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

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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 ver- sion 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
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