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