Streptomyces endocoffea sp. nov. , an endophytic actinomycete isolated from Coffea arabica (L.)

An aerobic, non-motile, Gram-stain positive actinomycete, designated strain CA3R110 T , was isolated from the surface-sterilised root of Coffea arabica L. collected from Lampang Province, Thailand. 16S rRNA gene sequence analysis indicated that strain CA3R110 T is a member of the genus Streptomyces and showed the closest similarities to Streptomyces rapamycinicus NRRL B-5491 T (99.1%), followed by S. iranensis HM 35 T (99.0%). Strain CA3R110 T contained LL-diaminopimelic acid in cell peptidoglycan, MK-9(H 6 ), and MK-9(H 8 ) as major menaquinone, iso-C 16:0, iso-C 15:0, C 16:0 as major fatty acids. Diphosphatidylglycerol, phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositolmannoside were detected in the cell. The chemotaxonomic characteristics possessed the typical properties of the genus Streptomyces. A low digital DNA–DNA hybridization (< 55.6%) and average nucleotide identity-blast (ANIb) (< 92.2%) values revealed that strain CA3R110 T could be distinguished from any known Streptomyces species. With the differences in phenotypic and genotypic data, strain CA3R110 T represents a novel species of genus Streptomyces, for which the name Streptomyces endocoffea sp. nov. is proposed. The type strain is CA3R110 T (= TBRC 11245 T = NBRC 114296 T ). indole, redox-cofactor, PKS-like, heterocyst glycolipid synthase-like PKS (hglE-KS), siderophore, ectoine, non-alpha poly-amino e-Polylysin homoserine RRE-element cluster, Other synthesised post-translationally modied (RiPP-like), gene % biosynthetic gene gene cluster (100 %), azalomycin biosynthetic gene cluster (100 biosynthetic 2-methylisoborneol biosynthetic gene cluster (100 A biosynthetic gene pristinol biosynthetic gene cluster (100 desferrioxamine biosynthetic gene cluster geosmin biosynthetic gene cluster (100 ectoine (100 coelibactin gene cluster (100 elaiophylin biosynthetic gene cluster (87 feglymycin biosynthetic cluster (78 biosynthetic gene cluster (69 mediomycin biosynthetic gene sceliphrolactam glycinocin gene biosynthetic gene


Introduction
An attempt to investigate the novel actinomycete in an unexplored environment is one strategy to discover a new bioactive compound from the microbial resource. It is known that plants are major sources for recovering secondary metabolites. They always accumulate the secondary metabolites in their tissue. Therefore the environment inside the plant tissue may differ signi cantly from other habitats.
Hence the microorganisms residing in the plant tissue are expected to be different from those of soil microorganisms. Many promising endophytic microbes have been reported as candidate biocontrol agents against pathogens. Endophytic actinomycetes, especially Streptomyces spp., have been recognized as the promising producer for biocontrol agents to soilborne plant diseases (Solecka et al. 2012). Many reports, for example, 3-acetonylidene-7-prenylindolin-2-one from mycelial cake extracted with EtOH of Streptomyces sp. NEAU-D50 showed cytotoxic activities against human adenocarcinoma cell line A549 and antifungal activity against the tested phytopathogenic fungi Colletotrichum orbiculare, Phytophthora capsici, Corynespora cassiicola and Fusarium oxysporum (Zhang et al. 2014). 6-Prenylindole from the fermented broth of Streptomyces sp. TP-A0595 exhibited against plant fungal pathogens, Alternaria brassicicola and Fusarium oxysporum (Sasaki et al. 2002;Singh and Dubey 2018). Azalomycin B from the fermentation broth of Streptomyces sp. HAAG3-15 showed signi cant antifungal activity against Fusarium oxysporum f. sp. cucumerinum (Cao et al. 2020). To date, many new species of endophytic Streptomyces have continuously been reported. For example, Streptomyces typhae sp. nov. (Peng et al. 2021), Streptomyces albidus sp. nov. (Kaewkla and Franco 2021), Streptomyces dioscori sp. nov. (Wang et al. 2018) were reported as novel endophytic Streptomyces species which were isolated from the root of Typha angustifolia L., Callitris preissii, and the Bulbil of Dioscorea bulbifera L., respectively.
In the course of our investigation of endophytic actinomycetes from the coffee plant, strain CA3R110 T was isolated from the surface-sterilised root of Coffea arabica L. collected from Lampang Province, Thailand. The polyphasic taxonomic details of strain CA3R110 T were reported in this study.

Materials And Methods
Actinobacterium isolation and cultural conditions The strain CA3R110 T was isolated from the surface-sterilized root of Coffea arabica L., collected from Ratchaburi province, Thailand. The root sample was gently washed in tap water to remove the dirt and cut into small pieces (1 cm each). After cutting, the sample was washed in sterile 0.1 % tween 20 for 30 sec and then washed twice in sterile distilled water. The sample was surface-sterilized with 95 % ethanol for 10 min and subsequently with 1 % sodium hypochlorite for 6 min, followed by washing in sterile distilled water for three times. Finally, the surface-sterilized sample was ground with sterile mortar and pestle with 0.5 ml sterile distilled water, and the suspension (200 µl) was spread on starch casein agar (SCA) [10 g soluble starch, 2 g KNO 3 , 0.3 g casein, 2 g K 2 HPO 4 , 0.05 g MgSO 4 .7H 2 O, 2 g NaCl, 0.02 g CaCO 3 , 0.01 g FeSO 4 .7H 2 O, 15 g agar, 1000 ml distilled water, pH: 7.0] supplemented with 25 mg l -1 nalidixic acid, and 50 mg l -1 nystatin and incubated at 30 o C for 30 days. The nal washing water was spread on the same agar medium as the control plates. The pure culture was preserved by freezing at -80 o C and freeze-drying.
Morphology, cultural, biochemical, and physiological tests Spore chain morphology and spore-surface ornamentation of strain CA3R110 T were observed after growing on a half strength-ISP2 medium at 30 o C for 30 days by a scanning electron microscope (model JSM-6610 LV; JEOL). The cultural characteristic was performed by cultivation the strain on various International Streptomyces Project (ISP) media (ISP2-7) (Shirling and Gottlieb 1966), czapek's sucrose agar (Waksman 1961), glucose-asparagine agar (ISP5 with 1% glucose replacing glycerol), and nutrient agar (Difco) at 30 o C for 14 days. The colours of the substrate and aerial mycelium and diffusible pigments were evaluated by comparison with the ISCC-NBS color charts (Kelly 1964). Growth at different temperatures (10-50 o C), and 0-10%, w/v NaCl (at increment of 1%) were evaluated on ISP2 agar after incubation for 14 days. The effect of pH on growth (4.0-11.0 at an increment of 0.5 pH unit) was determined by cultivation at 30 °C in ISP2 broth for 14 days. Catalase activity was observed by bubble production after applying 3% (v/v) hydrogen peroxide solution. Test for oxidase activity was carried out by oxidation of 1% N, N, N',N'-tetramethyl-p-phenylenediamine dihydrochloride. Carbon and nitrogen utilisations were examined according to the standard method of Shirling andGottlieb 1966 andGordon et al. (1974), respectively. Nitrate reduction, the acid production, the decomposition of insoluble compounds, e.g., xanthine, adenine, hypoxanthine and tyrosine, gelatin liquefaction, hydrolysis of casein (1 %, w/v), and starch (1%, w/v) were evaluated by using the methods of Arai (1975), Williams and Cross (1971) and Gordon et al. (1974). The enzyme activities were determined using the API ZYM system (bioMérieux).

Chemotaxonomy.
Biomass used for chemotaxonomic analyses was collected from cell culture grown in ISP 2 broth on a rotary shaker (200 r.p.m.) at 30˚C for ve days.
The diaminopimelic acid isomers and whole-cell sugars were evaluated based on the method of Hasegawa et al. (1983), and Komagata and Suzuki (1987), respectively. The polar lipids in the cell membrane were extracted and analyzed by the methods proposed by Minnikin et al. (1984) and analyzed by the two-dimension TLC technique of Collins and Jones (1980). Menaquinones were extracted as described by Collins et al. (1977) and were analyzed by high-performance liquid chromatography equipped with a Cosmosil 5C18 column (4.6 x 150 mm, Nacalai Tesque) (Tamaoka et al. 1983). Fatty acids were extracted, methylated and analyzed using the Sherlock Microbial Identi cation (MIDI) system and the ACTIN version 6 database (Sasser 1990), and Kämpfer and Kroppenstedt (1996).

DNA extraction, 16S rRNA gene analysis
Genomic DNA was extracted using the protocol of Tamaoka (1994). The 16S rRNA gene ampli cation and sequencing were conducted using the method suggested by Thawai (2015). The 16S rRNA gene similarity was calculated using the EzTaxon-e server (www.ezbiocloud.net) (Yoon et al. 2017). To reconstruct the phylogenetic tree, the 16S rRNA gene sequence was multiple-aligned with selected sequences available from the GenBank/EMBL/DDBJ databases by using CLUSTAL W multiple alignment mode within BioEdit program version 7.1.3.0 (Hall 1999). Phylogenetic trees were reconstructed by using neighbor-joining (NJ) (Saitou and Nei 1987), and maximum-likelihood (ML) (Felsenstein 1981) algorithms in MEGA version X program (Kumar et al. 2018). The evolutionary distances were calculated by using Kimura's 2-parameter model (Kimura 1980). The Tamura-Nei model (Tamura and Nei 1993) was applied to the maximum-likelihood analysis using the Subtree-Pruning-Regrafting-Extensive (SPR level 5) program. The con dence values of nodes were evaluated by using the bootstrap resampling method with 1,000 replicates (Felsenstein 1985).

Genome sequencing and analysis
Genomic DNA for whole-genome sequencing of strain CA3R110 T was extracted from 3-day old cultures grown in ISP2 broth at 30 o C and puri ed following the GeneJET Genomic DNA protocol puri cation Kit (Thermo Scienti c). The genomic DNA of strain CA3R110 T was sequenced using an Illumina HiSeq 4000 platform (Chulalongkorn University, Thailand). Genome assembly were performed using SPAdes (Bankevich et al. 2012). The online server, Rapid Annotations using Subsystems Technology (RAST) (http://rast.nmpdr.org/), was used for genome annotation (Aziz et al. 2008;Overbeek et al. 2014). The genome was applied to the JSpecies Web Server for calculating the average nucleotide identity (ANI) values (Richter et al. 2016;Richter and Rosselló-Móra 2009). The digital DNA-DNA hybridization (dDDH) values between the genome of strain CA3R110 T , and the most closely related species were calculated using the genome-to-genome distance calculator (GGDC 2.1; blast+method) in which formula 2 (identities/HSP length) was applied to the incomplete draft genome (Meier-Kolthoff et al. 2013). The phylogenomic tree was reconstructed using the Type (strain) Genome Server (TYGS) (Meier-Kolthoff and Göker 2019). Anti-SMASH program was used to evaluate the secondary metabolite biosynthesis gene clusters in the bacterial genome (Blin et al. 2019).

Results And Discussion
Morphology and cultural characteristic of strain CA3R110 T Strain CA3R110 T formed well-developed and nonfragmented branched substrate mycelia and grew well on ISP2, ISP4, ISP5, ISP6, and czapek's sucrose agar. Moderate growth was observed on ISP3, ISP7, nutrient agar, and glucose-asparagine agar. The strain could produce pale greenish-yellow to dark brownish-gray substrate mycelia on the above media tested. Greyish-white aerial mycelium was easily formed on all media tested after 21 days of cultivation at 30 o C. The brilliant greenish-yellow diffusible pigment was observed on ISP5 and ISP7 (Table S1). The spiral chain of spore with the rugose surface was detected (Fig. S1). Morphological detail revealed that it formed the same morphological characteristics as other members of the genus Streptomyces (Waksman and Henrici 1943;Pridham et al. 1958).

16S rRNA gene and genome analyses
According to the result from the EzBioCloud (http://www.ezbiocloud.net/) server (Yoon et al. 2017), strain CA3R110 T related to the genus Streptomyces. The strain is closely related to S. rapamycinicus NRRL B-5491 T (99.1%), followed by S. iranensis HM 35 T (99.0%). Strain CA3R110 T forms a distinct monophyletic line within the genus Streptomyces, supported by the two treeing algorithms, maximum-likelihood (ML), and neighbor-joining (NJ), but low bootstrap values (<50%) were observed on both phylogenetic trees ( Fig. 1 and Fig. S3). To con rm the taxonomic position of strain CA3R110 T at the species level, genomebased taxonomic characterization of strain CA3R110 T was performed. Genome sequencing of strain CA3R110 T resulted in 269 contigs, an N50 of 328 kb, genome coverage of 138x, and comprised 13128250 bp. The genomic DNA G+C content of this strain was 70.9 mol%, which was in the range of the genus Streptomyces (Pridham et al. 1958). The draft genome of the strain showed 10797 protein-coding genes, 94 tRNA genes, 1 tmRNA gene and 46 copies of misc_RNA genes.
The genomic comparison between strain CA3R110 T and the type strain of the genus Streptomyces indicated that strain CA3R110 T shared the highest ANIb and ANIm values (92.2 and 93.8%, respectively) with S. rapamycinicus NRRL B-5491 T followed by S. iranensis HM 35 T (92.1% and 93.9%, respectively) which were below an ANI threshold range (95-96 %) for species demarcation (Richter and Rosselló-Móra 2009;Chun et al. 2018) (Table S3). The phylogenomic tree indicated that strain CA3R110 T formed a stable clade with S. rapamycinicus NRRL B-5491 T and S. iranensis HM 35 T that was supported by 95 % bootstrap value (Fig. S4). As evident from genomic analysis, S. rapamycinicus NRRL B-5491 T and S.
iranensis HM 35 T should be selected for phenotypically comparative study.

Differential characteristics between strain CA3R110 T and the closest neighbors
To clarify the novelty at species level of strain CA3R110 T , the phenotypic properties of strain CA3R110 T and its closest relatives, S. rapamycinicus DSM 41530 T and S. iranensis DSM 41954 T were compared in the same culture conditions. Unlike S. rapamycinicus DSM 41530 T and S. iranensis DSM 41954 T , strain CA3R110 T grew in the presence of up to 3 % NaCl. The pH and temperature for cell growth were in the range of 6-9 and 20-40 o C, respectively. Physiological and biochemical properties showed that strain CA3R110 T signi cantly differed from S. rapamycinicus DSM 41530 T and S. iranensis DSM 41954 T . For example, the utilization of L-arabinose, D-mannitol, D-xylose, D-melibiose, D-mannose, L-ra nose, Dribose, dextran, sucrose, and xylitol as sole carbon sources and the utilization of L-histidine, 4hydroxyproline, and L-proline were the different points for discriminating between strain CA3R110 T and both type strains. It can also be distinguishable based on the ability to degrade compounds such as adenine, hypoxanthine tyrosine, and gelatin and the ability to produce acid from inulin, lactose, and Dmelezitose. Moreover, the composition of sugar of the strain CA3R110 T made it different from diagnostic sugars found in S. rapamycinicus DSM 41530 T and S. iranensis DSM 41954 T . Unlike S. rapamycinicus NRRL B-54917 T and S. iranensis DSM 41954 T , strain CA3R110 T was found to have galactose, glucose and ribose, while the closest relatives contained only glucose and ribose in cell-hydrolysates. In addition, the levels of digital DNA-DNA relatedness between strain CA3R110 T and S. rapamycinicus NRRL B-5491 T , S. iranensis HM 35 T were 55.6% and 57.0%, respectively. These values are below the threshold value of 70 %, as suggested by Wayne et al. (1987) for determining novel species for bacterial strains (Table S3).
These phenotypic, chemotaxonomic, and genotypic data indicated that strain CA3R110 T merit classi cation as a novel species of the genus Streptomyces, for which we propose the name Streptomyces endocoffea sp. nov.
Description of Streptomyces endocoffea sp. nov.
The type strain, CA3R110 T (= TBRC 11245 T = NBRC 114296 T ), is an endophytic actinobacterium isolated from the root of Coffea arabica L., which grows in Lampang province, Thailand. The GenBank/EMBL/ DDBJ accession number for the 16S rRNA gene sequence of strain CA3R110 T is MN116545. The wholegenome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession JAERRG000000000. The version described in this paper is version JAERRG010000000.

Supplementary Files
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