1    

Advance Publication

J. Gen. Appl. Microbiol.

doi 10.2323/jgam.2019.04.004

©2019 Applied Microbiology, Molecular and Cellular Biosciences Research Foundation

Title: Endophytic actinomycetes associated with Cinnamomum cassia Presl in Hoa Binh province, Vietnam: 1  

Distribution, antimicrobial activity and, genetic features 2  

Running title: Endophytic actinomycetes in Cinnamomum 3  

(Received January 9, 2019; Accepted April 16, 2019; J-STAGE Advance publication date: August 2, 2019) 4  

Thi Hanh Nguyen Vu1¶, Quang Huy Nguyen2,3,1¶, Thi My Linh Dinh1, Ngoc Tung Quach1, Thi Nhan Khieu4, 5  

Ha Hoang1, Son Chu-Ky5, Thu Trang Vu5, Hoang Ha Chu1,2, Jusung Lee6, Heonjoong Kang6,7, Wen-Jun 6  

Li8 and Quyet-Tien Phi1,2* 7  

8  

1Institute of Biotechnology (IBT), Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, 9  

Cau Giay, 10000 Hanoi, Vietnam 10  

2Graduate University of Science and Technology (GUST), Vietnam Academy of Science and Technology 11  

(VAST), 18 Hoang Quoc Viet, Cau Giay, 10000 Hanoi, Vietnam 12  

3University of Science and Technology of Hanoi (USTH), Vietnam Academy of Science and Technology 13  

(VAST), 18 Hoang Quoc Viet, Cau Giay, 10000 Hanoi, Vietnam 14  

4Department of Science, Technology and Environment, Ministry of Education and Training, 49 Dai Co Viet, Hai 15  

Ba Trung, 10000 Hanoi, Vietnam 16  

5School of Biotechnology and Food Technology (SBFT), Hanoi University of Science and Technology (HUST), 17  

1 Dai Co Viet, Hai Ba Trung, 10000 Hanoi, Vietnam 18  

6The Center for Marine Natural Products and Drug Discovery, School of Earth and Environmental Sciences, 19  

College of Natural Sciences, Seoul National University NS-80, Seoul 08826, Korea 20  

7Research Institute of Oceanography, Seoul National University NS-80, Seoul 08826, Korea 21  

8State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, School of 22  

Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China 23  

2     ¶: These authors contributed equally to this work. 24  

*Corresponding author: Quyet-Tien Phi, Institute of Biotechnology, Vietnam Academy of Science and 25  

Technology (VAST), 18 Hoang Quoc Viet, Cau Giay, 10000 Hanoi, Vietnam. Phone: (+84) 02437917973, Fax: 26  

(+84) 024.38363144, Email: tienpq@ibt.ac.vn; qtien.phi@gmail.com. 27  

 28  

SUMMARY 29  

Endophytic microbes associated with medicinal plants are considered to be potential producers of various 30  

bioactive secondary metabolites. The present study investigated the distribution, antimicrobial activity and 31  

genetic features of endophytic actinomycetes isolated from the medicinal plant Cinnamomum cassia Presl 32  

collected in Hoa Binh province of northern Vietnam. Based on phenotypic characteristics, 111 actinomycetes 33  

were isolated from roots, stems and leaves of the host plants by using nine selective media. The isolated 34  

actinomycetes were mainly recovered from stems (n=67, 60.4%), followed by roots (n=29, 26.1%) and leaves 35  

(n=15, 13.5%). The isolates were accordingly assigned into 5 color categories of aerial mycelium, of which gray 36  

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%) 37  

and green (n=3; 2.7%). Of the total endophytic actinomycetes tested, 38 strains (occupying 34.2%) showed 38  

antimicrobial activity against at least one of nine tested microbes and, among them, 26 actinomycetes (68.4%) 39  

revealed anthracycline-like antibiotics production. Analysis of 16S rRNA gene sequences deposited on GenBank 40  

(NCBI) of the antibiotic-producing actinomycetes identified 3 distinct genera, including Streptomyces, 41  

Microbacterium, and Nocardia, among which Streptomyces genus was the most dominant and represented 25 42  

different species. Further genetic investigation of the antibiotic-producing actinomycetes found that 28 (73.7%) 43  

and 11 (28.9%) strains possessed genes encoding polyketide synthase (pks) and nonribosomal peptide synthetase 44  

(nrps), respectively. The findings in the present study highlighted endophytic actinomycetes from C. cassia Presl 45  

which possessed broad-spectrum bioactivities with the potential for applications in the agricultural and 46  

pharmaceutical sectors. 47  

Keywords: Antimicrobial activity, anthracyclines, Cinnamomum cassia, endophytic actinomycetes, polyketide 48  

synthase, nonribosomal peptide synthetase. 49  

INTRODUCTION 50  

For thousands of years, medicinal plants have widely been used as natural medicines in the treatment of 51  

human diseases (Pan et al., 2014). Nevertheless, medicinal plants are also well known to be the hosts of 52  

3    

endophytic microorganisms (Qin et al., 2009; Golinska et al., 2015). Since this association has formed over the 53  

long-term, endophytic microbes might acquire and develop specific genetic determinants to produce bioactive 54  

compounds similar to those produced by plant hosts (Alvin et al., 2014; Golinska et al., 2015). Thus, endophytic 55  

actinomycetes associated with traditionally used medicinal plants, especially in the tropics, could be a rich source 56  

of functional metabolites (Golinska et al., 2015). In the literature, endophytic actinomycetes associated with 57  

medicinal plants have shown the ability to synthesize many valuable bioactive compounds, including anticancer, 58  

antiviral, antimicrobial, antifungal and antiparasitic agents (Nakashima et al., 2013; Zhang et al., 2014; Inahashi 59  

et al., 2015; Tanvir et al., 2016; Zin et al., 2017). A number of the secondary metabolites are novel in structure 60  

and have broad-spectrum bioactivities that could be potentially applicable in the pharmaceutical, medical and 61  

agricultural sectors (Golinska et al., 2015; Matsumoto and Takahashi, 2017). 62  

The diversity and bioactivity of endophytic actinomycetes are associated with the genetic background of 63  

actinomycetes, plant species and geographic areas (Qin et al., 2009; Gohain et al., 2015; Golinska et al., 2015; 64  

Matsumoto and Takahashi, 2017). Moreover, the distribution of the endophytic actinomycete population also 65  

varies according to the different tissues of host plants (Qin et al., 2009; Gohain et al., 2015; Salam et al., 2017). 66  

Recently, a number of novels and previously uncultured endophytic actinomycetes with diverse metabolic 67  

pathways have been isolated through new isolation approaches and media (Qin et al., 2009; Tanvir et al., 2016; 68  

Matsumoto and Takahashi, 2017). 69  

Vietnam has been recognized as one of the tropical countries with a very high biodiversity of medicinal 70  

plant species, accounting for approximately 11% of the 35.000 species of medicinal plants known worldwide 71  

(Khanh et al., 2005). Unfortunately, little is known about the distribution, and the potential to produce secondary 72  

metabolites, of endophytic actinomycetes associated with medicinal plants (Khieu et al., 2015; Salam et al., 2017). 73  

Cinnamomum cassia Presl, a commonly used medicinal plant, is one such example. The present study clarified 74  

the distribution and characterization of endophytic actinomycetes from Vietnamese C. cassia Presl. 75  

Actinomycetes which were isolated were then examined for antimicrobial activity against microbial pathogens 76  

and the production of anthracycline-like antibiotics. Furthermore, the presence of secondary metabolic 77  

biosynthetic genes encoding for polyketide synthase (pks-I and pks-II) and nonribosomal peptide synthetase 78  

(nrps) was also determined. 79  

MATERIALS AND METHODS 80  

Sample collection and isolation of endophytic actinomycetes from C. cassia Presl 81  

4    

The stem, root and leave segments from 5 C. cassia plants were randomly selected from different sites in 82  

Hoa Binh province (20°47’21’’N; 105°21’20’’E) of northern Vietnam. The samples were separately placed in 83  

plastic bags according to the different plant organs, transported to the laboratory and then processed within 4 84  

hours after collection. The plant voucher specimens were identified as C. cassia Presl species by the Institute of 85  

Ecology and Biological Resources, Vietnam Academy of Science and Technology. 86  

Surface-sterilization of plant organs and the isolation of endophytic actinomycetes have been described 87  

in previous studies (Qin et al., 2009; Li et al., 2012; Salam et al., 2017). Briefly, the plant organs were firstly 88  

washed with sterile distilled water (dH2O), cut into small pieces (1 – 2 cm), surface-sterilized for 5 min by 15% 89  

NaClO, rinsed in 2.0% Na2S2O3 for 2 min, and washed three times with dH2O. The pretreated samples were 90  

immersed in 70% ethanol for 7 min, followed by triple washes with dH2O, then dried under laminar flow 91  

conditions. Finally, for each plant organ, the sterilized segments were homogenized in sterile dH2O and used for 92  

endophytic actinomycete isolation. The supernatants were spread onto nine different agar media previously 93  

described (Qin et al., 2009; Li et al., 2012; Salam et al., 2017), including humic acid-vitamin B agar (HV), 94  

raffinose-histidine agar (RA), tap water-yeast extract agar (TWYE), International Streptomyces Project 5 (ISP5), 95  

trehalose-proline agar (TA), sodium succinate-asparagine agar (SA), starch agar (STA), citrate acid agar (CA) 96  

and sodium propionate agar (SPA). All media were supplemented with filter-sterilized mixtures of nalidixic acid 97  

(25 mg/mL), nystatin (50 mg/mL), and K2Cr2O7 (50 mg/mL) to inhibit the growth of bacteria and fungi. The 98  

culture media plates were incubated at 30oC for 6 - 8 weeks. The experiments were performed in triplicate. 99  

Apparent actinomycete colonies were rapidly picked up and streaked out on ISP2 medium. Pure isolates were 100  

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

Identification of actinomycetes based on morphological characteristics 102  

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

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

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

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

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

et al., 2009). 108  

Evaluation of antimicrobial activity 109  

5    

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

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

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

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

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

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

16S rRNA gene sequencing and phylogenetic analysis 116  

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

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

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

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

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

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

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

as an out-group. 124  

Screening for secondary metabolic biosynthetic genes 125  

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

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

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

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

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

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

Screening for anthracycline-producing actinomycetes 132  

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

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

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

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

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

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

6    

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

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

RESULTS 141  

Distribution of endophytic actinomycetes from C. cassia Presl 142  

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

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

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

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

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

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

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

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

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

Antimicrobial activity of endophytes against microbial pathogens 152  

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

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

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

(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%), 156  

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

2.6%). 158  

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

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

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

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

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

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

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

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

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

7    

Various reports previously summarized that anthracycline antibiotics biosynthesized by actinomycetes 168  

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

assigned accession numbers (Table 1). 184  

Screening of biosynthetic genes of antibiotic-producing endophytes 185  

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

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

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

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

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

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

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

DISCUSSION 193  

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

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

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

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

8    

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

2013). 226  

9    

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

2007). 252  

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

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

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

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

10    

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

11    

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

Reference 288  

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403  

Tables and Figures in the main text 404  

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

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

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

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

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

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

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

nucleotide. 412  

413  

Regular Table (Included in the main text) 414  

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

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

Presl 417  

14    

No Type strain (GenBank Accession number) Organ of the host plant

Activity against pathogenic microbes*

Biosynthetic genes# Anthracycline-

like activity# Gram-negative bacteria Gram-positive

bacteria Yeast Total

1 2 3 4 5 6 7 8 9 pks-I pks-II nrps

1 Streptomyces graminisoli HBQ33 (KM207770) Root ++ ++ - - - ++ - ++ ++ 5 + + - -

2 Streptomyces exfoliatus HBQ43 (MF796953) Root - - - - - + + - + 3 - - - -

3 Streptomyces sulphureus HBQ62 (KR076805) Root + + - - - - - + - 3 - - - +

4 Streptomyces fulvissimus HBQ75 (MF796970) Root + + - ++ - ++ + ++ - 6 - + + +

5 Microbacterium resistens HBQ76 (KR076806) Root ++ + - +++ - + ++ ++ - 6 - + + +

6 Streptomyces globisporus HBQ78 (MF796957) Root ++ + - ++ - + + + - 6 - - - +

7 Streptomyces pratensis HBQ79 (MH388021) Root ++ + - ++ - + + ++ - 6 - - - +

8 Streptomyces parvulus HBQ87 (KR076807) Root +++ +++ - +++ - ++ +++ ++ - 6 + + + +

9 Streptomyces lienomycini HBQ90 (MF796965) Root + + - + - + ++ + - 6 - - - +

10 Streptomyces cyaneofuscatus HBQ92 (MF796967) Root - - - + - - - - - 1 + + + +

11 Streptomyces scabrisporus HBQ93 (MF796971) Root - ++ - ++ - ++ + ++ - 5 - - - -

12 Streptomyces caniferus HBQ06 (KR076796) Stem - + - - - + + ++ + 4 + + - -

13 Streptomyces tubercidicus HBQ07 (KR076797) Stem + + - - - + + + + 6 + + - -

14 Streptomyces platensis HBQ08 (KR076798) Stem + + - - - + + + + 6 + + - +

15 Streptomyces angustmyceticus HBQ09 (KR076799) Stem + + - - - + + + + 6 + + - -

16 Streptomyces platensis HBQ10 (KR076800) Stem + + - - - + + + + 6 + + - -

17 Streptomyces nigrescens HBQ11 (KR076801) Stem + + - - - + + + + 6 + + - -

18 Streptomyces bingchenggensis HBQ16 (KR076802) Stem ++ - - - - - - + - 2 - - - -

19 Streptomyces angustmyceticus HBQ19 (KM207769) Stem + ++ ++ ++ - ++ +++ ++ +++ 8 + + - +

20 Streptomyces spongiae HBQ47 (MF796954) Stem + + - - - + + + + 6 + + + -

21 Streptomyces platensis HBQ49 (KR076803) Stem - + - ++ - - - - - 2 + - + +

15    

22 Streptomyces albidoflavus HBQ55 (KR076804) Stem - + - + - - - - - 2 + - + -

23 Streptomyces pratensis HBQ72 (MF796955) Stem - + - ++ - + + ++ - 5 + + + -

24 Streptomyces chattanoogensis HBQ77 (MF796956) Stem - - - - - - + - - 1 + - + +

25 Streptomyces puniceus HBQ80 (MF796959) Stem ++ + - + - + + + - 6 - - - +

26 Streptomyces bluensis HBQ81 (MF796960) Stem - - - + - - - - - 1 + - - +

27 Streptomyces sannanensis HBQ82 (MF796961) Stem - - - + - - - + - 2 + + - +

28 Nocardia jiangxiensis HBQ83 (MF796962) Stem - + - + - + ++ - - 4 - - - +

29 Streptomyces fulvissimus HBQ91 (MF796966) Stem + + - ++ - + + + - 6 - + + +

30 Streptomyces pratensis HBQ102 (MF796968) Stem ++ ++ - ++ - - ++ + - 5 - + - +

31 Streptomyces coelicoflavus HBQ109 (MF796969) Stem + ++ - + - ++ - + - 5 + + - +

32 Streptomyces cavourensis HBQ84 (MF796963) Leaf - - - + - - - - - 1 + + - +

33 Streptomyces globisporus HBQ86 (MF796964) Leaf + - - - - - - - - 1 - - - +

34 Streptomyces parvus HBQ94 (MF807158) Leaf - - - ++ - - - - - 1 - + - +

35 Streptomyces puniceus HBQ95 (MH388020) Leaf - - - - - - + - - 1 + - - +

36 Streptomyces ribosidificus HBQ104 (MF796972) Leaf + ++ - ++ - + ++ ++ - 6 + + - +

37 Streptomyces cyaneofuscatus HBQ106 (KR076810) Leaf + ++ - + - ++ ++ ++ - 6 - + + +

38 Streptomyces californicus HBQ107 (KR076811) Leaf + ++ - ++ - + + + - 6 + + - +

39 Total 24 28 1 24 0 25 26 27 10 22 23 11 26

* Microbes: (1) Escherichia coli ATCC 11105; (2) Proteus vulgaris ATCC 49132; (3) Salmonella enterica Typhimurium ATCC 14028; (4) Pseudomonas aeruginosa ATCC 418  9027; (5) Enterobacter aerogenes ATCC 13048; (6) Sarcina lutea ATCC 9341; (7) methicillin-resistant Staphylococcus epidermidis ATCC 35984; (8) Bacillus cereus ATCC 419  11778; (9) Candida albicans ATCC 10231. 420  

Antimicrobial activity: (-) negative inhibition, (+) positive inhibition; width of growth inhibition zone: +++ > 20 mm, ++ = 10 - 20 mm, + < 10 mm. 421  # PCR amplification of biosynthetic genes/anthracyclines-like antibiotic activity: a positive result (+); a negative result (-). 422  

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gray white yellow red green

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Figure 1

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

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Number of microbes inhibited

Figure 2

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Typewriter

Figure 3.