1 Advance Publication J. Gen. Appl. Microbiol. doi 10.2323/jgam.2019.04.004 ©2019 Applied Microbiology, Molecular and Cellular Biosciences Research Foundation 1 Title: Endophytic actinomycetes associated with Cinnamomum cassia Presl in Hoa Binh province, Vietnam: 2 Distribution, antimicrobial activity and, genetic features 3 Running title: Endophytic actinomycetes in Cinnamomum 4 (Received January 9, 2019; Accepted April 16, 2019; J-STAGE Advance publication date: August 2, 2019) 5 Thi Hanh Nguyen Vu1¶, Quang Huy Nguyen2,3,1¶, Thi My Linh Dinh1, Ngoc Tung Quach1, Thi Nhan Khieu4, 6 Ha Hoang1, Son Chu-Ky5, Thu Trang Vu5, Hoang Ha Chu1,2, Jusung Lee6, Heonjoong Kang6,7, Wen-Jun 7 Li8 and Quyet-Tien Phi1,2* 8 9 1Institute of Biotechnology (IBT), Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, 10 Cau Giay, 10000 Hanoi, Vietnam 11 2Graduate University of Science and Technology (GUST), Vietnam Academy of Science and Technology 12 (VAST), 18 Hoang Quoc Viet, Cau Giay, 10000 Hanoi, Vietnam 13 3University of Science and Technology of Hanoi (USTH), Vietnam Academy of Science and Technology 14 (VAST), 18 Hoang Quoc Viet, Cau Giay, 10000 Hanoi, Vietnam 15 4Department of Science, Technology and Environment, Ministry of Education and Training, 49 Dai Co Viet, Hai 16 Ba Trung, 10000 Hanoi, Vietnam 17 5School of Biotechnology and Food Technology (SBFT), Hanoi University of Science and Technology (HUST), 18 1 Dai Co Viet, Hai Ba Trung, 10000 Hanoi, Vietnam 19 6The Center for Marine Natural Products and Drug Discovery, School of Earth and Environmental Sciences, 20 College of Natural Sciences, Seoul National University NS-80, Seoul 08826, Korea 21 7Research Institute of Oceanography, Seoul National University NS-80, Seoul 08826, Korea 22 8State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, School of 23 Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China 2 24 ¶: These authors contributed equally to this work. 25 *Corresponding author: Quyet-Tien Phi, Institute of Biotechnology, Vietnam Academy of Science and 26 Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, 10000 Hanoi, Vietnam. Phone: (+84) 02437917973, Fax: 27 (+84) 024.38363144, Email: [email protected]; [email protected]. 28 29 SUMMARY 30 Endophytic microbes associated with medicinal plants are considered to be potential producers of various 31 bioactive secondary metabolites. The present study investigated the distribution, antimicrobial activity and 32 genetic features of endophytic actinomycetes isolated from the medicinal plant Cinnamomum cassia Presl 33 collected in Hoa Binh province of northern Vietnam. Based on phenotypic characteristics, 111 actinomycetes 34 were isolated from roots, stems and leaves of the host plants by using nine selective media. The isolated 35 actinomycetes were mainly recovered from stems (n=67, 60.4%), followed by roots (n=29, 26.1%) and leaves 36 (n=15, 13.5%). The isolates were accordingly assigned into 5 color categories of aerial mycelium, of which gray 37 is the most dominant (n=42, 37.8%), followed by white (n=33; 29.7%), yellow (n=25; 22,5%), red (n=8; 7.2%) 38 and green (n=3; 2.7%). Of the total endophytic actinomycetes tested, 38 strains (occupying 34.2%) showed 39 antimicrobial activity against at least one of nine tested microbes and, among them, 26 actinomycetes (68.4%) 40 revealed anthracycline-like antibiotics production. Analysis of 16S rRNA gene sequences deposited on GenBank 41 (NCBI) of the antibiotic-producing actinomycetes identified 3 distinct genera, including Streptomyces, 42 Microbacterium, and Nocardia, among which Streptomyces genus was the most dominant and represented 25 43 different species. Further genetic investigation of the antibiotic-producing actinomycetes found that 28 (73.7%) 44 and 11 (28.9%) strains possessed genes encoding polyketide synthase (pks) and nonribosomal peptide synthetase 45 (nrps), respectively. The findings in the present study highlighted endophytic actinomycetes from C. cassia Presl 46 which possessed broad-spectrum bioactivities with the potential for applications in the agricultural and 47 pharmaceutical sectors. 48 Keywords: Antimicrobial activity, anthracyclines, Cinnamomum cassia, endophytic actinomycetes, polyketide 49 synthase, nonribosomal peptide synthetase. 50 INTRODUCTION 51 For thousands of years, medicinal plants have widely been used as natural medicines in the treatment of 52 human diseases (Pan et al., 2014). Nevertheless, medicinal plants are also well known to be the hosts of 3 53 endophytic microorganisms (Qin et al., 2009; Golinska et al., 2015). Since this association has formed over the 54 long-term, endophytic microbes might acquire and develop specific genetic determinants to produce bioactive 55 compounds similar to those produced by plant hosts (Alvin et al., 2014; Golinska et al., 2015). Thus, endophytic 56 actinomycetes associated with traditionally used medicinal plants, especially in the tropics, could be a rich source 57 of functional metabolites (Golinska et al., 2015). In the literature, endophytic actinomycetes associated with 58 medicinal plants have shown the ability to synthesize many valuable bioactive compounds, including anticancer, 59 antiviral, antimicrobial, antifungal and antiparasitic agents (Nakashima et al., 2013; Zhang et al., 2014; Inahashi 60 et al., 2015; Tanvir et al., 2016; Zin et al., 2017). A number of the secondary metabolites are novel in structure 61 and have broad-spectrum bioactivities that could be potentially applicable in the pharmaceutical, medical and 62 agricultural sectors (Golinska et al., 2015; Matsumoto and Takahashi, 2017). 63 The diversity and bioactivity of endophytic actinomycetes are associated with the genetic background of 64 actinomycetes, plant species and geographic areas (Qin et al., 2009; Gohain et al., 2015; Golinska et al., 2015; 65 Matsumoto and Takahashi, 2017). Moreover, the distribution of the endophytic actinomycete population also 66 varies according to the different tissues of host plants (Qin et al., 2009; Gohain et al., 2015; Salam et al., 2017). 67 Recently, a number of novels and previously uncultured endophytic actinomycetes with diverse metabolic 68 pathways have been isolated through new isolation approaches and media (Qin et al., 2009; Tanvir et al., 2016; 69 Matsumoto and Takahashi, 2017). 70 Vietnam has been recognized as one of the tropical countries with a very high biodiversity of medicinal 71 plant species, accounting for approximately 11% of the 35.000 species of medicinal plants known worldwide 72 (Khanh et al., 2005). Unfortunately, little is known about the distribution, and the potential to produce secondary 73 metabolites, of endophytic actinomycetes associated with medicinal plants (Khieu et al., 2015; Salam et al., 2017). 74 Cinnamomum cassia Presl, a commonly used medicinal plant, is one such example. The present study clarified 75 the distribution and characterization of endophytic actinomycetes from Vietnamese C. cassia Presl. 76 Actinomycetes which were isolated were then examined for antimicrobial activity against microbial pathogens 77 and the production of anthracycline-like antibiotics. Furthermore, the presence of secondary metabolic 78 biosynthetic genes encoding for polyketide synthase (pks-I and pks-II) and nonribosomal peptide synthetase 79 (nrps) was also determined. 80 MATERIALS AND METHODS 81 Sample collection and isolation of endophytic actinomycetes from C. cassia Presl 4 82 The stem, root and leave segments from 5 C. cassia plants were randomly selected from different sites in 83 Hoa Binh province (20°47’21’’N; 105°21’20’’E) of northern Vietnam. The samples were separately placed in 84 plastic bags according to the different plant organs, transported to the laboratory and then processed within 4 85 hours after collection. The plant voucher specimens were identified as C. cassia Presl species by the Institute of 86 Ecology and Biological Resources, Vietnam Academy of Science and Technology. 87 Surface-sterilization of plant organs and the isolation of endophytic actinomycetes have been described 88 in previous studies (Qin et al., 2009; Li et al., 2012; Salam et al., 2017). Briefly, the plant organs were firstly 89 washed with sterile distilled water (dH2O), cut into small pieces (1 – 2 cm), surface-sterilized for 5 min by 15% 90 NaClO, rinsed in 2.0% Na2S2O3 for 2 min, and washed three times with dH2O. The pretreated samples were 91 immersed in 70% ethanol for 7 min, followed by triple washes with dH2O, then dried under laminar flow 92 conditions. Finally, for each plant organ, the sterilized segments were homogenized in sterile dH2O and used for 93 endophytic actinomycete isolation. The supernatants were spread onto nine different agar media previously 94 described (Qin et al., 2009; Li et al., 2012; Salam et al., 2017), including humic acid-vitamin B agar (HV), 95 raffinose-histidine agar (RA), tap water-yeast extract agar (TWYE), International Streptomyces Project 5 (ISP5), 96 trehalose-proline agar (TA), sodium succinate-asparagine agar (SA), starch agar (STA), citrate acid agar (CA) 97 and sodium propionate agar (SPA). All media were supplemented with filter-sterilized mixtures of nalidixic acid 98 (25 mg/mL), nystatin (50 mg/mL), and K2Cr2O7 (50 mg/mL) to inhibit the growth of bacteria and fungi. The 99 culture media plates were incubated at 30oC for 6 - 8 weeks. The experiments were performed in triplicate. 100 Apparent actinomycete colonies were rapidly picked up and streaked out