Micromonospora Parathelypteridis Sp. Nov., an Endophytic Actinomycete with Antifungal Activity Isolated from the Root of Parathelypteris Beddomei (Bak.) Ching

NOTE Zhao et al., Int J Syst Evol Microbiol 2017;67:268–274 DOI 10.1099/ijsem.0.001614

Micromonospora parathelypteridis sp. nov., an endophytic actinomycete with antifungal activity isolated from the root of Parathelypteris beddomei (Bak.) Ching

Shanshan Zhao,1 Chongxi Liu,1 Weiwei Zheng,1 Zhaoxu Ma,1 Tingting Cao,1 Junwei Zhao,1 Kai Yan,1 Wensheng Xiang1,2,* and Xiangjing Wang1,*

Abstract A novel endophytic actinomycete with antifungal activity, designated strain NEAU-JXY5T, was isolated from the root of Parathelypteris beddomei (Bak.) Ching. Strain NEAU-JXY5T showed closest 16S rRNA gene sequence similarity to Micromonospora luteifusca GUI2T (99.31 %), and phylogenetically clustered with Micromonospora noduli GUI43T (99.24 %), ’Micromonospora lycii’ NEAU-gq11 (99.19 %), ’Micromonospora zeae’ NEAU-gq9 (99.12 %), Micromonospora saelicesensis Lupac 09T (98.97 %), Micromonospora vinacea GUI63T (98.96 %), ’Micromonospora jinlongensis’ NEAU-GRX11 (98.91 %), Micromonospora profundi DS3010T (98.77 %), Micromonospora zamorensis CR38T (98.76 %), Micromonospora chokoriensis 2–19(6)T (98.71 %), Micromonospora lupini Lupac 14NT (98.69 %), Micromonospora ureilytica GUI23T (98.69 %), Micromonospora violae NEAU-zh8T (98.57 %) and Micromonospora taraxaci NEAU-P5T (98.37 %). Phylogenetic analysis based on gyrB gene sequences also indicated that the isolate clustered with the above strains except M. violae NEAU-zh8T. A combination of DNA–DNA hybridization results and some phenotypic characteristics indicated that the strain could be readily distinguished from these closest phylogenetic relatives. Therefore, it is concluded that strain NEAU-JXY5T represents a novel species of the genus Micromonospora, for which the name Micromonospora parathelypteridis sp. nov. is proposed. The type strain is NEAU-JXY5T (=CGMCC 4.7347T=DSM 103125T).

Micromonospora is the type genus of the family Micromono- polyketide, maklamicin, which showed antimicrobial activ- sporaceae that was described by Ørskov [1]. Members of ity against Gram-positive bacteria [12]. Micromonospora sp. this genus have been isolated from various sources, such as EN43, isolated from healthy wheat tissue, was able to sup- soil, insects, marine sediments and plants [2–5]. Further- press a number of pathogens both in vitro and in planta more, the genus Micromonospora has gradually been recog- [13]. Therefore, the exploration of endophytic actinobacte- nized as an important source of secondary metabolites. ria holds great promise for the discovery of novel biologi- Many antibiotics, including calicheamicin, gentamicin, cally active natural products. As part of a programme to megalomicin, telomycin and rosamicin have been isolated discover actinomycetes with novel antibiotic production from this genus [6–10]. Thus, the impact of the genus properties, an endophytic actinomycete with antifungal T Micromonospora on medicine is considerable. Endophytic activity, strain NEAU-JXY5 , was isolated. In this study, we species of the genus Micromonospora have recently been performed polyphasic taxonomy on this strain and propose reviewed with respect to their potential for use as antagonist that the isolate represents a novel species of the genus agents. For example, Micromonospora lupini, isolated from Micromonospora. root nodules, generated two novel anthraquinones, lupinaci- Strain NEAU-JXY5T was isolated from the root of Parathe- dins A and B with significant antitumour activity [11]. lypteris beddomei (Bak.) Ching collected from Harbin, Hei- Micromonospora sp. GMKU326, isolated from the root of a longjiang province, north China (45 45¢ N 126 41¢ E). leguminous plant, could produce a new spirotetronate-class The root specimen was processed as described by Wang

Author affiliations: 1Key Laboratory of Agriculture Biological Functional Gene of Heilongjiang Provincial Education Committee, Northeast Agricultural University, No. 59 Mucai Street, Xiangfang District, Harbin 150030, PR China; 2State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, PR China. *Correspondence: Wensheng Xiang, [email protected]; Xiangjing Wang, [email protected] Keywords: Micromonospora parathelypteridis sp. nov.; Parathelypteris beddomei (Bak.) Ching; polyphasic taxonomy; 16S rRNA gene; antifungal activity. Abbreviation: ISP, International Streptomyces Project. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA and gyrB gene sequences of strain NEAU-JXY5T are KU997023 and KX008605, respectively. Five supplementary figures and two supplementary tables are available with the online Supplementary Material.

001614 ã 2017 IUMS Downloaded from www.microbiologyresearch.org by IP: 219.142.90.210268 On: Fri, 08 Dec 2017 02:06:11 Zhao et al., Int J Syst Evol Microbiol 2017;67:268–274 et al. [14] and placed on a plate of Gause’s synthetic agar provided by the manufacturer. An almost full-length 16S no. 1 [15] supplemented with cycloheximide (50 mg lÀ1) rRNA gene sequence of strain NEAU-JXY5T (1507 bp) was and nalidixic acid (50 mg lÀ1). After 2 weeks of aerobic obtained and aligned with multiple sequences obtained incubation at 28 C, colonies were transferred and purified from the GenBank/EMBL/DDBJ databases using CLUSTAL X on International Streptomyces Project (ISP) medium 3 1.83 software. Phylogenetic trees were reconstructed by the [16] and maintained as glycerol suspensions (20 %, v/v) at neighbour-joining [37] and maximum-likelihood [38] tree- À80 C. making algorithms by using the software package MEGA version 6.06 [39]. Confidence values of branch nodes were Morphological characteristics were observed by light evaluated using the bootstrap resampling method with 1000 microscopy (ECLIPSE E200; Nikon) and scanning electron replications [40]. A distance matrix was generated using microscopy (S-3400N; Hitachi) after cultivation on ISP 3 ’ medium at 28 C for 4 weeks. Motility was assessed by light Kimura s two-parameter model [41]. All positions contain- microscopic (ECLIPSE E200; Nikon) observation of cells ing gaps and missing data were eliminated from the dataset suspended in phosphate buffer (pH 7.0, 1 mM). Cultural (complete deletion option). Pairwise alignment analysis of characteristics were determined after 3 weeks at 28 C using 16S rRNA gene sequence similarities between strains were ISP 2–7, SA1 agar, N-Z amine agar and Bennett’s agar [16– calculated on the EzTaxon-e server [42]. PCR amplifications 19]. ISCC-NBS colour charts Standard Samples No 2106 and sequencing of the gyrase B subunit (gyrB) gene were was used to determine the colour of colonies and soluble carried out using primers GYF1 and GYR3B [43]. Sequenc- pigments [20]. The utilization of sole carbon and nitrogen ing and phylogenetic analysis was performed as described sources (0.5 % w/v), decomposition of cellulose, hydrolysis above. of starch and aesculin, reduction of nitrate, peptonization The G+C contents of the genomic DNA were determined of milk, liquefaction of gelatin and production of H2S were using the thermal denaturation (Tm) method [44] with examined as described previously [21, 22]. Production of Escherichia coli JM109 DNA used as the reference. catalase, esterase and urease were tested as described by DNA–DNA relatedness tests between strain NEAU-JXY5T Smibert and Krieg [23]. Growth at different temperatures T and Micromonospora luteifusca GUI2 , Micromonospora (4, 10, 15, 20, 28, 32, 35, 37 and 40 C) was determined on noduli GUI43T, ‘Micromonospora lycii’ NEAU-gq11, ISP 3 agar after incubation for 14 days. Tolerance of pH ‘Micromonospora zeae’ NEAU-gq9, Micromonospora saelice- range (pH 4, 5, 6, 7, 8, 9, 10 and 11), using the buffer sys- sensis Lupac 09T, Micromonospora vinacea GUI63T, tem described by Xu et al. [24] and NaCl tolerance (0, 1, 2, ‘Micromonospora jinlongensis’ NEAU-GRX11, Micromono- 3, 4, 5, 6 and 7 %, w/v) for growth were determined after spora profundi DS3010T, Micromonospora zamorensis CR38T, incubation for 14 days in ISP 2 broth in shake flasks – T Micromonospora chokoriensis 2 19(6) , Micromonospora (250 r.p.m.) at 28 C. lupini Lupac 14NT, Micromonospora ureilytica GUI23T, T The freeze-dried cells used for chemotaxonomic analysis Micromonospora violae NEAU-zh8 and Micromonospora T were obtained from cultures grown in GY [25] medium on taraxaci NEAU-P5 were carried out as described by De Ley a rotary shaker for 4 days at 28 C. The isomer of diamino- et al. [45] under consideration of the modifications described pimelic acid in the cell-wall hydrolysates was derivatized by Huss et al. [46], using a model Cary 100 Bio UV/VIS-spec- according to McKerrow et al. [26] and analysed by the trophotometer equipped with a Peltier-thermostatted 6Â6 HPLC method described by Yu et al. [27]. The whole-cell multicell changer and a temperature controller with in situ sugars were analysed according to the procedures developed temperature probe (Varian). The DNA samples used for by Lechevalier and Lechevalier [28]. Cellular menaquinones hybridization were diluted to OD260 around 1.0 using were extracted and purified as described by Collins et al. 0.1ÂSSC (saline sodium citrate buffer), then sheared using a [29] and were analysed by HPLC [30]. Polar lipids were JY92-II ultrasonic cell disruptor (ultrasonic time 3 s, interval quantified, examined via two-dimensional TLC and identi- time 4 s, 90 times). The DNA renaturation rates were deter- fied by using established procedures [31]. The presence of mined in 2ÂSSC at 70 C. The experiments were performed mycolic acids was checked by the acid methanolysis method with three replications and the DNA–DNA relatedness value as described previously [32]. Following the technique was expressed as a mean value. described by Gao et al. [33], fatty acid methyl esters were The antifungal activity bioassay was carried out according extracted from the biomass which was obtained in ISP 2 to the procedure described by Bai et al. [47]. The fungal broth at 28 C for 7 days, and analysed by GC-MS using the strain Sclerotinia sclerotiorum was kindly provided by Soy- method of Xiang et al. [34]. bean Research Institute of Northeast Agricultural University Extraction of genomic DNA and PCR-mediated amplifica- (Harbin, China). Corynespora cassiicola, Alternaria solani, tion of the 16S rRNA gene were carried out using a standard Curvularia lunata, Setosphaeriaturcica turcicaf, Helmintho- procedure [35, 36]. The PCR product was separated by gel sporium maydis, Sphacelotheca reiliana, Thanatephorus electrophoresis, then purified and cloned into the pMD19-T cucumeris, Fusarium oxysporum and Colletotrichum orbicu- vector (Takara). Sequencing was performed by using an lare were kindly provided by the Institute of Vegetables and Applied Biosystems DNA sequencer (model 3730XL) and Flowers, Chinese Academy of Agricultural Sciences (Beijing, analysis of the data was performed using the software China). All the tested strains were incubated on potato

Downloaded from www.microbiologyresearch.org by IP: 219.142.90.210269 On: Fri, 08 Dec 2017 02:06:11 Zhao et al., Int J Syst Evol Microbiol 2017;67:268–274 dextrose agar (PDA; potato 200 g, dextrose 20 g, agar 20 g, (98.91 %), M. profundi DS3010T (98.77 %), M. zamorensis distilled water 1 l) at 28 C. CR38T (98.76 %), M. chokoriensis 2–19(6)T (98.71 %), M. lupini Lupac 14NT (98.69 %), M. ureilytica GUI23T (98.69 %), Morphological observation of 4-week-old cultures of strain M. violae NEAU-zh8T (98.57 %) and M. taraxaci NEAU-P5T NEAU-JXY5T grown on ISP 3 medium revealed that it has (98.37 %), which was supported by a bootstrap value of 56 % the typical characteristics of the members of the genus in the neighbour-joining tree (Fig. 1) and also recovered with Micromonospora [48]. It produced well-developed and the maximum-likelihood algorithm (Fig. S4). Partial sequence branched substrate mycelium, but lacked aerial mycelium. T (1162 bp) of the gyrB gene of strain NEAU-JXY5 was Non-motile and elliptical spores (0.7–0.8Â0.8–0.9 µm) were obtained and the phylogenetic analysis of gyrB gene sequences borne singly on the substrate mycelium and the spore sur- T (Fig. S5) supported that strain NEAU-JXY5 was placed in face was smooth (Fig. S1, available in the online Supplemen- T the genus Micromonospora, also clustering with with the tary Material). Strain NEAU-JXY5 grew well on N-Z T above strains except M. violae NEAU-zh8 . DNA–DNA amine, Bennett’s, SA 1, ISP 2 and ISP 3 media, moderately hybridization was employed to further clarify the relatedness on ISP 4 and ISP 6 media, and poorly on ISP 5 and ISP 7 T between the isolate and M. luteifusca GUI2 , M. noduli media. The colour of colonies was vivid yellow on N-Z T ‘ ’ ‘ ’ ’ GUI43 , M. lycii NEAU-gq11, M. zeae NEAU-gq9, M. saeli- amine, Bennett s and SA 1 agar, brilliant orange yellow on cesensis Lupac 09T, M. vinacea GUI63T, ‘M. jinlongensis’ ISP 2 agar, and brilliant greenish-yellow on ISP 3, ISP 4, ISP NEAU-GRX11, M. profundi DS3010T, M. zamorensis CR38T, 5, ISP 6 and ISP 7 agar (Table S1). No diffusible pigments M. chokoriensis 2–19(6)T, M. lupini Lupac 14NT, M. ureilytica or melanin were observed on any of the tested media. T T T T – GUI23 , M. violae NEAU-zh8 and M. taraxaci NEAU-P5 , Growth of strain NEAU- JXY5 was observed at 15 37 C and the levels of DNA–DNA relatedness between them were (optimum 28 C), at pH 5.0–9.0 (optimum pH 7.0) and in – 52.4±0.2, 50.9±0.3, 52.4±0.7, 60.2±0.3, 56.3±1.1, 58.9±0.5, 58.9 the presence of 0 3 % NaCl (w/v). Detailed physiological ±0.7, 58.7±0.3, 43.8±0.3, 48.4±0.6, 50.9±0.9, 49.3±0.4, 41.3 characteristics of strain NEAU- JXY5T are presented in the T ±0.8 and 46.0±0.5 %, respectively. These values were below the species description. In addition, strain NEAU-JXY5 has threshold value of 70 % recommended by Wayne et al. [54] antifungal activity against the tested fungi (Fig. S2). for assigning strains to the same species. The DNA G+C con- T Chemotaxonomic characteristics of strain NEAU-JXY5T tent of the strain NEAU-JXY5 is 72.3±0.3 mol%. also supported its classification as a member of the genus Furthermore, strain NEAU-JXY5T could be unambiguously Micromonospora. It contained meso-diaminopimelic acid as distinguished from its closely related strains by physiologi- the cell-wall diamino acid and whole-cell sugars included cal and biochemical characteristics as summarized in xylose and glucose. The phospholipid profile consisted Table 1, such as its ability to grow at pH 5.0 and 15 C, and of diphosphatidylglycerol, phosphatidylethanolamine and the differences in coagulation and peptonization of milk, phosphatidylinositol (Fig. S3), phospholipid type PII decomposition of cellulose, production of catalase and ure- according to Lechevalier et al. [49]. The menaquinones ase, hydrolysis of starch and Tweens 20, 40 and 80, and pat- detected were MK-9(H8) (57.5 %), MK-10(H2) (27.1 %), terns of carbon and nitrogen utilization. Meanwhile, the MK-10(H6) (12.8 %) and MK-9(H4) (2.6 %). The cellular T ! colony colours of strain NEAU-JXY5 could be distin- fatty acid profile was composed of C17 : 0 (27.0 %), C18 : 1 9c guished from other closely related species on various (15.0 %), C15 : 0 (12.6 %), iso-C16 : 0 (11.8 %), anteiso-C15 : 0 ! ! medium and no diffusible pigments or melanin were (11.4 %), C17 : 1 7c (10.7 %), C18 : 0 (8.1 %), C16 : 1 7c (1.7 %) observed on ISP 3 and Bennet (Table S1). Most notably, and 10-methyl C (1.7 %) (Table S2). Mycolic acids were T 18 : 0 strain NEAU-JXY5 could not utilize raffinose, while the not detected. closely related species could. The fatty acid composition of Comparative 16S rRNA gene sequence analysis showed that strain NEAU-JXY5Talso differed from other closely related T strain NEAU-JXY5 was phylogenetically related to members strains. For instance, iso-C15 : 0 was a major constituent of of the genus Micromonospora. The species was determined to many related strains, such as M. luteifusca GUI2T, M. noduli be most closely related to M. luteifusca GUI2T (99.31 % 16S GUI43T, M. profundi DS3010T, M. chokoriensis 2–19(6)T, rRNA gene sequence similarity), M. noduli GUI43T (99.24 %), M. ureilytica GUI23T and M. taraxaci NEAU-P5T, but the ‘M. lycii’ NEAU-gq11 (99.19 %) and ‘M. zeae’ NEAU-gq9 same fatty acid was not detected in the isolate. In addition, ‘ ’ (99.12 %) which were obtained from nodules and roots of dif- iso-C16 : 0 was also a predominant feature of strains M. lycii ferent plants [50–53]. The 16S rRNA gene sequence similari- NEAU-gq11, ‘M. zeae’ NEAU-gq9, M. saelicesensis Lupac ties between strain NEAU-JXY5T and strains of other 09T, M. vinacea GUI63T, ‘M. jinlongensis’ NEAU-GRX11 members of the genus Micromonospora were less than 99.0 %. and M. lupini Lupac 14NT, however, it was not the most Phylogenetic analysis based on the 16S rRNA gene sequence abundant component of strain NEAU-JXY5T, which sup- indicated that strain NEAU-JXY5T and M. luteifusca GUI2T plied a marked difference between the isolate and closely (99.31 % 16S rRNA gene sequence similarity) formed a mono- related species. Furthermore, strain NEAU-JXY5T was dis- phyletic clade, and clustered with M. noduli GUI43T tinguished from the most closely related strains by increased (99.24 %), ‘M. lycii’ NEAU-gq11 (99.19 %), ‘M. zeae’ NEAU- levels of C18 : 1 !9c, while other fatty acids were much less gq9 (99.12 %), M. saelicesensis Lupac 09T (98.97 %), M. vina- notable. Besides, many phylogenetically related species T ‘ ’ cea GUI63 (98.96 %), M. jinlongensis NEAU-GRX11 shared significant amounts of MK-10(H4), but this was

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T 61 ∗ M. rhizosphaerae 211018 (FJ261956) ∗ ‘M. yasonensi’ DS3186 (JN989295) ∗ 55 M. olivasterospora DSM 43868T (X92613) ∗ 0.005 M. avicenniae 268506T (JQ867183) ∗ M. equina Y22T (JF912511) ∗ M. viridifaciens DSM 43909T (X92623) M. echinaurantiaca DSM 43904T (X92618) ∗ M. kangleipakensis MBRL 34T (JN560152) 55 M. soli SL3-70 T (AB981051) ∗ M. inositola DSM 43819T (X92610) M. pallida DSM 43817T (X92608) 86 M. costi CS1-12 T (AB981048) ∗ M. fulviviridis DSM 43906T (X92620) M. echinospora DSM 43816T (X92607) 71 ∗ M. inyonensis DSM 46123T (X92629) 76 M. sagamiensis DSM 43912T (X92624) ∗ M. narathiwatensis BTG4-1 T (AB193559) ∗ ‘M. spongicola’ S3-1 (AB645957) ∗ M. nigra DSM 43818 T (X92609) ∗ M. yangpuensis FXJ6.011T (GU002071) ∗ ∗ M. eburnea LK2-10 T (AB107231) M. ﬂuostatini PWB-003T (LC033898) M. chaiyaphumensis MC5-1T (AB196710) M. halotolerans CR18T (FN658652) 99 M. cremea CR30T (FN658654) ∗ M. coriariae NAR01T (AJ784008) M. endolithica DSM 44398T (AJ560635) ∗ ∗ M. chalcea DSM 43026T (X92594) M. tulbaghiae TVU1T (EU196562) ∗ M. rosaria DSM 803T (X92631) M. chersina DSM 44151T (X92628) M. citrea DSM 43903T (X92617) ∗ M. echinofusca DSM 43913 T (X92625) ∗ M. coerulea DSM 43143T (X92598) M. auratinigra TT1-11 T (AB159779) ∗ M. peucetia DSM 43363T (X92603) ∗ ‘M. endophytica’ DCWR9-8-2 (AB981049) M. zhanjiangensis 2902at01T (KJ742700) ∗ ∗ M. pisi GUI 15T (AM944497) ∗ M. pattaloongensis TJ2-2T (AB275607) T 87 ∗ M. ovatispora 2701SIM06 (JQ836686) 50 ∗ M. sonneratiae 274745T (JQ619535) 88 M. polyrhachis NEAU-ycm2T (KC139400) ∗ M. marina JSM1-1T (AB196712) M. sediminicola SH2-13 T (AB609325) ∗ M. aurantiaca DSM 43813T (X92604) M. maritima D10-9-5 T (HQ704071) M. halophytica DSM 43171T (X92601) M. coxensis 2-30-b(28) T (AB241455) M. purpureochromogenes DSM 43821T (X92611) M. humi P0402T (GU459068) 99 M. harpali NEAU-JC6T (KJ609003) ∗ M. oryzae CP2R9-1T (AB981052) M. haikouensis 232617T (GU130129) M. mangrovi 2803GPT1-18 T (JQ836668) ∗ ∗ ∗ M. rifamycinica AM105T (AY561829) 65 M. wenchangensis 2602GPT1-05 T (JQ768361) ∗ M. krabiensis MA-2T (AB196716) 59 M. carbonacea DSM 43168T (X92599) M. schwarzwaldensis HKI0641T (KC517406) M. sediminis CH3-3T (AB889541) 72 M. palomenae NEAU-CX1T (KF887911) ∗ M. nickelidurans K55T (HQ174560) ∗ T ∗ M. matsumotoense IMSNU 22003 (AF152109) 50 ∗ ‘M. maoerensis’ NEAU-MES19 (KC469639) 98 M. vulcania NEAU-JM2 T (KF956808) ∗ M. mirobrigensis WA201T (AJ626950) M. siamensis TT2-4T (AB193565) T 63 ∗ M. violae NEAU-zh8 (KC161209) M. chokoriensis 2-19(6) T (AB241454) ∗ 94 M. taraxaci NEAU-P5 T (KC439463) 64 M. lupini Lupac 14NT (AJ783996) 71 M. parathelypteridis NEAU-JXY5T (KU997023) ∗ ∗ 56 M. luteifusca GUI2T (FN658633) ∗ M. saelicesensis Lupac 09T (AJ783993) M. ureilytica GUI23T (FN658641) M. profundi. DS3010T (KF494813) M. noduli GUI43T (FN658649) 73 ‘M. lycii ’ NEAU-gq11 (KC193249) ∗ M. vinacea GUI63T (FN658651) 87 ‘M. jinlongensis’ NEAU-GRX11 (KC134254) ∗ ∗ ‘M. zeae’ NEAU-gq9 (KC287242) 52 M. zamorensis CR38T (FN658656) C. citrea subsp. citrea DSM 44097T (NR041763)

Fig. 1. Neighbour-joining tree based on nearly complete 16S rRNA gene sequences showing the relationship between strain NEAU- JXY5T and members of the genus Micromonospora. The outgroup used was Catellatospora citrea subsp. citrea DSM 44097T. Asterisks denote branches that were also recovered using the maximum-likelihood method. Bootstrap values 50 % (based on 1000 replications) are shown at branch points. Bar, 0.005 substitutions per nucleotide position.

Downloaded from www.microbiologyresearch.org by IP: 219.142.90.210271 On: Fri, 08 Dec 2017 02:06:11 Zhao et al., Int J Syst Evol Microbiol 2017;67:268–274 ) ); 2 6,8 NEAU- ’ + + + + À À . All data T MK-10(H MK-9(H ) NEAU-P5 + + + 2,4,6 À ÀÀ ÀÀ M. jinlongensis MK-10 (H ‘ ; 8, ) T 4,6 + + + + ÀÀ À ÀÀÀ À M. taraxaci (H MK-10 GUI63 ; 15, ) T 4,6 + + + + + + + À MK- 10 (H M. vinacea NEAU-zh8 ) 4 + + + + + + + + + ; 7, À ÀÀÀÀ ÀÀÀÀÀ À ÀÀÀÀ ÀÀ À T MK- 10(H ) M. violae 4,6 + + + + + + + + + + + + + + + + À (H Lupac 09 MK-10 ; 14, T ) 4,6 + + + + + + + À À À À GUI23 (H MK-10 ) 4,6 + ÀÀÀ ÀÀ À ÀÀ À M. saelicesensis (H MK-9 M. ureilytica ); ) 6 4,6 ; 13, T + + + + + ÀÀÀ À MK-9(H NEAU-gq9; 6, MK-10(H ’ ) 4 Lupac 14N + + + + + ++ + + + + + + MK- M. zeae ‘ 10(H ) M. lupini 2,4,8 À ÀÀ À MK-10 (H ; 12, T ) ); NEAU-gq11; 5, 2,6 8 19(6) ’ – + + + + + + + + + + + + + + 2 ÀÀÀ ÀÀ MK-9(H MK-10(H M. lycii ‘ ; 4, ); T ) 4 4,6 À À À M. chokoriensis GUI43 MK-9(H MK-10(H and closely related species ; 11, T T ); ) 4 4 M. noduli CR38 + + + +++++++++ + + + + + + + + + + + ++++++++++++ + + + + + + + + + + + + + ÀÀÀÀÀÀÀÀÀ ÀÀ ÀÀÀÀ ; 3, T MK-9(H MK-10(H GUI2 , negative. ) À ); 2,6 8 M. zamorensis + À À À À À À À ÀÀÀÀÀ À ; 10, MK-10(H T M. luteifusca ; 2, T DS3010 C + + + + M. profundi Differential characteristics of strain NEAU-JXY5 -Xylose -Threonine -Glutamine -Tyrosine -Arabinose L L Catalase Urease + Starch D Raffinose nitrogen source L of milk Tween 20 carbon source L Tween 40Tween 80 + + + + + + + + + Menaquinones (>10 %) MK-9(H Growth at 15 Decomposition of cellulose + + Production of: CharacteristicGrowth at pH 5.0 1 + 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Hydrolysis of Utilization as sole Coagulation and peptonization Utilization as sole Strains: 1, NEAU-JXY5 Table 1. GRX11; 9, were obtained in this study. +, Positive;

Downloaded from www.microbiologyresearch.org by IP: 219.142.90.210272 On: Fri, 08 Dec 2017 02:06:11 Zhao et al., Int J Syst Evol Microbiol 2017;67:268–274 absent in strain NEAU-JXY5T; this phenomenon could eas- 2012BAD19B06), the National Natural Science Foundation of China (No. 31471832, 31500010, 31572070, 31672092 and 31372006) and ily differentiate the isolate from the phylogenetically related Chang Jiang Scholar Candidates Program for Provincial Universities in species (Table 1). Heilongjiang (CSCP). In conclusion, it is evident from the genotypic, chemotaxo- T Acknowledgements nomic and phenotypic data that strain NEAU-JXY5 repre- We are grateful to Professor Aharon Oren for helpful advice on the sents a novel species of the genus Micromonospora, for specific epithet. which the name Micromonospora parathelypteridis sp. nov. Conflicts of interest is proposed. The authors declare that there are no conflicts of interest.

DESCRIPTION OF MICROMONOSPORA References PARATHELYPTERIDIS SP. NOV. 1. Ørskov J. Investigations into the Morphology of the Ray Fungi. Copenhagen: Levin and Munksgaard; 1923. Micromonospora parathelypteridis (pa.ra.the.ly.pte¢ri.dis. N. 2. Li L, Hong K. Micromonospora ovatispora sp. nov. isolated from L. gen. n. parathelypteridis of Parathelypteris, referring to mangrove soil. Int J Syst Evol Microbiol 2016;66:889–893. the isolation of the type strain from Parathelypteris 3. Xiang W, Yu C, Liu C, Zhao J, Yang L et al. Micromonospora polyrhachis sp. nov., an actinomycete isolated from edible Chinese beddomei). black ant (Polyrhachis vicina Roger). Int J Syst Evol Microbiol 2014; – Aerobic, Gram-stain-positive actinobacterium that forms 64:495 500. well-developed and branched substrate hyphae. Aerial 4. Phongsopitanun W, Kudo T, Mori M, Shiomi K, Pittayakhajonwut P et al. Micromonospora fluostatini sp. nov., isolated from marine mycelium is absent. 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