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 on ISP2 medium. Pure isolates were

101 recovered and stored in 15% glycerol at - 80°C.

102 Identification of actinomycetes based on morphological characteristics

103 Actinomycete isolates were tentatively classified by traditional identification methods, according to

104 morphological characteristics, color of aerial mycelium and pigmentation produced on ISP media (Shirling and

105 Gottlieb, 1966; Goodfellow and Haynes, 1984). Isolates were grown on ISP2 agar plates for morphological

106 feature analysis of spores and spore-chains using a BH2 light microscope (Olympus Corporation, Tokyo, Japan)

107 with a magnification of 100X or 400X and a scanning electron microscope JSM-5410 (JEOL, Tokyo, Japan) (Li

108 et al., 2009).

109 Evaluation of antimicrobial activity 5

110 All endophytic actinomycetes were evaluated for inhibitory activity against 9 microbes, including Gram-

111 negative bacteria (Escherichia coli ATCC 11105, Proteus vulgaris ATCC 49132, Pseudomonas aeruginosa

112 ATCC 9027, Salmonella enterica Typhimurium ATCC 14028 and Enterobacter aerogenes ATCC 13048); Gram-

113 positive bacteria (Sarcina lutea ATCC 9341, Bacillus cereus ATCC 11778 and methicillin-resistant

114 Staphylococcus epidermidis ATCC 35984 (MRSE)); and yeast (Candida albicans ATCC 10231), using the agar

115 well diffusion method (Holder and Boyce, 1994). All experiments were performed in triplicate.

116 16S rRNA gene sequencing and phylogenetic analysis

117 The genomic DNA extraction and amplification of 16S rRNA gene were performed as previously

118 described (Phi et al., 2010; Salam et al., 2017). PCR amplicons were purified and sent for sequencing at 1st

119 Base Laboratories Sdn. Bhd., Malaysia. For each strain, the 16S rRNA gene sequence was treated and blasted on

120 GenBank database using Blast tool (http://www.blast.ncbi.nlm/nihgov/Blast.cgi) for the identification of the

121 homology species. A neighbor-joining phylogenetic tree based on 16S RNA gene sequences was computed using

122 MEGA7 software (Tamura et al., 2013). The tree branch was supported with a bootstrap of 1000 replications. The

123 phylogenetic tree was rooted using Bacillus thuringiensis ATCC 10792 (GenBank accession number CP020754)

124 as an out-group.

125 Screening for secondary metabolic biosynthetic genes

126 Three sets of degenerate primers: A3F (5’-GCS TAC SYS ATS TAC ACS TCS GG-3’) and A7R (5’-SAS

127 GTC VCC SGT SCG GTA S-3’), K1F (5’-TSA AGT CSA ACA TCG GBC A-3’) and M6R (5’-CGC AGG TTS

128 CSG TAC CAG TA-3’), KSaF (5’-TSG CST GCT TGG AYG CSA TC-3’) and KSaR (5’-TGG AAN CCG CCG

129 AAB CCG CT-3’) were used for amplification of the nrps, pks-I and pks-II genes, respectively (Metsä-Ketelä et

130 al., 1999; Ayuso-Sacido and Genilloud, 2005). PCR compositions and amplification conditions were performed

131 as previously described (Salam et al., 2017).

132 Screening for anthracycline-producing actinomycetes

133 The potential for the production of anthracycline-like antibiotics in actinomycetes was screened using the

134 pigment formulating test described previously (Trease and Evans, 1996). The principle of the method is based on

135 the presence of an anthraquinone ring in the chemical composition where by the color of anthracycline

136 compounds will be changed from orange to purple when their pH levels are altered from acid to alkaline,

137 respectively. Briefly, actinomycete isolates were grown on ISP2 agar plates at 30oC for 5- 6 days. After that, two

138 wells of 6mm diameter were punched carefully on the culture plate. The first well was loaded with 25 µl of 1 N 6

139 HCl, while the second one was loaded with 25 µl of 2 N NaOH, then the plates were incubated at 30oC. The

140 change of color around the wells was observed after 10 – 30 min.

141 RESULTS

142 Distribution of endophytic actinomycetes from C. cassia Presl

143 On the basis of colony morphology and mycelium color, a total of 111 endophytic actinomycetes were

144 obtained on 9 selective media, among which strains were mainly retrieved from stems (n=67; 60.4%), followed

145 by roots (n=29; 26.1%) and leaves (n=15; 13.5%) (Fig. 1A). According to the isolation media, the greatest

146 proportion of endophytic actinomycetes was obtained on CA medium (n=28; 25.2%), and the lowest number

147 were isolated on HV medium (n=1; 0.9%) (Fig. 1B). Overall, four media CA, SA, TA and TWYE were generally

148 appropriate for the isolation of endophytic actinomycetes from C. cassia Presl, accounting for 77.5% of total the

149 isolates (n=86). All the strains were categorized into 5 out of 7 mycelium color series in which the grey color

150 group was the most prevalent (n=42; 37.8%), followed by white (n=33; 29.7%), yellow (n=25; 22.5%), red (n=8;

151 7.2%) and green (n=3; 2.7%) (Fig. 1C).

152 Antimicrobial activity of endophytes against microbial pathogens

153 Thirty-eight (34.2%) of the 111 isolates exhibited inhibitory activities against at least one of nine tested

154 microbes, and none of the actinomycetes showed bactericidal activity against E. aerogenes (Table 1). The

155 inhibitory effect against bacterial pathogens was highest for P. vulgaris (n=28; 73.7%), followed by B. cereus

156 (n=27; 71.1%), MRSE (n=26; 68.4%), S. lutea (n=25 of each; 65.8%), P. aeruginosa and E. coli (n=24; 63.2%),

157 and C. albicans (n=10; 26.3%), whereas the results were less for growth inhibition against S. Typhimurium (n=1;

158 2.6%).

159 Inhibitory patterns of endophytes against the pathogens were very diverse (Table 1), ranging from one to

160 eight microbes (Fig. 2). Nine isolates showed antagonistic activity against all three groups of pathogenic

161 microbes, notably, the HBQ19 isolate revealed remarkable inhibitory activity against eight microbes. Seventeen

162 isolates exhibited antimicrobial activities against six pathogens, among which 11 exhibited growth inhibition

163 against both Gram-negative/-positive bacteria, but no antimicrobial activity against C. albicans was recorded.

164 Meanwhile, six other isolates exhibited inhibitory activities against three pathogenic groups and none of all

165 isolates possessed antibacterial activity against P. aeruginosa. Thirteen isolates exhibited inhibitory effects

166 against two to five microbes, while seven isolates were active against only one microbe.

167 Biosynthesis of anthracycline-like compounds from antibiotics-producing actinomycetes 7

168 Various reports previously summarized that anthracycline antibiotics biosynthesized by actinomycetes

169 have numerously shown cytotoxicity against human tumors or tumor cell lines (Metsä-Ketelä et al., 2007; Zhang

170 et al., 2017). Therefore, anthracycline antibiotics from actinomycetes have been so far, one of the most effective

171 substances widely used in the treatment of many types of cancer (Minotti et al., 2004). The present study showed

172 that of the 38 antimicrobial-producing actinomycetes, 26 (68.4%) isolates possessed capacity for biosynthesis of

173 anthracycline-like antibiotics (Table 1). Among those, 17 isolates interestingly exhibited antimicrobial activity

174 against at least 3 tested microbes (Table 1).

175 Analysis of 16S rRNA gene sequence of antibiotic-producing actinomycetes

176 By analysis of 16S rRNA gene sequence, 38 antibiotic-producing actinomycetes could be classified into

177 three different genera, among which Streptomyces was the most common genus (n=36; 94.7%), followed by

178 Microbacterium (n=1; 2.6%) and Nocardia (n=1; 2.6%) (Table 1). Overall, comparison of 16S rRNA gene

179 sequences of endophytic actinomycetes and type strains show high similarity levels (96 – 100%). Accordingly,

180 phylogenetic tree analysis revealed a main cluster of Streptomyces genus and two monophyletic branches

181 corresponding to 2 rare actinomycetes genera (Fig. 3). Notably, among the Streptomyces cluster, isolates were

182 distributed in multiple branches. Moreover, 36 Streptomyces isolates could be assigned into 25 different species

183 (Table 1). All 16S rRNA gene sequences of 38 actinomycetes were deposited on the GenBank (NCBI) with

184 assigned accession numbers (Table 1).

185 Screening of biosynthetic genes of antibiotic-producing endophytes

186 Three common genes pks-I, pks-II and nrps coding enzymes involved in secondary metabolite-

187 biosynthesis have been widely proved to associate with the prediction of antibiotic biosynthetic pathways

188 (Ayuso-Sacido and Genilloud, 2005; Metsä-Ketelä et al., 2007). Investigation of three genes in 38 antibiotic-

189 producing actinomycetes found 28 isolates (73.7%) possessing pks-I and/or pks-II gene in which 17 isolates

190 (44.7%) hold both genes; 11 isolates (28.9%) out of 38 isolates harbored nrps gene (Table 1). Taken together, 4

191 isolates hold all three biosynthetic genes and were identified as 4 different species of Streptomyces genus of

192 which 3 inhibited the growth of at least 5 microbes.

193 DISCUSSION

194 The present study revealed considerable diversity of endophytic actinomycetes associated with the

195 medicinal plant C. cassia Presl collected from a mountainous region in Hoa Binh province of Vietnam. Analysis

196 of morphological features revealed a high proportion of 111 isolated actinomycetes from the C. cassia host plant.

197 In general, the distribution of isolated endophytic actinomycetes remarkably varies according to the geographic 8

198 region, plant species, specific plant tissues and isolation media (Qin et al., 2009; Janso and Carter, 2010; Kaewkla

199 and Franco, 2013; Gohain et al., 2015; Salam et al., 2017). For instance, a recent study reported the diversity of

200 endophytic actinomycetes in Dracaena cochinchinensis Lour from 4 distinct provinces of China and Vietnam

201 (Salam et al., 2017). The distribution of endophytic actinomycetes was different among the provinces in each

202 country and between the countries. In a large surveillance, Qin et al. (2009) obtained 2,174 endophytic

203 actinomycetes from nearly 90 plants in the tropical rain forest in Xishuangbanna of China (approximately 24

204 isolates/plant), while Janso and Carter (2010) isolated only 123 actinomycete strains from 113 plants of coastal

205 tropical forests in Papua New Guinea and Mborokua Islands (1 strain/plant). Another study from China, Li et al.

206 (2012) successfully isolated 228 endophytic actinomycetes from Artemisia annua plant.

207 Regarding the distribution of actinomycetes according to the plant tissues, we found the greatest number

208 of endophytic actinomycetes from the stems of C. cassia Presl (60.4%) The current findings were different from

209 other studies in which endophytic actinomycetes were mainly recovered from roots compared with stems and

210 leaves (Kaewkla and Franco, 2013; Gohain et al., 2015). This could be explained by different features of the

211 medicinal plants studied. Nevertheless, the proportion of actinomycetes exhibited board-spectrum antimicrobial

212 activity was the highest in the roots (90.9%), followed by the stems (70.0%) and the leaves of C. cassia (42.8%).

213 The proportion is significantly different between roots and leaves (p<0.03). Thus, stem and root of C. cassia Presl

214 are important sources for recovering valuable antibiotic-producing endophytic actinomycetes.

215 The media and reliable methodologies for the isolation of endophytes play a very important role in

216 recovering culturable actinomycetes (Qin et al., 2009; Kaewkla and Franco, 2013). In the present study,

217 endophytes were mainly recovered from the 4 isolated media CA, SA, TA and TWYE, which accounted for

218 77.5% of the total actinomycetes, suggesting that the four above media are mostly suitable for the isolation of

219 endophytic actinomycetes from C. cassia Presl tissues. On the contrary, other media such as RA, STA, ISP5 and

220 SPA were less suitable, particularly the HV medium contributing only 1 isolate. The results obtained in the

221 present study were different compared with previous studies where the HV and RA media were suitable for the

222 isolation of endophytic actinomycetes from other medicinal plants (Li et al., 2012; Kaewkla and Franco, 2013).

223 The differences can result from the different isolation methodologies and media used accordingly. These findings

224 suggest that the use of multiple media could be essential for increasing the number of isolated endophytic

225 actinomycetes and, in particular, for acquiring novel and/or rare strains (Qin et al., 2009; Kaewkla and Franco,

226 2013). 9

227 The antimicrobial activities were systematically evaluated for all of the isolated endophytic

228 actinomycetes. Twenty-seven (71.1%) of 38 antibiotic-producing actinomycetes showed antimicrobial activity

229 patterns against at least three microbes, suggesting that these endophytes could have broad-spectrum

230 antimicrobial activities. Besides, previous studies have led to theories suggesting that potential anti-tumor

231 substances synthesized by actinomycetes are largely due to anthracycline antibiotics (Igarashi et al., 2007;

232 Abdelfattah, 2008; Lu et al., 2017). Here, 26 (68.4%) of antibiotic-producing actinomycetes exhibited the

233 capability of producing anthracycline-like metabolites. Natural anthracycline antibiotics are reported to be

234 produced by the polyketide biosynthesis pathway. We also found high proportions of actinomycetes harboring

235 secondary metabolite biosynthetic genes pks (73.7%) and nrps (28.9%), compared with previous studies (Zhao et

236 al., 2011; Li et al., 2012; Salam et al., 2017). In fact, the polyketide synthases and nonribosomal peptide

237 synthetases consist of a highly-conserved modular enzymatic structure that is encoded by highly similar tandem

238 repeat sequences, spanning 600 - 700 bp for pks-II, and 1200 – 1400 bp for both pks-I and nrps genes (Metsä-

239 Ketelä et al., 1999; Ayuso-Sacido and Genilloud, 2005). In many cases, these repeated sequences cause some

240 difficulty for amplification by available primer pairs. This may explain why the pks-I sequence was detected in

241 only 19 of the 26 anthracycline-like metabolites producing isolates.

242 In agreement with global studies on actinomycetes from different plants, Streptomyces serves as the

243 dominant genus that accounts for 90% of the total isolates from the C. cassia Presl (Qin et al., 2009).

244 Nevertheless, other studies found less than 30% of endophytic actinomycetes are recovered from different

245 medicinal plants belonging to Streptomyces genus (Zhao et al., 2011; Li et al., 2012). All these findings suggest

246 that the genetic diversity of endophytic actinomycetes is strongly associated with specific plant species and thus,

247 endophytes could have a different capacity to acquire important hereditary features from the plant hosts. In the

248 present study, the Streptomyces genus was dominant, 25 distinct Streptomyces species were assigned, suggesting

249 the high diversity among isolates from this genus. The phylogenetic tree exhibited multiple-phylogenetic

250 branches supporting theories that the isolates have evolved from different ancestors. Importantly, the

251 Streptomyces genus accounts for about 70% of the natural products in the pharmaceutical market (Bull and Stach,

252 2007).

253 Since Cinnamomum species are mainly distributed in India, China and South-East Asia countries, recent

254 studies have focused on the study of isolation and antimicrobial activity of endophytes from the plant. For

255 instance, a study from Malaysia found that an endophytic fungus Phoma sp., isolated from Cinnamomum

256 mollissimum, exhibited antifungal activity and cytotoxicity against P388 cancer cells (Santiago et al. 2012). The 10

257 compound 5-hydroxyramulosin isolated from the Phoma fungus showed strong antifungal activity against

258 Aspergillus niger (IC50 of 1.56 µg/ml) and was cytotoxic against murine leukemia cells (IC50 of 2.10 µg/ml).

259 Another study has found a new endophytic actinomycete strain designated as Streptomyces rochei Ch1 from

260 Cinnamomum sp. in Cherapunji rainforest, North-East India (Joy and Banerjee 2015). Although the strain

261 exhibited broad-spectrum antibacterial activity against eight pathogens of both Gram-positive and Gram-negative

262 bacteria, nevertheless, bioactive compounds were not isolated yet. Similarly, a study from Philippines reported

263 that an endophytic fungus Fusarium sp. 2 isolated from Cinnamomum mercadoi possessed antimicrobial activity

264 against four different pathogenic bacteria (Marcellano et al. 2017). Recently, Vu et al (2018) isolated and

265 elucidated structures of 5 bioactive metabolites from endophytic Streptomyces cavourensis YBQ59 associated

266 with Cinnamomum sp. in Yen Bai, Vietnam. The compounds revealed not only remarkably antimicrobial activity

267 against MRSA, but also a strong cytotoxicity effect against human cancer cell lines (Vu et al. 2018).

268 In the last decade, many novel antibiotics have been isolated from endophytic actinomycetes, such as

269 munumbicins which have been isolated from a culture broth of Streptomyces sp. NRRL 30562 (Castillo et al.,

270 2002), alnumycin from Streptomyces sp. DSM 11575 (Bieber et al., 1998) , spoxazomicins from

271 Streptosporangium oxazolinicum K07-0460T (Inahashi et al., 2011), stenothricin and bagremycin from Nocardia

272 caishijiensis SORS 64b (Tanvir et al., 2016). Interestingly, some of the novel antibiotics exhibited strong effects

273 against multiple-drug resistant infectious pathogens (Castillo et al., 2002; Tanvir et al., 2016). In addition, novel

274 anthracycline-like antibiotics have been isolated from endophytic actinomycetes (Igarashi et al., 2007;

275 Abdelfattah, 2008; Lu et al., 2017). So far, clinical anti-tumor drugs produced by actinomycetes officially

276 released to the market consisting of doxorubicin, aclarubicin, daunomycin and doxorubicin are antibiotics

277 belonging to anthracyclines. The results from the present study highlight the potential for the isolation of novel

278 and valuable secondary metabolites from the actinomycetes in C. cassia Presl.

279 In conclusion, the present study is the first report about the distribution and several bioactivities of

280 endophytic actinomycetes associated with the medicinal plant C. cassia Presl, collected from Hoa Binh province,

281 Vietnam. Many of these endophytes displayed broad-spectrum antimicrobial activities, which implies a potential

282 for agricultural, pharmaceutical and medicinal applications. Further studies focus on the isolation and

283 determination of chemical structures of bioactive compounds produced by potential actinomycete strains.

284 Acknowledgements: This work is financially supported by the grant GUST.STS.ĐT2017-SH03,

285 Graduate University of Science and Technology, VAST. We thank the National Key Laboratory of Gene

286 Technology, Institute of Biotechnology (IBT) for supporting equipment. 11

287 Conflict of interest: The authors declare no competing interest.

288 Reference

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403

404 Tables and Figures in the main text

405 Figure 1. Distribution of 111 endophytic actinomycetes isolated according to: (A) the three different plant tissues,

406 (B) the isolation media selected (see materials and methods), and (C) the different mycelium color series.

407 Figure 2. Inhibitory patterns and number of endophytic actinomycetes against microbes

408 Figure 3. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences of 38 endophytic

409 actinomycetes showing the homology with closest type strain sequences. Bacillus thuringiensis strain ATCC

410 10792 was used as an out-group. Numbers at branches indicate bootstrap values in 1000 replications. Only

411 bootstrap values greater than 50% are shown in the tree. The bar represents the distance of 0.02 substitutions per

412 nucleotide.

413

414 Regular Table (Included in the main text)

415 Table 1. Classification, inhibitory effect against microbes, amplification of biosynthetic genes and production

416 capacity of anthracyclines-like antibiotics of 38 endophytic actinomycetes from the medicinal plant C. cassia

417 Presl 14

Activity against pathogenic microbes* Biosynthetic genes# Organ of the Gram-positive Anthracycline- No Type strain (GenBank Accession number) Gram-negative bacteria Yeast host plant bacteria like activity# Total 1 2 3 4 5 6 7 8 9 pks-I pks-II nrps Streptomyces graminisoli HBQ33 (KM207770) Root ++ ++ - - - ++ - ++ ++ + + - - 1 5 Streptomyces exfoliatus HBQ43 (MF796953) Root - - - - - + + - + - - - - 2 3 Streptomyces sulphureus HBQ62 (KR076805) Root + + - - - - - + - - - - + 3 3 Streptomyces fulvissimus HBQ75 (MF796970) Root + + - ++ - ++ + ++ - - + + + 4 6 Microbacterium resistens HBQ76 (KR076806) Root ++ + - +++ - + ++ ++ - - + + + 5 6 Streptomyces globisporus HBQ78 (MF796957) Root ++ + - ++ - + + + - - - - + 6 6 Streptomyces pratensis HBQ79 (MH388021) Root ++ + - ++ - + + ++ - - - - + 7 6 Streptomyces parvulus HBQ87 (KR076807) Root +++ +++ - +++ - ++ +++ ++ - + + + + 8 6 Streptomyces lienomycini HBQ90 (MF796965) Root + + - + - + ++ + - - - - + 9 6 Streptomyces cyaneofuscatus HBQ92 (MF796967) Root - - - + - - - - - + + + + 10 1 Streptomyces scabrisporus HBQ93 (MF796971) Root - ++ - ++ - ++ + ++ - - - - - 11 5 Streptomyces caniferus HBQ06 (KR076796) Stem - + - - - + + ++ + + + - - 12 4 Streptomyces tubercidicus HBQ07 (KR076797) Stem + + - - - + + + + + + - - 13 6 Streptomyces platensis HBQ08 (KR076798) Stem + + - - - + + + + + + - + 14 6 Streptomyces angustmyceticus HBQ09 (KR076799) Stem + + - - - + + + + + + - - 15 6 Stem + + - - - + + + + + + - - 16 Streptomyces platensis HBQ10 (KR076800) 6 Streptomyces nigrescens HBQ11 (KR076801) Stem + + - - - + + + + + + - - 17 6 Streptomyces bingchenggensis HBQ16 (KR076802) Stem ++ ------+ - - - - - 18 2 Streptomyces angustmyceticus HBQ19 (KM207769) Stem + ++ ++ ++ - ++ +++ ++ +++ + + - + 19 8 Streptomyces spongiae HBQ47 (MF796954) Stem + + - - - + + + + + + + - 20 6 Streptomyces platensis HBQ49 (KR076803) Stem - + - ++ - - - - - + - + + 21 2 15

Streptomyces albidoflavus HBQ55 (KR076804) Stem - + - + - - - - - + - + - 22 2 Streptomyces pratensis HBQ72 (MF796955) Stem - + - ++ - + + ++ - + + + - 23 5 Streptomyces chattanoogensis HBQ77 (MF796956) Stem ------+ - - + - + + 24 1 Streptomyces puniceus HBQ80 (MF796959) Stem ++ + - + - + + + - - - - + 25 6 Streptomyces bluensis HBQ81 (MF796960) Stem - - - + - - - - - + - - + 26 1 Streptomyces sannanensis HBQ82 (MF796961) Stem - - - + - - - + - + + - + 27 2 Nocardia jiangxiensis HBQ83 (MF796962) Stem - + - + - + ++ - - - - - + 28 4 Streptomyces fulvissimus HBQ91 (MF796966) Stem + + - ++ - + + + - - + + + 29 6 Streptomyces pratensis HBQ102 (MF796968) Stem ++ ++ - ++ - - ++ + - - + - + 30 5 Streptomyces coelicoflavus HBQ109 (MF796969) Stem + ++ - + - ++ - + - + + - + 31 5 Streptomyces cavourensis HBQ84 (MF796963) Leaf - - - + - - - - - + + - + 32 1 Streptomyces globisporus HBQ86 (MF796964) Leaf + ------+ 33 1 Streptomyces parvus HBQ94 (MF807158) Leaf - - - ++ ------+ - + 34 1 Streptomyces puniceus HBQ95 (MH388020) Leaf ------+ - - + - - + 35 1 Streptomyces ribosidificus HBQ104 (MF796972) Leaf + ++ - ++ - + ++ ++ - + + - + 36 6 Streptomyces cyaneofuscatus HBQ106 (KR076810) Leaf + ++ - + - ++ ++ ++ - - + + + 37 6 Streptomyces californicus HBQ107 (KR076811) Leaf + ++ - ++ - + + + - + + - + 38 6 Total 24 28 1 24 0 25 26 27 10 22 23 11 26 39 418 * Microbes: (1) Escherichia coli ATCC 11105; (2) Proteus vulgaris ATCC 49132; (3) Salmonella enterica Typhimurium ATCC 14028; (4) Pseudomonas aeruginosa ATCC 419 9027; (5) Enterobacter aerogenes ATCC 13048; (6) Sarcina lutea ATCC 9341; (7) methicillin-resistant Staphylococcus epidermidis ATCC 35984; (8) Bacillus cereus ATCC 420 11778; (9) Candida albicans ATCC 10231. 421 Antimicrobial activity: (-) negative inhibition, (+) positive inhibition; width of growth inhibition zone: +++ > 20 mm, ++ = 10 - 20 mm, + < 10 mm.

422 # PCR amplification of biosynthetic genes/anthracyclines-like antibiotic activity: a positive result (+); a negative result (-). A 15 30 28 29 B

25

20 20 20 18

15 11 10

Number of isolates of Number 5 5 67 5 3 1 0 CA TA TWYE SA RA ISP5 STA SPA HV roots stems leaves Isolation medium

45 42 40 35 33 C 30 25 25 20 15 10 8 5 3 Figure 1 actinomycetes of Number 0 gray white yellow red green

Mycelium color 18 17

16

14

12

10

8 7

6

5 Number of actinomycetes of Number 4 4

2 2 2 1

0 1 microbe 2 microbes 3 microbes 4 microbes 5 microbes 6 microbes 8 microbes

Number of microbes inhibited

Figure 2