Streptomyces Spinosus Sp. Nov. And Streptomyces Shenzenensis Subsp. Endophyticus Subsp. Nov. Endophytic Actinobacteria Isolated From Jasmine Rice and Their Genome Mining Correlate With Potential as Antibiotic Producers and Plant Growth Promoters

Two endophytic actinobacteria, strains SBTS01 T and W18L9 T , were isolated from leaf sheath tissue and leaf sample of Jasmine rice (Oryza sativa KDML 105), respectively, grown in a rice paddy eld in Roiet Province, Thailand. The polyphasic study showed that both strains belonged to the genus Streptomyces; they are aerobe, with well-developed substrate mycelia and aerial mycelia to form long chains of spores. Strain SBTS01 T shared the highest 16S rRNA gene sequence similarity with Streptomyces rochei NRRL B-2410 T (99.0%) and Streptomyces naganishii NRRL ISP-5282 T (99.0%). Strain W18L9 T shared the highest 16S rRNA gene sequence similarity with Streptomyces shenzhenensis DSM 42034 T . The genotypic and phenotypic data of strains SBTS01 T and W18L9 T distinguished these two strains with the closely related species with valid names. The genome analysis showed the dDDH, ANIb and ANIm values of the draft genome between strain SBTS01 T and its related species; S. rochei NRRL B-2410 T (29.4, 81.1, 86.5%) and W18L9 T and its closest species; S. shenzhenensis DSM 42034 T (72.5, 95.1, 97.0%). The name proposed for the new species of type strain SBTS01 T is Streptomyces spinosus (=NRRL B-65636 T =TBRC 15052 T ). The name proposed for the novel subspecies of strain W18L9 T is Streptomyces shenzenensis subsp. endophyticus (=NRRL B-65635 T =TBRC 15051 T ). Both strains could produce antibiotics to inhibit pathogens and contained plant growth promoting (PGP) traits. Genome mining of these two strains revealed that they comprised many Biosynthesis Gene Clusters (BGCs); terpene, type 1, 2 and 3 polyketide synthase, Non-ribosomal peptide synthetase (NRPS), and lanthipeptide including genes encoding PGP traits; nitrogen xation, ACC (1-aminocyclopropane-1-carboxylate) deaminase and siderophore production. T , S. graminisoli NBRC 108883 T , Streptomyces jiujiangensis DSM 42115 T , and Streptomyces hyaluromycini DSM 100105 T . These type strains were obtained from ARS type culture collection, USA (NRRL strain), German Culture Collection (DSMZ), Germany (DSM strains) and Biological Resource Center, National Institute of Technology and Evaluation (NITE), Japan (NBRC strains). All these cultures were grown for 7 days at 27 o C in Tryptic Soya Broth (Oxoid) in an Erlenmeyer ask at 150 rpm and harvested by centrifugation. Washed cells were saponied, methylated and extracted for FAME analysis. The Sherlock software version 6.4 was used for analysis. 61689, a fungi was grown on Potato Dextrose agar (PDA, Oxoid) at pH 6.0 for 7 days. A plug was cut at the edge of the radial growth of fungi by using a 6 mm cork borer and placed at the centre of a new PDA plate at pH 6.0 and incubated at 27 o C for 4 days or until the diameter of the fungi was about 2.5 cm. Then, six wells were made by using a 6 mm cork borer with the centre of the wells approximately 1.5 cm away from the edge of the PDA plate. Fifty microliters of each extracted compound were put into the wells. Each sample was repeated in triplicate. Methanol was used as a negative control. Also, plate of fungi without making wells was used as a negative control. The growth of fungi was measured toward the direction of each well in 4 days after the test. properties: bioactive compound production, plant growth promoting traits, degradable enzymes and bioremediation. The closest similarity of microorganisms was acquired by applying blastp on the UniProt database with matrix; blosum62 (The UniProt 2021). were also mined. Strain W18L9 T contained ve genes encoding xenobiotic degradable enzymes; alkanesulfonate monooxygenase, nitroalkane oxidase, nitroreductase, epoxide hydrolase and phenol 2-monooxygenase.


Introduction
The genus Streptomyces belongs to the family Streptomycetaceae which was proposed by Waksman and Henrici (1943). The genus contains more than 900 validly published species and 50 validly published subspecies (https://lpsn.dsmz.de/genus/streptomyces) (Parte et al. 2020). Most of them were isolated from soil as well as from various environments such as marine sediments, seawater, fresh water, caves, animals and plant tissues (Kämpfer 2012). Streptomyces spp. are well recognized as producers of bioactive compounds, especially antibiotics, as more than 70 percent of currently known antibiotics are produced by this genus. Also, there are many reports of its application as plant growth promoters by producing phytohormones, solubilize phosphate, inhibiting plant pathogens and inducing the plant immune system (Conn et al. 2008;Kämpfer 2012). There have been many studies of isolation and application of endophytes belonging to the genus Streptomyces as biocontrol agents and plant growth promoters (Barnett et Abdelmohsen et al. 2015). Also, many BGCs are known as "silent" or "cryptic" which may not express to produce bioactive compounds in a laboratory or require some inducers (van Bergeijk et al. 2020).
Jasmine rice (Oryza sativa KDML 105) or Khao Dawk Mali of Thailand containing aroma compound, 2-acetyl-1-pyrroline (2AP) has expanded wide popularity in Asia and the Middle East (Wakte et al. 2017). However, this rice variety is susceptible to blast causing by Pyricularia oryzae and bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae (Kampapongsa and Kaewkla, 2016). Recently, there were some studies of endophytic actinobacteria from rice and their application to inhibit rice pathogens and promote rice growth (Naik et  and Bora, 2021). Currently, there are two valid species of the genus Streptomyces isolated from rice tissue; Streptomyces oryza (Mingma et al. 2015) and Streptomyces roietensis (Kaewkla and Franco, 2017).
In this study, the taxonomic position of endophytic actinobacteria isolated from Jasmine rice, strains SBTS01 T and W18L9 T were determined by using a polyphasic taxonomic approach, including morphological, physiological, chemotaxonomic, phylogenetic and genomic characteristics. Antibiotic production against rice and human pathogens and plant growth promoting studies were conducted. Genome data mining of these two strains was also reported and showed the correlation with these properties.

Materials And Methods
Isolation of strains SBTS01 T and W18L9 T Strains SBTS01 T and W18L9 T were obtained from a project which focused on the biodiversity of endophytic actinobacteria from Jasmine rice plants and their application as plant growth promoters and biocontrol agents to inhibit plant pathogens. Jasmine rice (Oryza sativa KDML 105) was collected from the grounds of the Rice Research Centre paddy eld, Roi Et province, Thailand (15.54937N 103.80975E) and surface sterilized by applying the previously described method (Kampapongsa and Kaewkla 2016).
Surface-sterilized leaf, leaf sheath, stem and root tissue were placed onto three different media (Kampapongsa and Kaewkla 2016). Plates were kept in sealed plastic boxes, which were moistened with wet paper towels, and incubated at 27 o C for 12 weeks.
Actinobacterial strains were puri ed by using half strength potato dextrose agar (HPDA) and stored on HPDA slants at 4 o C and in 20% glycerol at -80 o C.
Phylogenetic analysis of 16S rRNA gene Genomic DNA of strains SBTS01 T and W18L9 T was extracted by using GenElute™ (Sigma), and the 16S rRNA gene was ampli ed and sequenced as described previously (Kaewkla and Franco 2013). The 16S rRNA gene sequence of these two strains was examined by using EzbioCloud server (Yoon et al. 2017), and subsequently aligned with the 16S rRNA gene sequences (available from GenBank/EMBL) of the closely related members of the genus Streptomyces with validly published names, using CLUSTAL X (Thompson et al. 1997) with Nocardia africana DSM 44491 T as the out-group.
The phylogenetic trees were constructed by the neighbor-joining (NJ) and maximum parsimony (MP) algorithms using the software package MEGA version X (Kumar et al. 2018). The Jukes-Cantor method (Jukes and Cantor 1969) was applied to the analysis with the NJ algorithm (Saitou and Nei 1987). The MP tree was obtained using the min-mini heuristic algorithm with a search factor of 1 (Nei and Kumar 2000). The topology of the tree was evaluated by performing a bootstrap analysis based on 1000 replications (Felsenstein 1985).

Sequencing, assembly, annotation
Genomic DNA of strain W18L9 T for whole genome sequencing was extracted by using GenElute TM (Sigma). For genome sequencing of this strain, library was prepared by using short insert size library and samples were sequenced by using Illumina Hiseq X-ten platform (Illumina) (2x150 bp paired-end reads) at the Beijing Genome Institute (BGI), Hongkong.
For whole genome sequencing of strain SBTS01 T and the type strain, S. shenzenensis DSM 42031 T , genomic DNA was extracted according to the previously described protocol (Saito and Miura 1963). A library was prepared by using microbial short insert size library (PCR free library) and samples were sequenced by using the Novaseq 6000 systems, Hiseq PE (Illumina) (2x150 bp paired-end reads) at the Singapore Joint Venture and Sequencing Center, Novogene AIT.
The reads were De novo assembled by using Unicycler (0.4.8) (Wick et al. 2017). The draft assemblies of genomes of strains SBTS01 T , W18L9 T and S. shenzenensis DSM 42031 T have been submitted to GenBank as accession number JAINRC000000000, JACBWY000000000, and JAJONF000000000, respectively. Genomes of strains SBTS01 T and W18L9 T were annotated by using Prokka version 1.14.5 (Seemann 2014) and the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al. 2016).

Genome comparison
The phylogenomic tree of strains SBTS01 T , W18L9 T and related taxa was constructed by using the Type (Strain) Genome Server (TYGS) ( (Kornerup and Wanscher 1978). Catalase production, assimilation of six organic acids and utilization of four phenolic compounds as sole carbon source, hydrolysis of skim milk and starch were tested according to the previously described procedures (Kurup and Schmitt 1973). Decomposition of L-tyrosine, adenine, xanthine, urea, esculin, hippurate and acid production from eighteen carbohydrates were carried out according to the previously described methods (Gordon et al. 1974). Growth at different NaCl concentrations (1, 3, 5, 10, 15 and 20 %, w/v), temperatures (4,15,27,37,45 and 55 o C), and pH between 4 and 10 (in 1 pH unit intervals and pH was adjusted aseptically after autoclaving) were evaluated after incubation for 14 days on ISP 2 medium (Kurup and Schmitt 1973 In vitro antibacterial and antifungal activity Page 5/24 The bacteria used for the antibacterial bioassay were a rice pathogen; Xanthomonas oryzae pv. oryzae (Xoo)  The fungal assay was carried out according to the dual culture method previously described protocol (Kampapongsa and Kaewkla 2016).

Antibiotics production assay
Strain SBTS01 T and W18L9 T were tested for antibiotics production by agar diffusion assay. Both strains were cultured on ISP 2 agar and HPDA for 7 days and whole agar plate was cut to small pieces and extracted with 100% methanol and shaken for three hours. The methanol extract was evaporated by using an evaporator. Then, extracted compound was freeze dried by using a lyophilizer. The dried samples were dissolved with 70% methanol and tested for antibiotics production against S. aureus ATCC 29213, MRSA 03120385 and Xoo PXO 86 according to the agar diffusion assay described by Kaewkla and Franco (2021).
For antifungal assay against P. grisea BRIP 61689, a fungi was grown on Potato Dextrose agar (PDA, Oxoid) at pH 6.0 for 7 days. A plug was cut at the edge of the radial growth of fungi by using a 6 mm cork borer and placed at the centre of a new PDA plate at pH 6.0 and incubated at 27 o C for 4 days or until the diameter of the fungi was about 2.5 cm. Then, six wells were made by using a 6 mm cork borer with the centre of the wells approximately 1.5 cm away from the edge of the PDA plate. Fifty microliters of each extracted compound were put into the wells. Each sample was repeated in triplicate. Methanol was used as a negative control.
Also, plate of fungi without making wells was used as a negative control. The growth of fungi was measured toward the direction of each well in 4 days after the test.

Indole acetic acid (IAA) production
The production of IAA was studied according to the method of Khamna et al. (2009) with some modi cations. One actinobacterium disc (8 mm) of 7-day growth on HPDA was inoculated into ISP 2 broth supplemented with 0.2% (w/v) Ltryptophan, on a rotary shaker at 150 rpm for 7 days in the dark at 28 °C. The cultures were centrifuged at 6000 g for 10 min. The supernatant was analyzed for IAA production according to the method of Glickmann and Dessaux (1995) by using Salkowski's reagent.

Cytokinin production
The cytokinin production of strain SBTS01 T and W18L9 T was carried out according to the previously described method (Hussain and Husnain 2009). One actinobacterium disc (8 mm) was inoculated into Yeast malt extract casamino acid broth (Malt extract 10 g, Yeast extract 1 g, Glucose 4 g, casamino acid 3 g, Na 2 HPO 4 0.5 g, DO water 1 L) on a rotary shaker at 150 rpm for 7 days in the dark at 28 °C. The cultures were centrifuged at 10000 g for 10 min. The supernatant was analyzed for cytokinins by using reverse phase HPLC (Prontosil, Hypersorb ODS 5.0 μm, photodiode array; 265 nm), eluted with a ow rate of 1 ml/min with gradient elution of acetonitrile and 0.1 M acetic acid in 5% acetonitrite, at the Central Laboratory, Faculty of Agriculture, Chiang Mai University, Chiang Mai, Thailand. Total cytokinins were analyzed as free cytokinins; zeatin, zeatin riboside, N 6 (D 2 -isopentenyl) adenine and N 6 (D 2 -isopentenyl) adenosine.

Siderophore production
Siderophore production of strain SBTS01 T and W18L9 T was tested according to the previously described method (Pérez-Miranda et al. 2007). Brie y, actinobacterium was streaked on HPDA and grown for 7 days. Chrome azurol S (CAS) medium added agarose as a solidi ed agent was poured at the top of the HPDA plate and left for 15 min. The positive result with changing from blue to purple indicated catechol type or from blue to orange indicated hydroxamates type.

Phosphate solubilization
The ability of strain SBTS01 T and W18L9 T for phosphate solubilization was tested by applying the standard method (Castagno et al. 2011). NBRIP agar was used and a positive result was observed by measuring the halo zone around colonies of these two strains after incubation for 14 days.
1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production The ability of strains SBTS01 T and W18L9 T to produce ACC deaminase was tested by using the DF agar (Penrose and Glick 2003).
Growth on DF medium added ACC as nitrogen source was compared to growth on DF medium with and without nitrogen source as negative and positive controls, respectively.

Nitrogen xation assay
The method of nitrogen xation of strains SBTS01 T and W18L9 T in vitro was applied by the following methods (Baldani et al. 1997). Nitrogen-free semi solid (NFb) agar was used and a positive result was detected by the color of the indicator changing from light blue to dark blue.

Hydrogen cyanide production
The method of hydrogen cyanide production of two strains was applied by the following procedure (Bakker and Schippers, 1987).

Cellulase production
Strains SBTS01 T and W18L9 T were tested for cellulase production on carboxymethyl cellulose (CMC) agar plates and the positive result was detected by a congo red method according to the previously described protocol (Islam and Roy 2018).

Secondary metabolite and biosynthesis gene cluster prediction
Secondary metabolite Analysis Shell (anti-SMASH) version 6.0 (Blin et al. 2021) was used to predict biosynthetic gene clusters (BGCs) for secondary metabolite synthesis of strains SBTS01 T and W18L9 T . The genomes of strains SBTS01 T and W18L9 T were examined by in silico approach to search genes encoding metabolite products relating to various properties: bioactive compound production, plant growth promoting traits, degradable enzymes and bioremediation. The closest similarity of microorganisms was acquired by applying blastp on the UniProt database with matrix; blosum62 (The UniProt 2021).

Results & Discussion
Isolation of strains SBTS01 T and W18L9 T Strain SBTS01 T was isolated from Jasmine rice (Oryza sativa KDML 105) and arose as a dark green colony on leaf sheath tissue after incubation for nine weeks on Humic acid vitamin B agar (HVA) (Hayakawa and Nonomura 1987). Strain W18L9 T was also isolated from Jasmine rice and emerged as a white colony on leaf tissue after incubation for two weeks on Tap Water Yeast extract agar (TWYA) (Coombs and Franco 2003).

Phylogenetic analysis of the 16S rRNA gene
The phylogenetic evaluation of strains SBTS01 T and W18L9 T with the closely related members of the genus Streptomyces showed that both strains belonged to genus Streptomyces and were encompassed by other members of this genus (Fig 1 and Fig S1).
Strain SBTS01 T shared 16S rRNA gene similarity at 98.6% with strain W18L9 T . Both strains were placed in different clusters in both trees. Based on these results, both strains were considered as different species.
The closest type strains which shared the highest 16S rRNA gene sequence similarity with strain SBTS01 T were S. rochei NRRL B-2410 T and S. naganishii NBRC 12892 T , both at 99.0%. However, strain SBTS01 T formed a different cluster with these type strains and formed a single phylogenic lineage in both trees (Fig 1 and Fig S1). The type strains which shared the highest 16S rRNA gene sequence similarity with strain W18L9 T were S. shenzhenensis DSM 42034 T , S. graminisoli NBRC 108883 T , S. jiujiangensis DSM 42115 T , and S. hyaluromycini DSM 100105 T at 99.7, 99.2, 98.9, and 98.9%, respectively. The closest neighbor of strain W18L9 T positioning in the same clade was S. shenzhenensis DSM 42034 T in both trees with high bootstrap support at 81 and 67, respectively ( Fig. 1 and Fig. S1).
Based on the percentage of 16S rRNA gene similarity and phylogenetic position of strains SBTS01 T and W18L9 T , two closely related type strains of strain SBTS01 T and four closely related type strains of strain W18L9 T as mentioned above were selected for a side-by-side comparison study.

Genome analysis
Genome comparison between strains SBTS01 T and W18L9 T The phylogenetic tree based on TYGS revealed that strain SBTS01 T was placed in the different species cluster with strain W18L9 T  Table S1.
Genome comparison of strain SBTS01 T The draft genome sequence of strain SBTS01 T was 9.17 Mb with DNA G+C content determined by in silico genome sequence as 72.5 mol%. The phylogenetic tree based on TYGS revealed the relationship between strain SBTS01 T and the related type strains.
The phylogenomic tree showed that strain SBTS01 T was placed in the different species cluster from all related type strains ( The genome analysis showed the dDDH, ANIb and ANIm values between strain SBTS01 T and two related species which shared 16S rRNA gene similarity at 99.0%; S. rochei NRRL B-2410 T were 29.4, 81.1, 86.5%, and S. naganishii NBRC 12892 T were 26.9, 82.1, 86.9%, respectively. Strain SBTS01 T and its closest neighbour on the phylogenomic tree; S. corchorusii DSM 40340 T had the highest dDDH, ANIb and ANIm values at 54.1, 92.6, 94.3%, respectively. However, strain SBTS01 T and S. corchorusii DSM 40340 T shared a 16S rRNA gene similarity of only 97.0%. Based on the previous report, the cutoff value at the species level was evaluated at 98.7% (Stackebrandt and Ebers 2006). Therefore, strain SBTS01 T and this type strain belonged to different species. Hence, there is no requirement to compare physiological and biochemical characteristics between strain SBTS01 T and this type strain. Based on the genome comparison, strain SBTS01 T is a novel species of the genus Streptomyces.
Genome comparison of strain W18L9 T The draft genome sequence of strain W18L9 T was 10.13 Mb with DNA G+C content determined by in silico genome sequence as 70.9 mol%. The phylogenetic tree based on TYGS revealed that strain W18L9 T was placed in the same species cluster as its closest neighbour, S. shenzhenensis DSM 42034 T but in a different subspecies cluster (Fig. S2) Chun et al. 2018). Based on these data, strain W18L9 T belonged to the same species of S. shenzhenensis DSM 42034 T . According to Nouioui et al. (2018), dDDH values between 70 and 79% de ned subspecies delineation. Based on the dDDH value between strain W18L9 T and S. shenzhenensis DSM 42034 T at 72.5% and the position at different subspecies clusters on the phylogenomic tree, strain W18L9 T belonged to the same species of S. shenzhenensis DSM 42034 T but it was de ned as a novel subspecies.

Chemotaxonomic characteristics
The LL-isomer of DAP was detected in both strains SBTS01 T and W18L9 T . The whole-cell sugars of strain SBTS01 T contained galactose, glucose and mannose and strain W18L9 T contained galactose, glucose, mannose and xylose. The polar lipids of strain SBTS01 T contained phosphatidylethanolamine (PE), phosphatidylinositol (PI), three aminoglycolipids and one glycolipid and strain W18L9 T contained phosphatidylethanolamine (PE), phosphatidylinositol (PI), six glycolipids, one aminolipid, and four unknown lipids which correspond to phospholipid type II (Lechevalier et al. 1977) (Fig. S3).

Phenotypic and physiological characterization
Strain SBTS01 T The colony morphology of strain SBTS01 T is described in Table S3. Strain SBTS01 T showed morphological characteristics with well-developed substrate mycelium and aerial mycelia on most media used. Melanin pigment was produced on ISP 7. Electron micrographs showed that strain SBTS01 T formed round rod-shaped spores with spiny surface and spore chains in loop forms (approximately 1 micron length x 1 micron diameter) ( Fig. 2A). The physiological properties of strain SBTS01 T and its closest type strain; S. rochei NRRL B-2410 T were different. Strain SBTS01 T could assimilate benzoate and tartrate and grow at NaCl 5% (w/v) but the closest type strain could not assimilate these compounds and could grow weakly at this salt concentration. Also, strain SBTS01 T could grow weakly at 45 o C and produce melanin pigment on ISP 7 but the type strain could not. On the other hand, the type strain could hydrolyze L-tyrosine and xanthine but strain SBTS01 T could not (Table 1).

Strain W18L9 T
The colony morphology of strain W18L9 T is described in Table S3. Strain W18L9 T showed morphological characteristics with welldeveloped substrate mycelia and aerial mycelia on most media used. No melanin pigment was observed on ISP 7. Electron micrographs showed that strain W18L9 T formed oval rod-shaped spores (approximately 1.5 micron length x 1 microns diameter) with warty surface and formed spore chains in loops (Fig. 2B). The physiological properties of strain W18L9 T and its closest type strain, S. shenzhenensis DSM 42034 T were different. Strain W18L9 T could assimilate maltose, hydrolyze urea, assimilate tartrate and could grow at 15 o C and pH 10 but the type strain could not. On the other hand, the type strain could hydrolyze hippurate and grow weakly at 5% NaCl (w/v) but strain W18L9 T could not (Table 2).
Based on the results of polyphasic study, strain SBTS01 T is proposed as a representative of a novel species of the genus Streptomyces named Streptomyces spinosus sp. nov. and strain W18L9 T is proposed as a novel subspecies of Streptomyces shenzenensis DSM 42034 T named Streptomyces shenzenensis subsp. endophyticus subsp. nov.

In vitro antibacterial and antifungal activities
Strains SBTS01 T and W18L9 T showed strong and moderate activity against a bacterial rice pathogen; Xoo PXO 86, respectively.
Strains SBTS01 T and W18L9 T showed moderate and strong activity against P. grisea BRIP 61689, respectively. These results were obtained by the dual culture method. Both strains showed strong activity against S. aureus ATCC 29213 and MRSA 03120385 on both dual culture and agar extraction methods. The antifungal assay against other fungal pathogens was tested by using only a dual culture method. Strain SBTS01 T showed weak activity against R. solani AG8 while strain W18L9 T had strong activity against this fungus. Both strains showed moderate activity against a rice fungus; C. lunata BCC 15558. Strains SBTS01 T and W18L9 T showed moderate and weak activity against H. oryzae DOAC 1570, respectively (Table 3). HPDA was the best medium for antimicrobial activity of both strains tested by dual culture and agar extraction methods. HPDA and ISP 2 contained 10 and 4 g of dextrose, respectively. Then, it was not an effect of carbon catabolite regulation (CCR) which is a regulatory mechanism that limits production due to a negative effect applied by high concentrations of dextrose (Sanchez and Demain 2002). ISP 2 contained malt extract and yeast extract at 10 and 4 g/L while HPDA comprised potato infusion. It was reported that some nitrogen sources may decrease antibiotic production in different microorganisms including Streptomyces (Ra eenia 2013). Based on the result of antibiotic production, it is necessary to select at least two media with different compositions to screen for antimicrobial activity.

Plant growth promoting traits
Only strain W18L9 T could grow on DF agar with ACC deaminase as a nitrogen source, x nitrogen on NFB medium, solubilize inorganic phosphate and produce cellulase enzyme (Table 4). Strain SBTS01 T had negative results for these tests. Both strains could not produce HCN but they could produce siderophore (hydroxymate type). Strains SBTS01 T and W18L9 T produced IAA at 71 and 150 ug/mL and produced cytokinins at 0.165 and 0.169 mg/L, respectively (Table 4).

Secondary metabolites and biosynthesis gene cluster prediction
There were many BGCs of strains SBTS01 T and W18L9 T derived from "antiSMASH" database. There were six groups of metabolites; terpene, type 1 polyketide synthase (T1PKS), type2 PKS (T2PKS), type3 PKS (T3PKS), Non-ribosomal peptide synthetase cluster (NRPS), lanthipeptide cluster and other compounds (Table 5). Both strains contained terpene clusters; geosmin and alba avenone (100% similarity). Geosmin is recognized as an earthy odor that was generally produced by many genera of actinobacteria including Streptomyces species (Jiang et al. 2007). Alba avenone, a tricyclic sesquiterpene antibiotic, which was produced by many Streptomyces sp. possessed an antibacterial activity (Moody et al. 2011). Also, strains SBTS01 T and W18L9 T contained gene clusters encoding hopene at 76 and 96% similarity, respectively. Hopene is a precursor for producing bioactive hopanoids, a group of pentacyclic triterpenoids such as diploterol which is toxic to mouse leukemia cells (Schmerk et al. 2015; Sohlenkamp and Geiger 2016). Strain SBTS01 T contained isorenieratene (62% similarity) which is a carotenoid light-harvesting pigment produced by green sulfur bacteria; Chlorobium spp. There were some reports of isorenieratene biosynthesis by actinobacteria but the function of this compound is still unknown and it may relate to blue or UV light resistance (Maresca et al. In correlation with the genome data mining, strain SBTS01 T also showed good activity against Xoo PXO 86, S. aureus ATCC 29213 and MRSA 03120385 in which this strain may be likely to produce novel antibiotics. Finally, strain SBTS01 T contained a gene cluster, melanin (71% similarity). Melanin pigment produced from microorganisms could help to resist chemicals and oxidizing agents, thermal, and biochemical stresses (El-Naggar and El-Ewasy, 2017). This gene nding correlated with in vitro study in which strain SBTS01 T produced melanin pigment on ISP 7. In addition, this strain contained a gene cluster encoding spore pigment which bene ts bacteria to tolerate UV and other environmental stresses (El-Naggar and El-Ewasy, 2017). As strain SBTS01 T was isolated from a rice paddy eld which applied many pesticides such as fungicide, herbicide, and insecticide, this strain tended to adapt for resisting to survive from chemicals (personal communication with Dr. Ronnachai Changsri, a researcher at rice department, Thailand).
Feglymycin, a novel peptide antibiotics was reported to inhibit the replication of the human immunode ciency virus (HIV) in vitro (Vértesy et al. 1999). Furthermore, strain W18L9 T comprised NRPS/NRPS like clusters; xenotetrapeptide, tolaasin I / tolaasin F (100 and 50% similarity). The novel xenotetrapeptides were nonribosomally synthesized peptides which have been still unknown function produced from Xenorhabdus nematophila, bacteria reside as endosymbionts in the foreguts of soil nematodes in genus Steinernema (Khandelwal and Banerjee-Bhatnagar 2003). In correlation with the genome data mining, strain W18L9 T showed good activity against S. aureus ATCC 29213, MRSA 03120385, P. grisea BRIP 61689 and R. solani AG8 in vitro. The properties of strain W18L9 T to inhibit rice pathogens are correspondent with its ecophysiology inside rice tissue which bene ts plant to inhibit pathogens.
In addition, the result showed that both strains contained gene cluster to synthesize siderophore, desferrioxamine E which plays an important role in the growth and development of Streptomyces (Codd et al. 2018). It was correspondent with the result of PGP traits of both strains which could produce siderophore (hydroxymate type) in vitro.

In silico gene prediction
Other genes of strains SBTS01 T and W18L9 T encoding other bioactive compounds relating to plant growth promoting, degradable enzymes and bioremediation were scanned. The result showed that these strains contained a variety of genes encoding drought tolerant and stress response proteins and iron uptake protein relating to plant growth promoting properties (Table S4 and S5). For drought and salt tolerant proteins, both strains contained genes encoding ectoine production, glycine betaine/L-proline transporter, glycine, betaine and proline production, Ca 2+ : H + antiporter and chloride channel protein. Only strain SBTS01 T contained sodium solute symporter and potassium channel family protein which can support plants to tolerate salt stress (Horn et al. 2006).
Only strains W18L9 T contained the gene encoding ACC deaminase which supports plants in stress condition such as drought, ooding, nutrient starvation, temperature, salt and oxidative stresses including toxic agents (Gupta and Pandey 2019). Strain W18L9 T contained genes relating to nitrogen xation; ADP-ribosyl-dinitrogen reductase. These genes correlated with the results in which this strain could produce ACC deaminase and had ability to x nitrogen in vitro. Also, the gene encoding indole-3-glycerol phosphate synthase relating to indole acetic acid (IAA) production was detected in the genome of strain SBTS01 T . IAA is a derivative of a plant hormone, auxin which promotes plant growth and support plant growth in salt stress condition (Keswani et al. 2020). In correlation with this gene nding, strain SBTS01 T and W18L9 were able to produce IAA in vitro. Furthermore, there were many genes encoding universal stress proteins (usp) which were detected in both genomes. It was reported that bacteria containing usp genes play a role in maintaining internal and external protection mechanisms in biotic and abiotic stresses (Kim et al. 2012). Strain W18L9 T and strain SBTS01 T comprised genes encoding exopolyphosphatase and inorganic pyrophosphatase while strain SBTS01 T also contained phosphatase and alkaline phosphatase. However, only strain W18L9 T showed an ability to solubilize inorganic phosphate in vitro. From this result, it can conclude that strain SBTS01 T contained cryptic genes which required gene induction to express phosphate solubilization. The rice paddy eld which plant source collected is in Suvannabhum district, Roiet province. This area is known as Thug Gura Ronghai which contains poor soil, ne-loamy soil type and moderate salt (EC 4-8 dS/m). The subsoil contains a lot of rock salt and potash (Sahunalu 2003). In correlation with the ecophysiology, strains SBTS01 T and W18L9 T contained genes encoding plant growth promoting traits to promote rice growth in poor soil and salinity condition.
Strain SBTS01 T and W18L9 T comprised many genes encoding bene cial enzymes which can be applied in a variety of industries. There were several types of enzymes; alpha-N-arabinofuranosidase, amylase, β-glucosidase,β-xylosidase, and β-xylanase which could degrade hemicellulose, polysaccharide, or plant residue to obtain fermentable sugars for applying in bioethanol production (Podkaminer et al. 2012). Genes encoding chitinase were also detected in both strains. It was reported that bacteria producing chitinolytic enzymes could inhibit fungal pathogens by degrading fungal cell walls and degrading waste containing chitin (Brzezinska et al. 2014). Also, these two strains contained genes encoding lipase and various types of proteases which can be applied in many industries such as detergent, food and feed additive, leather and textile industries.
Alkanesulfonate monooxygenase participates in alkanesulfonate degradation. Nitroalkane oxidase relates to nitroalkane degradation in which nitroalkane compounds are used as solvents and explosive compounds and as herbicide and pesticide agents in agriculture and 2-nitropropane was reported as a carcinogen (Li et al. 2008). Nitroreductase can reduce pollutants such as 2,4-dinitrotoluene and 2,4,6-trinitrotoluene, as well as key chemical intermediates, 3-nitrophthalimide, 4-nitrophthalimide and 4nitro-1,8-naphthalic anhydride. Also, strain W18L9 T contained gene encoding tellurium resistance protein. Gene encoding a latex clearing protein was detected in this strain which can apply to degrade waste containing rubber such as used gloves and tires.
This will be valuable to degrade xenobiotic waste in the environment. As stated above, the rice paddy eld was contaminated with many pesticides applied in the area for many years. Strain W18L9 T may adapt to survive in the environment contaminated with these compounds and contains genes encoding enzymes for degrading pesticides.
Strain SBTS01 T contained genes encoding aldehyde dehydrogenase family protein, 2-nitropropane dioxygenase and haloacid dehalogenase type II. This strain also contained genes encoding ion detoxi cation protein; selenium-binding protein, tellurium For anticancer compound, strain W18L9 T comprised gene encoding immune checkpoint inhibitors (ICIs) which are monoclonal antibodies that block inhibitors of T-cell activation and function. The ICIs have potential effects on more than 14 different cancers which were reported to have effects in some patients for more than 5 years (20% -40%; 5-year survival rate in patients with melanoma) (Johnson et al. 2018). Based on the genome data mining, strain W18L9 T has the potential to produce anti-HIV and anticancer compounds which will be fruitful to study this strain in-depth for novel bioactive compounds in the future.

Conclusion
In conclusion, strains SBTS01 T and W18L9 T can be differentiated from other species of the genus Streptomyces by ANI and dDDH values, a phylogenetic tree of the genome, and their phenotypic and chemotaxonomic features. The name, Streptomyces spinosus is proposed for this novel species and Streptomyces shenzenensis subsp. endophyticus is proposed for this novel subspecies. These strains could produce antibiotics to inhibit bacterial pathogens, X. oryzae pv. oryzae PXO 86, S. aureus ATCC 29213, MRSA deaminase, produce cellulase enzyme and x nitrogen. Both strains could produce siderophore, IAA and cytokinin. Genome mining revealed that these strains have the potential to produce novel metabolites. The genome data mining of these strains comprised several genes relating to plant growth promotion under stress conditions. Genome mining correlated with properties of these strains in vitro. Also, both strains contained versatile genes encoding bene cial enzymes and xenobiotic degradation which can be used in many industries and remedy polluted environments for further study.
Description of Streptomyces spinosus sp. nov.