain and strain 8ZJF-21 using the online OrthoANI (Yoon et al., 2017). The closest homolog was considered as the type strain according to the phylogenetic tree. Its genome sequence was downloaded from the database of EzBioCloud.³

Strain 8ZJF-21 was cultured in 100 ml of a soybean liquid medium (SLM, 15 g of corn flour, 10 g of glucose, 0.5 g of K₂HPO₄, 0.5 g of NaCl, 0.5 g of MgSO₄, 3 g of beef extract, 10 g of yeast extract, 10 g of soluble starch, 2g of CaCO₃, pH 7.2-7.4) with shaking at 180 rpm for 7 days at 28°C. The fermentation broth was filtered through a Whatman No.1 filter. After centrifugation at 10,000 rpm for 15 min, the supernatant was extracted twice in the ratio of 1:1 (culture supernatant: different gradient methanol). To remove the impurities, the suspension went through a silica-gel chromatography column (5.5 cm × 80 cm, inner diameter × length). The elution with gradient methanol solutions was filtered through a 0.22 μm sterile filter (Millipore, Bedford, MA, United States) (Li et al., 2021). The organic solvent was concentrated using a rotary vacuum evaporator (N-1300, EYELA, Ailang Instrument Co., Ltd., Shanghai, China). The obtained extract was redissolved in 10% (v/v) of dimethyl sulfoxide (DMSO) with a final concentration of 20 mg ml^–1.

Sterilized PDA agar media containing final extract concentrations (1.563, 3.125, 6.25, 12.50, 25, 50, or 100 mg L^–1) were prepared by a serial dilution method (Yun et al., 2021). Ten percent (v/v) of DMSO was used as a control. A 5-mm-diameter disc of Foc TR4 was placed on the center of the plate. The growth diameter of Foc TR4 was recorded until the mycelia reaching the plate edge in the control group. The half-maximal effective concentration (EC₅₀) of the extract against Foc TR4 was calculated according to Hoekstra and Van Ewijk (1993). All experiments were repeated with three biological replicates.

To investigate the potentiality of strain 8ZJF-21 to control Foc TR4 and promote plant-growth traits, a pot experiment was carried out in a completely randomized design. Foc TR4-GFP strain overexpressing a green fluorescent protein (GFP) gene was selected to detect the infection in banana roots (Yun et al., 2021). Foc TR4-GFP was prepared by incubation in PDB (potato dextrose broth) in a rotary shaker (180 rpm) for 5 days at 28°C. The liquid culture was then filtered through four layers of sterile gauze. The spores were enumerated by hemocytometer under a light microscope (Axio Scope A1, Carl ZEISS, Germany) and were then diluted to 1.0 × 10⁵ cfu/mL with sterile water. Strain 8ZJF-21 was inoculated in one liter of an Erlenmeyer flask containing 300 ml of sterilized SLM at 200 rpm and 28°C for 7 days. The suspension was diluted to the final concentration of 1 × 10⁵ cfu/ml. Spore suspension of Foc TR4-GFP (100 ml) and strain 8ZJF-21 (100 ml) was completely mixed with 10 g of autoclaved soil. The banana seedlings (Musa AAA group, Cavendish cv. Brazil) with four to five leaves were transferred to the pots (12 cm in diameter). These banana seedlings were kept in a glasshouse under natural light at 28°C ± 2°C. Three experiment groups were set including sterilized SLM + Foc TR4-GFP (1 × 10⁶ spores/g soil) (G1), fermentation broth of strain 8ZJF-21 (1 × 10⁶ spores/g soil) + Foc TR4-GFP (1 × 10⁶ spores/g soil) (G2), and sterilized SLM (G3). All experiments were performed in triplicates. Each group contained 60 pots with three replicates. After 0.5, 1, 2, 3, 4, and 5 days post inoculation (dpi), the root samples of banana seedlings were collected for determining the expression levels of defense-related and antioxidant enzyme genes. The chlorotic symptom of banana leaves was monitored at 30 dpi. The disease indexes were recorded according to Li et al. (2021). Foc TR4-GFP infection in banana roots was detected by a confocal microscope (FV1000-IX81, Olympus, Japan). The physiological parameters of banana seedlings were measured at 30 dpi, including stem diameter, chlorophyll content, leaf area, dry weight, fresh weight, plant height, and leaf thickness (Zhang et al., 2021).

As above mentioned, roots treated with strain 8ZJF-21 and/or Foc TR4 were collected at 0.5, 1, 2, 3, 4, and 5 dpi. H₂O₂ was measured according to Ferguson et al. (1983). One gram of frozen sample was ground in 5 ml of pre-cooled acetone. After centrifugation at 10,000 rpm for 20 min at 4°C, the supernatant was mixed with 0.5 ml of TiCl₄ (20% v/v TiCl₄ in concentrated HCl). And then, 3.5 ml of NH₄OH was added dropwise with thorough mixing. Following centrifugation, the precipitates were redissolved in 25 ml of H₂SO₄ (2 mol/L). The absorbance was recorded at 415 nm. A blank without the addition of sample was made through the same procedure. The standards ranging from 0.15 to 0.75 mol L^–1 H₂O₂ were also reacted with TiC14. Malondialdehyde (MDA) was determined using the reaction method of thiobarbituric acid. Four grams of frozen samples were ground in 15 ml of trichloroacetic acid (5%, w/v). After centrifugation at 6,000 rpm for 10 min, 1.5 ml of the supernatant were mixed with 2.5 ml of thiobarbituric acid (0.5%, w/v) in 15% of trichloroacetic acid. The mixture was incubated at 100°C for 20 min. Absorbance of the supernatant was recorded at 532 nm and corrected using non-specific turbidity by subtracting the absorbance at 600 nm. The content of MDA was calculated according to Pongprasert et al. (2011) and expressed as mol g^–1 FW.

The total RNA of banana roots was extracted using the method of Trizol (Wang et al., 2014). The quality and quantity of RNA were measured by Nanodrop (Thermo Scientific, United States). The first-strand cDNA was synthesized using the Prime Script™ RT Reagent Kit with gDNA Eraser (Takara, Dalian, China). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed in a LightCycler^® 480 System (Roche Diagnostics, Mannheim, Germany) with the SYBR Premix Ex Taq II kit (Takara, Dalian, Liaoning, China). Four defense-related marker genes such as β-1,3-glucanase (Maβ-1,3-Glu, GenBank ID: AF001523), mitogen-activated protein kinase 1 (MaMAPK1, GenBank ID: XM018826311), phenylalanine ammonia lyase (MaPAL, GenBank ID: XM009403673), and pathogen-related protein 1 (MaPR-1, GenBank ID: XM009388962) were selected. The primer sequences were listed in Supplementary Table 1. The reaction system of qRT-PCR was described in our previous study (Zhang et al., 2019). The house-keeping gene of 18S rRNA (GenBank ID: U42083) was used as a reference gene to normalize the expression levels of target genes using the 2^–ΔΔCt method (Wang et al., 2014). All experiments were repeated in triplicates with at least three biological replicates of each sample.

Foc TR4 was cultured in the potato dextrose broth (PBD) at 200 rpm for 7 days at 28°C. After filtration through six layers of gauze to remove hyphae, spores were collected at 1,000 rpm for 10 min and washed using sterile water four times. The spore suspension (1 × 10⁶ spores/mL) of Foc TR4 was prepared using sterile water (Wang et al., 2012). The effect of the extract on spore germination of Foc TR4 was evaluated according to Wei et al. (2020). Briefly, different concentration extracts (1 × EC₅₀, 2 × EC₅₀, 4 × EC₅₀ and 8 × EC₅₀) of strain 8ZJF-21 and Foc TR4 (1 × 10⁶ spores/mL) were mixed completely and added to the concavity of slide. Foc TR4 spores treated with 10% (v/v) of DMSO were used as a control. After 16 h of incubation at 25°C, the spore germinated of Foc TR4 conidia were counted using a light microscope (Axio Scope A1, Carl ZEISS, Germany). Conidia were considered as germinated when germ tube began to appear. Five replicates were used in each treatment and at least 200 conidia were measured per replicate.

Foc TR4 was grown on the PDA medium with 4 × EC₅₀ of extract at 28°C for 5 days. The collected mycelia were fixed with 2.5% (v/v) of glutaraldehyde overnight at 4°C. DMSO (10%, v/v) treatment was used as a control. Agar plugs (5 mm in diameter) with Foc TR4 were cut from the edge of a 3-day-old fungal medium. The mycelial sections were prepared according to Jing et al. (2020). Morphological characteristics of Foc TR4 mycelia were detected using SEM. The effect of strain 8ZJF-21 extract on the cellular ultrastructure of Foc TR4 was observed by a transmission electron microscope (TEM, JEM-1400 Flash, Hitachi Limited, Tokyo, Japan) according to Wei et al. (2020). Four replicates were used per treatment and each experiment was repeated three times.

The volatile organic compounds in strain 8ZJF-21 extract were identified using GC-MS as our previous description (Li et al., 2021). Extract of strain 8ZJF-21 was first dissolved in the chromatographic grade methanol and was 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 chromatograph (5973 Inert XL MSD, Agilent, United States). Helium was used as a carrier gas with a flow rate of 1 ml min^–1. The column temperatures were set as follows: initial column temperature at 70°C for 3 min, followed by an increment of 5°C/min up to 100°C and 10°C/min up to 250°C. The final temperature was kept at 300°C for 5 min. The mass spectrometer was operated in the electron ionization mode at 70 eV with a continuous scan from 50 to 800 m/z (Jing et al., 2020). The peaks were identified by matching the mass spectra with the National Institute of Standards and Technology (NIST, United States) library.

All the experiments were implemented using a completely randomized design. Data were obtained from at least three biological replicates and were expressed as the mean ± standard deviation (SD). Data processing and statistical analysis were performed with the SPSS statistical software package (SPSS Inc., Cary, NC, United States, v.22). The significance was determined by Duncan’s multiple range tests (P < 0.05).

Antifungal activity of the selected endophytic actinomycetes was further tested against Foc TR4. Strain 8ZJF-21 isolated from the roots of medicinal plant C. capitulata exhibited a strong antagonistic activity (Figure 1A). Strain 8ZJF-21 can grow well on PDA and various ISP media (ISP2-ISP7). Different morphological characteristics of the colony were displayed in Supplementary Table 2. The strain can develop the branched substrate and aerial mycelia. Cream aerial mycelia and milky-white substrate hyphae were observed on all the selected ISP media. The grayish-white color of aerial and substrate mycelia was displayed on PDA. Diffusible pigment was not detected on the selected media except for ISP7, which was a typical profile of melanin pigment produced by Streptomyces (Shirling and Gottlieb, 1966). The spiral spore chains with rough surface were finally generated (Figure 1B).

In comparison with different culture conditions in the ISP2 medium, strain 8ZJF-21 can grow well at temperatures from 20 to 40°C (optimum at 30°C), NaCl up to 3% (w/v, optimal concentration 1%) and pH from 6.0 to 9.0 (optimum pH 7.0). It could produce extracellular enzymes such as amylase, cellulase, protease, and urease as well as reduce nitrate. It had no capacity to produce H₂S and respond to gelatin degradation. In addition, strain 8ZJF-21 was able to utilize all the tested sugars as sole carbon source and most of nitrogen source except for NH₄NO₃, arginine, and glutamic acid (Table 1). Compared with the reference Streptomyces strains (Qi et al., 2019; Jing et al., 2020; Wei et al., 2020), strain 8ZJF-21 was considered as a member of the genus Streptomyces.

To further identify strain 8ZJF-21, the whole genome was sequenced. A 1,639 bp-length sequence of 16S rDNA was extracted from the genome sequences. One-hundred percent of nucleotide similarity was found with 16S rDNA of S. malaysiensis DSM 4137 (GenBank ID: NZ_CP023992). The phylogenetic tree showed that strain 8ZJF-21 was located in a well-delineated subclade with S. malaysiensis (Figure 1C). Compared with the genomes of strain 8ZJF-21 to the typal genome of S. malaysiensis DSM 4137 (Supplementary Figure 1), the calculated ANI value was 98.49 above the threshold value of 95-96% for species delineation (Richter and Rosselló-Móra, 2009). Therefore, strain 8ZJF-21 was identified as S. malaysiensis.

The antifungal assay showed that S. malaysiensis 8ZJF-21 significantly inhibited the mycelial growth of all tested fungi (Figure 2). The inhibition rates ranged from 40.15 to 77.83%. The strongest antifungal activity was detected against the causal agent of strawberry anthracnose (C. fragariae, 77.83 ± 2.68), followed by F. oxysporum f. sp. cucumerinum (67.48 ± 1.32), C. gloeosporioides (63.96 ± 2.01), and F. graminearum Sehw (60.86 ± 1.32). In addition, strain 8ZJF-21 had also strong inhibition activities against A. tenuissima (59.73 ± 3.2), C. lunata (42.37 ± 2.31), F. oxysporum f. sp. cubense race 1 (42.88 ± 1.58), C. gloeosporioides (Penz) Penz and Sacc 1884 (43.33 ± 2.18), P. oryzae (48.51 ± 2.01), and C. acutatum (40.15 ± 1.32). It suggested that S. malaysiensis 8ZJF-21 had a broad-spectrum antifungal activity.

Streptomyces malaysiensis 8ZJF-21 inhibited the growth of different phytopathogenic fungi in vitro. It promoted us to evaluate its biocontrol efficiency against Foc TR4 using the pot experiment. The disease symptoms on banana seedlings were detected at 30 dpi. In the treatment group of Foc TR4 (G1), banana seedlings showed an obvious chlorotic symptom at the bottom of the leaves (Figure 3A). Compared with the control group, no obvious disease symptom was detected in the group of S. malaysiensis 8ZJF-21 + Foc TR4 (G2), suggesting that the protective treatment with S. malaysiensis 8ZJF-21 effectively prevented the infection of Foc TR4. The results were supported by the lack of obvious black symptoms in the split corms of banana seedlings treated with S. malaysiensis 8ZJF-21 (G2). We also evaluated Foc TR4-GFP infection in the roots of banana seedlings. The colony-forming units of Foc TR4-GFP in S. malaysiensis 8ZJF-21-treated roots were much lower than that in Foc TR4-GFP-treated roots (Figure 3B). The disease index was 65.37% in the G1 group, while only 18.07% were recorded in the G2 group (Figure 3C).

Compared to different agronomic traits of banana seedlings in different treatment groups (Figures 3D–J), Foc TR4 infection inhibited the growth of banana seedlings. Although no obvious difference of stem diameter was observed among the three treatment groups (Figure 3D), S. malaysiensis 8ZJF-21 significantly increased (p = 0.0036) the plant height and reached 46.73 ± 2.01 cm at 30 dpi (Figure 3E). Compared to the agronomic indicators in the G1 group, a significant increase was detected in the leaf area, leaf thickness, dry weight, and fresh weight in the G2 and G3 groups (Figures 3F–I). Chlorophyll content was sharply decreased in the Foc TR4-treated leaves due to chlorotic symptom (Figure 3I). Hence, S. malaysiensis 8ZJF-21 not only reduced the disease symptoms, but also promoted the growth of banana seedlings.

Biotic and abiotic stresses induce accumulation of reactive oxygen species in plant cells, thereby causing oxidative damage (Li et al., 2015). The oxidative damage expressed as the form of H₂O₂ was first determined in banana roots of different groups. Foc TR4 infection resulted in a rapid increase of H₂O₂ and reached a peak at 3 dpi (Figure 4A). S. malaysiensis 8ZJF-21 reduced the accumulation of H₂O₂ in Foc TR4-infected roots. It was supported by the changes of MDA contents in the G2 group, a marker for monitoring lipid peroxidation caused by oxidative damage (Figure 4B). The MDA contents in Foc TR4-inoculated roots dramatically increased from 0.5 dpi and reached the highest value at 4 dpi, which was four-fold higher than that in the G3 group. However, the increase of MDA contents in roots treated with S. malaysiensis 8ZJF-21 was obviously inhibited. The maximum was detected at 4dpi with the decrease of 69.04% in comparison with the G1 group.

To assay whether S. malaysiensis 8ZJF-21 could induce activities of antioxidant enzymes (such as CAT, SOD, PPO, and POD), the expression levels of these genes were investigated. The transcripts of MaCAT in banana roots treated with S. malaysiensis 8ZJF-21 increased gradually until 5 dpi (Figure 4C). In Foc TR4-treated roots, the expression peak was detected at 4 dpi. No obvious increase (P < 0.01) among different time points except for 2 dpi was detected in the G3 group. Similarly, S. malaysiensis 8ZJF-21 upregulated significantly (P < 0.05) the expression levels of MaSOD and MaPOD. Their transcripts reached the maximum at 4 dpi with two-fold higher than those in the G1 group (Figures 4D,E). Although Foc TR4 induced obviously the transcript accumulation of MaPPO, the expression levels were higher in the G2 group and increased by 21% at 3 dpi in comparison with Foc TR4-treated roots (Figure 4F).

To determine whether the defensive system was activated in response to S. malaysiensis 8ZJF-21 and/or Foc TR4, four defense-related genes (Mab-1,3-Glu, MaPAL, MaMAPK1, and MaPR1) were selected. By contrast, S. malaysiensis 8ZJF-21 and/or Foc TR4 significantly increased the transcripts of four defense-related genes with varying patterns (Figure 5). The transcript level of MaPR1 in roots treated with S. malaysiensis 8ZJF-21 increased significantly at 0.5 dpi and reached the highest peak at 2 dpi, which was 1.3-fold and 7.0-fold higher than that in the G1 and G3 groups, respectively (Figure 5A). The transcript level of MaPAL was also upregulated by S. malaysiensis 8ZJF-21 and the expression peak was detected at 2 dpi with 1.6-fold higher than that in the G1 group (Figure 5B). A similar expression pattern of Maβ-1,3-Glu was observed in the treated roots of S. malaysiensis 8ZJF-21. The transcripts of Maβ-1,3-Glu in the G2 group showed an increase of 1.64-fold at 1 dpi and 1.35-fold at 2 dpi in comparison with that in the G1 group (Figure 5C). The expression levels of MaMAPK1 reached their maximum values at 2 dpi in the G1 group and at 1 dpi in the G2 group. High transcripts were maintained by S. malaysiensis 8ZJF-21 until 4 dpi (Figure 5D). It suggested that S. malaysiensis 8ZJF-21 could improve the plant resistance to Foc TR4 by activating the MAPK-mediated signaling pathway of defense response.

Foc TR4 was inoculated on the PDA plate containing different concentration extracts of S. malaysiensis 8ZJF-21. The inhibition of mycelial growth was measured, until hypha reached the edge of the plate in the control group (10% of DMSO treatment). The mycelial growth of Foc TR4 was inhibited dramatically along with the increase of extract concentration. More than 12.50 μg/ml of extract almost completely restricted the mycelial growth of Foc TR4 (Figure 6A). The EC₅₀ value was 6.11 μg/ml (Supplementary Figure 2). Similarly, the extracts significantly reduced the germination rate of conidia and the length reduction of germ tubes (Figures 6B,C). All spore germination was almost completely inhibited by 4 × EC₅₀ of extract. No obvious inhibition of Foc TR4 growth and spore germination was observed in the control group. In addition, the extract-treated hyphae became deformed, shrunk, ruptured, and swollen (Figure 6D). The normal hyphae with a smooth surface appeared to be uniform in thickness. For cellular ultrastructure of Foc TR4, 4 × EC₅₀ of the extract caused vacuolization and organelle degradation. Mitochondria and cell nucleus gradually disappeared. High dense components were formed in treated cells (Figure 6E).

After sequencing and assembly, the genome of S. malaysiensis 8ZJF-21 consisted of 11,434,537 bp and had 71.09% of GC content. The genome contained 8 rRNA genes, 63 tRNA genes, and 9,787 coding sequences (Figure 7A). By annotation, 34.24, 48.33, and 75.44% of genes were assigned to three categories of KEGG, GO, and COG, respectively. In KEGG annotation, 2,491 of genes participated into the regulation of cellular processes (193), metabolism (2,008), human diseases (142), genetic information processing (216), environmental information processing (298), and organismal systems (44) (Supplementary Figure 3). For COG annotation, the top five categories contained transcription (833), nucleotide transport and metabolism (584), carbohydrate transport and metabolism (575), energy production and conversion (484) as well as inorganic ion transport and metabolism (400). Notably, 2,342 of genes were clustered into unknown function category (Figure 7B). A total of 4,730 genes were annotated into biological process (1,728), cellular component (1,657), and molecular function (3,950) using the GO database (Supplementary Figure 4).

Moreover, several BGCs in the genome of S. malaysiensis 8ZJF-21 were involved in secondary metabolism. By alignment with antiSMASH, the predicted 52 BGCs included NRPS (non-ribosomal peptide synthetase), PKS (polyketide synthase) type 1 and 2, siderophore, terpene, indole, butyrolactone, and betalactone (Supplementary Table 3). Fourteen BGCs exhibited more than 70% of similarity with the submitted BGCs in the database. Seven BGCs showed more than 100% of similarity with coelichelin, ectoine, nigericin B, ectoine, desferrioxamin B, echoside A/B, geosmin, and pristinol (Figure 7C). Six BGCs participated in the biosynthesis of antimicrobial compounds such as hopene, elaiophylin, coelichelin, ectoine, nigericin, and geldanamycin. Two BGCs probably regulated the biosynthesis of anticancer agent (hygrocin A/B and azalomycin F3a). Two siderophore molecules were encoded by cluster 21 and cluster 39. Gene clusters 67 and 78 were responsible for the biosynthesis of terpene. Clusters 66 and 71 were involved in the production of pigment. Notably, a high portion of unknown BGCs suggests that several novel secondary metabolites could be produced by S. malaysiensis 8ZJF-21.

Gas Chromatography-Mass Spectrometry (GC-MS) was used to identify the bioactive compounds in S. malaysiensis 8ZJF-21 extract. Compared to mass spectra with the NIST library, 19 volatile organic compounds were obtained according to retention time and molecular weight (Table 2). They contained acetophenone (1), chloroacetic acid, 3-tetradecyl ester (2), 2,4-furandicarboxylic acid, dimethyl ester (3), formic acid, trans-4-methylcyclohexyl ester (4), 5-hydroxymethylfurfural (5), cyclohexane (6), pyrazoline (7), pentacosanoic acid, methyl ester (8), hexadecanoic acid, ethyl ester (9), borneol, dimethyl(pentafluorophenyl)silyl ether (10), pentacosanoic acid, methyl ester (11), 1,2-benzenedicarboxylic acid, butyl 2-methylpropyl ester (12), 4-(3-methyl-2-butenyl)-1H-indole (13), 1,2-bis(p-(cis-styryl)phenyl)-trans-ethylene (14), colchiceinamide (15), 1,2-Bis(p-(cis-styryl)phenyl)-trans-ethylene (16), voaluteine (17), 4’-(3-(6-Methyl-3-pyridyl)-1-(p-tolyl)-2-pyrazolin-5-yl)acetanilide (18), and bufotalin (19). The peak area represented the relative proportion. Benzenedicarboxylic acid and 1H-Indole, 4-(3-methyl-2-butenyl) were two dominant components in S. malaysiensis 8ZJF-21 extract.