rare actinobacteria from medicinal plants of tropic al rainforests
TRANSCRIPT
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Rare actinobacteria from medicinal plants of tropical rainforests, 1
Xishuangbanna: isolation, diversity and antimicrobial activity 2
Sheng Qin1, Jie Li
1, Hua-Hong Chen
1, 2, Guo-Zhen Zhao
1, Wen-Yong Zhu
1, 3
Cheng-Lin Jiang 1
, Li-Hua Xu1, Wen-Jun Li
1* 4
1. The Key Laboratory for Microbial Resources of the Ministry of Education, P. R. 5
China, and Laboratory for Conservation and Utilization of Bio-Resources, Yunnan 6
Institute of Microbiology, Yunnan University, Kunming, 650091, P. R. China 7
2. Department of Chemistry, Chuxiong Normal College, Chuxiong, Yunnan, 675000, P. 8
R. China 9
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*Author for correspondence: Wen-Jun Li 11
Tel & Fax: +86 871 5033335 12
E-Mail: [email protected] ; [email protected] 13
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Running title: Rare actinobacteria from medicinal plants of tropical rainforests 16
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Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.01034-09 AEM Accepts, published online ahead of print on 31 July 2009
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Abstract 23
Endophytic actinobacteria are relatively unexplored as potential sources of novel 24
species and novel natural products for medical and commercial exploitation. 25
Xishuangbanna is well recognized in the world for its diverse flora, especially the 26
rainforest plants, many of which have indigenous pharmaceutical history. However, 27
little is known about the endophytic actinobacteria from this tropical area. In this 28
work, we studied the diversity of actinobacteria isolated from medicinal plants 29
collected from tropical rainforests in Xishuangbanna. Due to the use of different 30
selective isolation media and methods, a total of 2174 actinobacteria were isolated. 46 31
isolates were selected on the basis of their morphology on different media and were 32
further characterized by 16S rRNA gene sequencing. The results showed that they 33
represent an unexpected variety of 32 different genera. To our knowledge, this is the 34
first report describing isolation of Saccharopolyspora, Dietzia, Blastococcus, 35
Dactylosporangium, Promicromonospora, Oerskovia, Actinocorallia and Jiangella 36
species from the endophytic environments. At least 19 isolates are considered as novel 37
taxa by our current research. In addition, all of the 46 isolates were tested for 38
antimicrobial activity and screened for the presence of genes encoding polyketide 39
synthetases (PKS) and nonribosomal peptide synthetases (NRPS). These results 40
confirm that medicinal plants from Xishuangbanna represent an extremely rich 41
reservoir for the isolation of significant diversity of actinobacteria and novel species 42
which are potential sources for discovery of biologically active compounds. 43
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Keywords: Actinobacteria, Endophytic, Tropical rainforests, Medicinal plants, 45
Diversity 46
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Introduction 51
The class Actinobacteria present a high proportion of soil microbial biomass and 52
contain the most economically significant prokaryotes, producing over half of the 53
bioactive compounds in the Antibiotic Literature Database (47), notably antibiotics 54
(7), immunosuppressive agents (57), antitumor agents (20), and enzymes (65), 55
especially those belonging to the genus Streptomyces, which are the excellent 56
producers. The advent of drug resistance in many bacterial pathogens and the current 57
increase in the number of fungal infections has caused a resurgence of interest in 58
finding other reserves of biologically active compounds (64). As the search for novel 59
natural products continues, it becomes apparent that the rate of discovery of new 60
compounds from soil streptomycetes has decreased, whereas the rate of re-isolation of 61
known compounds has increased (29). Recent evidence is being accumulated that rare 62
actinomycete species, which are often very difficult to isolate and cultivate, might 63
represent a unique source of novel biologically active compounds (5). On the other 64
hand, new microbial habitats need to be examined, in the search for novel bioactive 65
compounds. One biologically important but relatively overlooked niche is the inner 66
tissues of higher plants. Early studies have demonstrated that some actinobacteria can 67
form intimate associations with plants and colonize their inner tissues. Frankia 68
species and Streptomyces scabies can penetrate their host and establish either 69
pathogenic or endophytic associations (6, 25). The actinomycete bacteria that reside 70
in the tissue of living plants and do not visibly harm the plants are known as 71
endophytic actinobacteria (37). These actinobacteria are relatively unstudied and are 72
potential sources of novel natural products for exploitation in medicine, agriculture, 73
and industry (74). 74
Endophytic actinobacteria have attracted attention in recent years, with increasing 75
reports of isolates from a range of plant types, including crop plants, cereals, such as 76
wheat, rice, potato, carrots, tomato and citrus (3, 18, 63, 72, 75, 81) and medicinal 77
plants (76, 89) The culturable endophytic actinobacteria from these plants were found 78
to fall within a narrow species distribution, with Streptomyces spp. as the most 79
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predominant species and Microbispora, Micromonospora, Nocardioides, Nocardia 80
and Streptosporangium as the common genera. Endophytic actinobacteria have been 81
demonstrated to improve and promote growth of host plants as well as to reduce 82
disease symptoms caused by plant pathogens through various mechanisms, including 83
the production of secondary metabolites, which are used in direct antagonism against 84
pests and diseases (10, 11, 13), changes in host physiology (42) and induction of 85
systemic acquired resistance in plants (17). Another significant function of these 86
actinobacteria was found to exhibit antibiotic activity, suggesting that endophytic 87
actinobacteria can be an interesting source for bioprospecting. New antibiotics 88
Alnumycin, Munumbacin A-D and Coronamycin have been reported from endophytic 89
Streptomyces spp. (8, 12). Recently, two novel antitumor anthraquinones, lupinacidins 90
A and B were isolated from a new endophytic Micromonospora sp. (43). Moreover, 91
new species of endophytic actinobacteria have been increasingly reported (26, 35). 92
Thus, endophytic actinobacteria are expected to be potential sources of new species 93
and new bioactive agents. 94
Of the myriad of ecosystems on earth, those having the greatest general 95
biodiversity of life seem to be the ones also having the greatest number and most 96
diverse endophytes (74). Tropical and temperate rainforests are the most biologically 97
diverse terrestrial ecosystems on earth that hold the greatest possible resource for 98
acquiring novel microorganisms and their products (74). One of the areas of 99
enormous plant biodiversity is Xishuangbanna, located in P. R. China at the boarder 100
to Myanmar, at the ecotone between the Asian tropics and subtropics, and is 101
dominated by tropical seasonal rain forests (88). It contains over 5000 species of 102
vascular plants, comprising 16 percent of China’s total plant diversity, with more than 103
3000 being endemic species (54, 61), many of which have ethnobotanical history. 104
Until this time, only few researches were carried out on the isolation of endophytic 105
actinobacteria and their secondary metabolites from Xishuangbanna (36, 87). As our 106
long term study on endophytic actinobacterial diversity and bioactive metabolites 107
from tropical rainforest medicinal plants in Xishuangbanna, many bioactive 108
endophytic Streptomyces have been isolated (50). However, this is insufficient to 109
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provide a general understanding of tropical rainforest endophytic actinobacteria 110
regarding to their diversity, distribution, and ecology, as well as for further 111
exploitation of diverse functions of this novel microbial source. 112
In the present study, the diversity of rare endophytic actinobacteria associated 113
with medicinal plants from tropical rainforest in Xishuangbanna was investigated by 114
combined special culturing techniques. The selected isolates were also identified by 115
16S rRNA gene analysis. The overall aims of this study were (i) to analyze 116
actinobacterial community and reveal whether the investigated rainforest in 117
Xishuangbanna represents a valuable source for abundant endophytic actinobacteria 118
and new species, (ii) to evaluate their antimicrobial activities and biosynthetic 119
potential of related secondary metabolites, and (iii) to study the relationships between 120
taxa of these endophytic actinobacteria and applied isolation methods. 121
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Materials and methods 123
Sample collection 124
An extensive collection of various plant materials was set up in the tropical 125
seasonal rainforest in Xishuangbanna for two years (from November 2005 to May 126
2006 and from September 2006 to June 2007) in the Menglun Nature Reserve 127
(21◦56’N, 101
◦11’S) and Jinghong Nature Reserve (22
◦01’-22
◦19’N, 100
◦47’ 128
-100◦57’S ). Each plant selected for endophyte isolation was based on its local 129
ethnobotanical properties including its properties to antibacterial, insecticidal, 130
antitumour, heal wounds and others. No history indicates that the plants had ever been 131
previously studied for endophytic actinobacteria. Healthy root, stem and leaf samples 132
of each plant were placed in sterile plastic bags and taken to the laboratory, which 133
were subjected to isolation within 96 h. Some representative plants samples of the 134
nearly 90 selected for study are as follows: Phyllanthus urinaria, Kadsura heteroclite, 135
Maesa indica, Rauvolfia verticillata, Paris yunnanensis, Maytenus austroyunnanensis, 136
Gloriosa superba, Scoparia dulcis, Tadehagi triquetrum, Goniothalamus sp., 137
Cephalotaxus sp., and Azadirachta sp. 138
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Selective isolation producers and media 140
Samples were air dried for 48 h at room temperature and then washed with an 141
ultrasonic step (160W, 15mim) to remove the surface soils and adhered epiphytes 142
thoroughly. After drying, the samples were subjected to a five-step surface 143
sterilization procedure: a 4 to 10-min wash in 5% NaOCl, followed by a 10-min wash 144
in 2.5% Na2S2O3, a 5-min wash in 75% ethanol, then wash in sterile water, and a final 145
rinse in 10% NaHCO3 for 10-min. After thoroughly drying in sterile conditions, the 146
surface-sterilized tissues were continuously subjected to drying at 100 ºC for 15 147
minutes. Surface treated tissues were then pretreated by a variety of methods 148
described below. 149
Method 1. Most of the samples were aseptically crumbled into small fragments and 150
directly placed on the selective media and incubated at 28 ºC for 2-8 weeks. 151
Method 2. Some samples (1.0 g) treated as above were mixed in a mortar with 0.5 g 152
of sterile powdered calcium carbonate and placed in a Petri dish. 2 ml of sterilized tap 153
water was added to the samples to give a moisture environment. The Petri dish was 154
maintained at 28 ºC for 2 weeks and then air-dried at room temperature to a constant 155
weight. Parts of the samples were directly diluted to 10-4
with sterile water and 156
spread-plated onto media. The other mixtured samples were continuously enriched 157
according to Otoguro et al. (66). Samples were placed in a glass vessel and flooded 158
with 50 ml of 10 mmol-1
phosphate buffer (pH 7.0) containing 10% plant extract or 159
soil extract at 28 ºC for 5 h to liberate actinomycete spores. A portion (10 ml) of the 160
flooding mixture was transferred into a centrifuge tube and centrifuged at 1500×g for 161
30 min. After settling for 30 min, a portion of the supernatant enriched with spores 162
was serially diluted with sterile tap water and plated onto media. 163
Method 3. Some plants, e.g. Maytenus austroyunnanensis, were aseptically 164
crumbled into small fragments and isolated using the combined enzymatic hydrolysis 165
and differential centrifugation method (44). The homogenate was centrifuged at 200 g 166
for 20 min at 4 ºC, and the supernatant was subsequently centrifuged at 3000 g for 30 167
min(4 ºC) to collect the sediments, which will be diluted to 10-2
with sterile water and 168
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spread-plated onto media. 169
200 µl each of the samples pretreated dilutions was spread over the surface of solid 170
media that were designed for cultivation of actinobacteria (Table 1). The inoculated 171
plates were incubated at 28 ºC for 2–4 weeks. The pH of all media was adjusted to 172
7.0-7.4. All media were amended with nalidixic acid (50 mg l-1
) and nystatin (100 mg 173
l-1
) to supress the Gram-negative bacterial and fungal gowth. As the colonies appeared 174
from the plates, they were observed and selected carefully according to their 175
characteristics. Special attention was given to the non-mycelium forming 176
actinobacteria. They were inoculated onto yeast extract-malt extract agar (ISP 2) 177
slants and incubated at 28 ºC for 2–3 weeks. 178
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Effectiveness of surface sterilization 180
Two experiments were carried out to check the validation of the sterilization 181
procedures. Firstly, the surface-sterilized tissue was imprinted onto the ISP 2 agar, 182
incubated at 28 ºC, then check for microbial growth. Secondly, the surface sterilized 183
samples were washed in sterile distilled water thrice and then soaked in 5ml sterile 184
water and stirred for 1min. An aliquot of 0.2ml suspension was then inoculated on to 185
ISP 2 agar plates, incubated at 28 ºC, and observed for microbial growth. If no 186
microbial growth occurs on the media surface, the sterilization is considered 187
complete. 188
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Preliminary identification of actinobacteria 190
Isolates were tentatively grouped and dereplicated by observing the morphological 191
and cultural characteristics, including the characteristics of colonies on the plates and 192
slants, the presence of aerial mycelium and substrate mycelium, spora mass colour, 193
distinctive reverse colony colour, diffusible pigment, and sporophore and spore chain 194
morphology. Many colonies that were similar in colour, shape and size, were 195
observed for hyphal length and structure with light microscopes, which allowed them 196
to be segregated into distinct isolates. Cell wall type was also determined on the base 197
of the occurrence of isomers of diaminopimelic acid to exclude the Streptomycetes 198
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from other spore-forming actinomycetes as whole-organism hydrolysates of 199
Streptomyces strains contain the LL-isomer and the latter the meso- diaminopimelic 200
acid. The diagnostic sugars of the representatives of each group were also detected 201
(38). Based on the preliminary grouping, 46 isolates were selected for further 202
research. 203
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DNA extraction, sequencing and analysis 205
The 46 selected isolates were subjected to 16S rRNA gene sequence analysis for 206
precise genera and species identification. The identity of the organisms was 207
determined based on partial or nearly full length of 16S rRNA gene sequence analysis. 208
Genomic DNA of each isolate was extracted using a method of Li et al. (53). The 16S 209
rRNA genes from pure cultures were amplified using the primer pair PA and PB 210
(Table 2). Polymerase chain reaction was carried out under the following conditions: 211
initial denaturation at 94°C for 4 min, followed by 30 cycles of 94°C for 1 min, 55°C 212
for 1 min and 72°C for 2 min, with a final extension 72°C for 10 min. The reaction 213
mixture (50 µl) contained dNTPs (0.25 mM each), 1×reaction buffer (20 mM Tris pH 214
8.4, KCl 50 mM), MgCl2 (3 mM), primers (1 µM each), Taq DNA polymerase (1.25 215
units) and 1 ng of template DNA (all PCR reagents were purchased from TaKaRa, 216
Dalian, China). The PCR products were separated by agarose gel electrophoresis and 217
purified using the QIA quick Gel Extraction Kits (Qiagen, Hilden, Germany) and 218
sequenced on the ABI PRISM 3730 sequencer. 219
The determined 16S rRNA gene sequences were compared with the 220
GenBank/EMBL/DDBJ databases by using the BLASTN (1) search program. A 221
phylogenetic tree was constructed with the neighbour-joining (71) method using the 222
software package MEGA 3.1 (46) after pairwise alignments using the CLUSTAL_X 223
1.8 program (80). The stability of relationships was assessed by performing bootstrap 224
analyses (28) of the neighbour-joining data based on 1000 resamplings. DNA 225
sequences were deposited to GenBank and the Accession numbers were shown in 226
Table 3. 227
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Detection of PKS-I, PKS-ⅡⅡⅡⅡand NRPS sequences 228
Three sets of degenerate primers for the amplification of genes encoding polyketide 229
synthases (PKS-І, PKS-Ⅱ) and nonribosomal peptide synthetase (NRPS) from the 230
tested isolates were carried out as recommended by Ayuso-Sacido and Genilloud (4) 231
and Ketela et al. (45) (Table 2). The reaction mixture contained 2.5 units of Taq DNA 232
polymerase, 1 mM MgCl2, 0.4 mM dNTPs, 2 µM of each primer and 5% DMSO in a 233
50 µl reaction volume. Control reactions, without the actinobacterial DNA template. 234
Thermocycling conditions consisted of one denaturation step of 94°C for 5 min 235
followed by 30 amplification cycles of 94°C for 1 min, 57°C (for K1F/M6R, 236
A3F/A7R) or 58°C (for KSα/ KSβ) for 1 min, 72°C for 2 min followed by a final 237
extension at 72°C for 5 min. 238
239
Fermentation, extraction and evaluation of the antimicrobial activity 240
Each isolate was cultured in soybean mannitol liquid medium [soybean flour 12 g 241
and mannitol 20 g in 1000 ml tap water (pH 7.2–7.4)] at 28°C and shaked at 180 242
rev/min). After 7-12 days of cultivation, the fermentation broth was extracted with 243
ethanol. Each organic solvent extract was then evaporated under reduced pressure to 244
yield an ethanol extract. The ethanol extracts were then used for the antimicrobial 245
activity screening. The inhibitory effect of the extract obtained from endophytic 246
actinobacteria was tested using the paper disk (7 mm diameter) assay method (59). 247
25µl of ethanol extract suspension were pipetted onto each disc and incubated at 37°C, 248
followed by measurement of the diameters of the inhibition zone after 24 h. 25µl 249
ethanol was used as the control. Pathogenic bacterial organisms Staphylococcus 250
aureus, Bacillus subtilis, Pseudomonas aeruginosa and yeast Candida albicans were 251
used as indicator organisms to determine the antimicrobial activity. The pathogenic 252
microorganisms were deposited in the Yunnan institute of microbiology, Yunnan 253
University. 254
Results and Discussion 255
Evaluation of surface sterilization protocol 256
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Surface sterilization is important for studying endophytes. Imprinted agar of ISP 2 257
plate from each surface-sterilized sample showed no microbial growth after 15 days 258
incubation at 28°C. In addition, ISP 2 agar plates spread with the last washing water 259
from plant samples also did not emerge colonies of microorganisms after two weeks 260
incubation. This indicated that the five steps surface sterilization protocol was 261
effective to kill the epiphytic microorganisms. Thus the subsequent isolates can be 262
considered as true endophytic actinobacteria. 263
A 2.5% sodium thiosulfate solution was used instead of sterilized water rinses in 264
this study in order to remove the chlorine cover left on the plants surface by 265
hypochlorite disinfection. This was based on the consideration that even though plant 266
tissues were rinsed thoroughly with sterilized water after treated with NaOCl, there 267
were still toxic compounds present on their surfaces, which may kill endophytes or at 268
least stressed them so much that they were unable to form colonies on the plates. By 269
our tentative experiments, the increase in the number of CFU was indeed obvious 270
compared to the control (supplementary Table S1). This was consistent with the result 271
of the effects of rice seed surface sterilization with 2.0% sodium thiosulfate instead of 272
water rinses (60), and the fact that thiosulfate can suppress the detrimental effects of 273
hypochlorite on skin (34). It is also known that the alkaline environment favors the 274
growth of actinomycetes but not for endophytic fungi. The growth of fungal 275
endophyte was effectively inhibited by soaking the plant tissues in 10% NaHCO3 276
solution in our study so that the actinobacterial endophytes could grow out of tissues 277
before fungi overgrow the samples and mask the actinobacteria. The present surface 278
sterilization method was effective to help us acquire endophytic actinobacteria. 279
280
Selective isolation of culturable endophytic actinobacteria from medicinal plants 281
On the basis of characteristic colonial morphology, notably the ability to form 282
aerial hyphae and substrate mycelia, organisms putatively identified as actinobacteria 283
were selected. In total, 2174 actinobacterial strains were isolated from nearly 90 284
selected plant samples. According to the preliminary morphological identification, the 285
most abundant genus was Streptomyces (87%), which was consistent with the other 286
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reports from different hosts (10, 18, 83). As the aim of our study was to isolate 287
non-Streptomyces rare actinobacterial genera, according to the morphological criteria, 288
280 strains that did not seem to belong to Streptomyces or in doubt were further 289
checked for cultural, micromorphological characteristics and examined for the 290
presence of isomers of diaminopimelic acid. Based on these results, Pseudonocardia 291
was the dominant genus (57%), followed by Nocardiopsis spp. (8.3%), 292
Micromonospora spp. (6.4%), Streptosporangium spp. (3.8%) and others (24.5%) that 293
could not be accurately identified to the genus level. Except for Pseudonocardia, the 294
genera Micromonospora, Streptosporangium and Nocardiopsis were also reported 295
frequently from a wide range of host plants, such as maize, lichens and wheat (2, 31, 296
33). Then, 46 representative strains of each group were subjected to 16S rRNA gene 297
sequence analysis. 298
We used several isolation procedures in this study in order to get endophytic 299
actinobacteria as much as possible. Compared to previous commonly used 300
pretreatment methods for isolating endophytic actinobacteria of cutting samples into 301
small pieces about 0.2×0.5cm (11, 83), method 1 enlarged the colonization area of 302
samples onto the plates agar, which could be helpful for recovering endophytes. 303
We used the combined calcium carbonate enrichment, rehydration and 304
centrifugation (RC) pretreatment procedure developed earlier in method 2. This 305
method allowed efficient isolation of Dactylosporangium, Kineosporia, Kineococcus, 306
Herbidospora, Jiangella and Promicromonospora species. Although at present the 307
precise rationale behind the calcium carbonate effect is not clear, it seems plausible 308
that it might involve the following effects: the pH of the isolation sources after mixing 309
with powdered calcium carbonate would be altered in favor of the growth of 310
actinomycete propagules (82); calcium ions have the ability of stimulating the 311
formation of aerial mycelia by several actinomycete cultures (62). Previous isolation 312
of soil samples with calcium carbonate have demonstrated significantly increase of 313
the relative plate counts of actinomycete population (82). The centrifugation stage 314
greatly eliminated streptomycetes and other non-motile actinomycetes from the liquid 315
phase, thereby facilitating selective growth of rare, especially motile actinomycetes on 316
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the isolation plates subsequent to inoculation (39). Depending on the combined 317
calcium carbonate and RC procedures, Otoguro et al. (66) have successfully isolated 318
diverse Actinokineospora spp and other actinomycetes from soils and plant litters. Our 319
present study demonstrated that the enrichment stages with calcium carbonate and RC 320
procedure were also suitable for isolation zoosporic and other rare actinobacteria from 321
endophytic habitats. 322
Pretreatment method 3 was especially useful for isolating endophytic actinobacteria. 323
Besides the predominant Streptomyces, Pseudonocardia, Nocardiopsis and 324
Micromonospora species, other genera, including Amycolatopsis, Nocardia, 325
Nonomuraea, Actinomadura, Gordonia, Promicromonospora and Mycobacterium 326
were also recovered. This procedure was based on the fact that endophytic bacteria 327
reside in plant tissues mainly in intercellular spaces, rarely in intracellular spaces and 328
inside vascular tissues (79). By mild treatment with enzymes hydrolyzing plant cell 329
walls and subsequent differential centrifugation, most of the microbes associating 330
with plant tissues were collected and simultaneously to maintain the microbial cells 331
intact, which could be demonstrated by scanning electron microscopy (24). 332
Centrifugation is commonly used to collect the intercellular (apoplastic) fluid of plant 333
tissue (9, 23), and also has been successfully applied for the isolation of endophytic 334
bacteria from sugarcane stems by 3000 g force (24). This method seems to be 335
time-consuming, requiring both surface sterilisation and centrifugation, but several 336
samples can be centrifuged at the same time. It was first successfully applied to the 337
culture isolation of diverse endophytic actinobacteria in our study. Recently, Wang et 338
al. (85) reported the diversity of uncultured plant-associated microbes by improved 339
DNA extraction method and increased differential centrifugation at 5000 g force, 340
actinobacteria were the most dominant microbial group, covering 37.7% in the 16S 341
rRNA gene library, which also demonstrated the feasibility and prospect of 342
pretreatment method 3 to be used for isolating endophytic actinobacteria. 343
Actinobacteria were isolated on all of the 11 selective isolation media. 344
Observations on the isolation frequency from method 1 suggested that most strains 345
were isolated from the relative simple nutrient media, such as TWYE, sodium 346
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propionate agar and HV, which were most effective for isolation of endophytic 347
actinobacteria. However, other media seemed to be less suitable, due to the fact that 348
its high nutrient concentration allowed fast growing bacteria to overgrow slower 349
growing actinobacteria. This was consistent with the result from Coombs and Franco 350
(18). It is interesting that the ISP 5, raffinose–histidine agar, trehalose-proline agar 351
and our designed sodium propionate, cellulose-proline and xylan-arginine media are 352
more effective for isolation from procedures 2 and 3. Much more total number of 353
colonies formed on these media than others (data not shown). Cirousse et al. (16) 354
have detected asparagine as the main amino acid (70%) in the phloem sap of alfalfa. 355
Additionally, Mazzafera and Goncalves (58) found in the xylem sap of coffee that 356
almost 90% of the nitrogen in the form of amino acids was represented by asparagine 357
and glutamine. Similar results were reported from Tejera et al. (78) that predominant 358
nitrogen compounds in the apoplastic sap of sugarcane stem were amino acids 359
(50–70% of N), including serine, proline, alanine and aspartic acid. These facts 360
reminded us of attempting to use amino acids as nitrogen sources for culture isolation 361
plausibly, nevertheless, the real principle for isolation and role of these substances in 362
relationship with the establishment of the symbiosis of endophytes need further 363
experimental analysis. 364
Diversity of endophytic actinobacteria by 16S rRNA gene analysis 365
The endophytic actinobacteria isolated in this study displayed considerable 366
diversity, which distributed under 10 suborders: Pseudonocardineae (8 strains), 367
Corynebacterineae (8 strains), Streptomycineae (2 strains), Frankineae (1 strain), 368
Micromonosporineae (5 strains), Kineosporiineae (2 strains), Micrococcineae (8 369
strains), Streptosporangineae (8 strains), Propionibacterineae (1 strain) and 370
Glycomycineae (3 strains) within the class Actinobacteria in the phylogenetic tree 371
(Fig. S1) including 32 genera and more than 40 species. Notably, some uncommon 372
genera from endophytic environment, such as Amycolatopsis, Lentzea, 373
Pseudonocardia, Rhodococcus, Nocardia, Tsukamurella, Mycobacterium, Dietzia, 374
Gordonia, Dactylosporangium, Kineosporia, Janibacter, Kineococcus, Arthrobacter, 375
Micrococcus, Nonomuraea, Herbidospora and Glycomyces were discovered from 376
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present study. The percentage of 16S rRNA gene sequence similarities (97.2-100%) 377
of these isolates to the closest type strains was presented in Table 3. As shown in 378
Table 4, 34 out of the 46 strains were isolated from woody plants and most of them 379
were from roots (20 isolates) and stems (14 isolates), although the detailed taxa of 380
strains had no corresponding relationships with the taxonomic affiliations (species, 381
genera, families) of host plants. 382
Further phylogenetic analyses of part isolates were shown in Fig. S2a-e. Pairwise 383
comparison of the 16S rRNA gene sequences from the three Pseudonocardia isolates 384
(YIM 56035, YIM 56051 and YIM 61043) showed relatively high similarities (98.4, 385
97.5 and 98.6%) to the type strains P. kongjuensis, P. antarctica and P. zijingensis. 386
However, the phylogenetic analysis indicated that they were diversely distributed 387
within this genus and clustered singly in the phylogenetic tree (Fig. S2a), suggesting 388
that isolates YIM 56035, YIM 56051 and YIM 61043 might belong to new species. 389
The Saccharopolyspora isolates (YIM 65359, YIM 61095 and YIM 60513) had 390
lineages that were distinct from each other and from members of the genus and 391
formed distinct subclades in the tree supported by high bootstrap values (Fig. S2b). 392
Strain YIM 61095 has been characterized as a new species of this genus (68). 16S 393
rRNA gene sequences from YIM 65359 and YIM 60513 showed 98.4% and 99.1% 394
identity to the nearest neighbours YIM 61095 and Saccharopolyspora gregorii, 395
respectively. DNA-DNA hybridizations, phenotypic comparisons and 396
chemotaxonomic analysis classified YIM 65359 and YIM 60513 as new species of 397
the genus Saccharopolyspora. The Glycomyces isolates (YIM 56134, YIM 61331 and 398
YIM 56256) formed distinct clusters within the Glycomyces 16S rRNA gene tree (Fig. 399
S2c), although shared highest 16S rRNA gene similarities within the range 98.9, 98.7 400
and 99.7% to the closest recognized strains. All the three isolates have been identified 401
as three different new species of the genus Glycomyces (67, 69). Although the 16S 402
rRNA gene sequences from two isolates, YIM 61515 and 56864 showed up to 99.0% 403
and 99.4% identity to the related type strains of the genus Promicromonospora (Fig. S 404
2d), DNA-DNA relatedness values (unpublished) were well below the 70% cut-off 405
point recommended by Wayne et al. (86) for the delineation of strains that belong to 406
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the same genomic species, indicating that they are still new species. It was particular 407
interesting to recover isolate YIM 61359. Comparison of the 16S rRNA gene 408
sequences of this isolate and the closest neighbours indicated that this strain was 409
closely related to the type strains of the genera Micromonospora (96.6%-98.1%, 410
similarity), Salinispora (97.7%, similarity) and Polymorphospora rubra (97.6%, 411
similarity). However, it formed a distinct monophyletic clade within the family 412
Micromonosporaceae (Fig. S2e) in the phylogenetic tree, most closely related to the 413
first obligate new marine actinomycete genus Salinispora, which produced antibiotic 414
Salinosporamide A (27, 56). This indicated that isolate YIM 61359 should represent a 415
new genus proposed as ‘Plantactinospora gen. nov’ of the family 416
Micromonosporaceae and merit secondary metabolites research. Although the isolates 417
YIM 65003 (Rhodococcus), YIM 65001 (Dietzia), YIM 65002 (Dietzia), YIM 60475 418
(Streptomyces), YIM 65188 (Streptomyces), YIM 56238 (Micrococcus) and YIM 419
61503 (Jiangella) had more than 97% 16S rRNA gene sequences similarities to the 420
nearest type strains respectively, they have already been identified as new species (14, 421
15, 49, 51, 52, 70) by using the polyphasic taxonomic analyses. Detailed 422
characterizations of other isolates are currently being done, in comparison with the 423
nearest type strains to determine whether they are new species. Taxonomic 424
description of these putative novel species will be further characterized elsewhere. 425
We isolated and cultured 46 representatives of 32 genera of actinobacteria from 426
medicinal plants. This is the first report to recover such a high diversity of culturable 427
actinobacteria from endophytic environments. In addition, to our knowledge, this is 428
the first time that strains of the rarely recovered genera Saccharopolyspora, Dietzia, 429
Blastococcus, Dactylosporangium, Promicromonospora, Oerskovia, Actinocorallia 430
and Jiangella have been isolated and cultured from the inner parts of plants. 431
Taechowisan et al. (76) isolated 330 isolates belonging to 4 different genera 432
(Streptomyces, Microbispora, Nocardia and Micromonospora) from 36 medicinal 433
plant species from Thailand. Tan et al. (77) found 619 actinomycetes isolated from 434
different cultivars of tomato, and all of them were Streptomyces spp. Recently, Lee et 435
al. (48) isolated 8 endophytic actinobacterial genera in only 81 isolates of Chinese 436
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cabbage roots, and Microbispora spp. (67%) were the most common isolates, 437
followed by Streptomyces spp. (12%) and Micromonospora spp. (11%). Obviously, 438
the actinobacterial richness and diversity from tropical rainforest in Xishuangbanna of 439
our present study is much higher comparison with other reports, and tropical 440
rainforest have potential as excellent source of actinobacteria and new species. 441
Therefore, it seems clear that endophytic communities are diverse and the extent of 442
diversity may vary between different sample collection regions and different plant 443
species, the diversity of isolated actinobacteria was largely dependent on the isolation 444
methods. 445
446
Antimicrobial activity and detection of the PKS and NRPS genes in the selected 447
actinobacteria 448
All the 46 isolates were tested against pathogenic bacteria Staphylococcus aureus, 449
Bacillus subtilis, Pseudomonas aeruginosa and yeast Candida albicans to evaluate 450
their production of antimicrobial activities. 24 out of 46 isolates (52.2%) exhibited 451
activity against at least one tested pathogenic microorganism. Activity against B. 452
subtilis was clearly the most frequent (12 isolates, 26.1%). Activity against P. 453
aeruginosa was the least frequent (10.9%), while 19.6% and 17.4% of the isolates 454
were active against S. aureus and C. albicans. Six isolates were found to inhibit two 455
pathogens. Two isolates, YIM 61515 and YIM 61333 appeared to have a broad 456
spectrum of antimicrobial activity. Four isolates, YIM 61341, YIM 61333, YIM 457
61105 and YIM 56134 exhibited relatively strong inhibitory effect against pathogenic 458
microorganisms C. albicans, S. aureus and P. aeruginosa. The strong inhibitory 459
activities of these strains against a variety of pathogens suggested that these 460
endophytic actinobacteria may be potential candidates for the production of bioactive 461
compounds. Chemical analysis of some of the strains is currently being carried out in 462
order to identify active compounds and to assess their novelty. 463
The 46 strains were screened for the presence of PKS-І, PKS-Ⅱ and NRPS 464
sequences by specific amplification of chromosomal DNA with the primers K1F/M6R, 465
KSα/ KSβ and A3F/A7R, respectively (Table 5) to evaluate the biosynthetic potential 466
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in terms of natural product drug discovery. NRPS sequences were detected in 12 467
isolates (26.1%) whereas PKS-І and PKS-Ⅱsequences were detected in only five and 468
seven out of the 46 strains (10.9% and 15.2%). Strains YIM 65293 and YIM 65377 469
gave positive amplification products with both the PKS-Ⅱ and NRPS primers. Both 470
PKS-І and NRPS gene sequences were amplified from strain YIM 61441. All the 471
three target genes were detected in strain YIM 61375. Several representative PKS-І, 472
PKS-Ⅱ and NRPS gene amplified products were cloned, sequenced, and their 473
subsequent analysis confirmed that they indeed encode parts of the expected 474
biosynthetic enzymes (see accession no. FJ615259). 475
The results of antimicrobial activity and the detection of functional genes seemed to 476
have no direct correlations in present study. Several isolates, such as YIM 61095 and 477
YIM 65377 had no antimicrobial activity, but could be successfully amplified target 478
genes. 28 strains (60.9%) were not detected the PKS and NRPS genes using the 479
selected degenerate primer pairs, which have been used in a broad survey of these 480
genes in a similar study (4). The absence of amplification products from some of the 481
strains may reflect the lack of PKS-І, PKS-Ⅱ and NRPS genes, though it is also 482
possible that these specific degenerate primer pairs might not be suitable to amplify 483
these genes. Furthermore, not all NRPS genes are involved in the biosynthesis of 484
bioactive secondary metabolites; indeed, the products of such genes may be involved 485
in functions such as iron metabolism or quorum sensing (30). It is also possible that 486
the genes detected by PCR are nonfunctional. Nevertheless, the strategy of 487
prescreening with PCR primers, targeting genes encoding the biosynthesis of 488
bioactive compounds is an effective approach for detecting novel and useful 489
secondary metabolites (19, 32, 41, 45). 490
491
Conclusions 492
In summary, this study demonstrates that an unexpected diversity of actinobacteria 493
colonize the interior of medicinal plants in Xishuangbanna tropical rainforests and 494
suggests that these strains might represent a valuable source of new species and 495
biological active compounds with antimicrobial activity and genes for their 496
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biosynthesis. The isolation procedure is critical and important step in studies on 497
endophytic actinobacteria. It is indeed need to improve traditional selective isolation 498
methods to recover the untapped majority of endophytic actinobacteria. Although 499
many questions remain to be answered regarding to the ecological function of 500
actinobacteria in the endophytic environment as well as their evolution and 501
biogeographic distribution. The increasing numbers of rare actinobacteria isolated 502
from medicinal plants indicate that plants are potentially unique sources of novel 503
actinobacteria with promising potential to produce highly bioactive metabolites and 504
their potential should not be overlooked. 505
506
507
Acknowledgments 508
This research was supported by National Basic Research Program of China (Project 509
no. 2004CB719601). W.-J. Li was supported by Program for New Century Excellent 510
Talents in University. 511
512
513
514
515
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70. Qin, S., G. Z. Zhao, J. Li, W. Y. Zhu, L. H. Xu, and W. J. Li. 2008. Jiangella 733
alba sp. nov., an endophytic actinomycete isolated from the stem of Maytenus 734
austroyunnanensis. Int. J. Syst. Evol. Microbiol. (In Press). 735
71. Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for 736
reconstructing phylogenetic tree. Mol. Biol. Evol. 4: 406-425. 737
72. Sessitsch, A., B. Reiter, and G. Berg. 2004. Endophytic bacterial communities of 738
field-grown potato plants and their plant-growth promoting and antagonistic 739
abilities. Can. J. Microbiol. 50: 239–249. 740
73. Shirling, E. B., and D. Gottlieb. 1966. Methods for characterization of 741
Streptomyces species. Int. J. Syst. Bacteriol. 16: 313–340. 742
74. Strobel, G., B. Daisy, U. Castillo, and J. Harper. 2004. Natural products from 743
endophytic microorganisms. J. Nat. Prod. 67: 257–268. 744
75. Surette, M. A., A. V. Sturz, R. R. Lada, and J. Nowak. 2003. Bacterial 745
endophytes in processing carrots (Daucus carota L. var. sativus): their localization, 746
population density, biodiversity and their effects on plant growth. Plant and Soil. 747
253: 381–390. 748
76. Taechowisan, T., J. F. Peberdy, and S. Lumyong. 2003. Isolation of endophytic 749
actinomycetes from selected plants and their antifungal activity. World. J. 750
Microbiol. Biotechnol. 19: 381-385. 751
77. Tan, H. M., L. X. Cao, Z. F. He, G. J. Su, B. Lin, and S. N. Zhou. 2006. 752
Isolation of endophytic actinomycetes from different cultivars of tomato and their 753
activities against Ralstonia solanacearum in vitro. World. J. Microbiol. 754
Biotechnol. 22: 1275-1280. 755
78. Tejera, N., E. Ortega, R. Rodes, and C. Lluch. 2006. Nitrogen compounds in 756
the apoplastic sap of sugarcane stem: Some implications in the association with 757
endophytes. J. Plant. Physiol. 163: 80-85. 758
79. Thomas, W. D., and R.W. Graham. 1952. Bacteria in apparently healthy pinto 759
beans. Phytopathology. 42: 214. 760
80. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 761
1997. The Clustal X windows interface: flexible strategies for multiple sequence 762
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81. Tian, X. L., L. X. Cao, H. M. Tan, W. Q. Han, M. Chen, Y. H. Liu, and S. N. 764
Zhou. 2007. Diversity of Cultivated and Uncultivated Actinobacterial Endophytes 765
in the Stems and Roots of Rice. Microbiol. Ecol. 53: 700-707. 766
82. Tsao, P. H., C. Leben, and G. W. Keitt. 1960. An enrichment method for 767
isolating actinomycetes that produce diffusible antifungal antibiotics. 768
Phytopathology. 50: 88-89. 769
83. Verma V. C., S. K. Gond, A. Kumar, A. Mishra, R. N. Kharwar, and A. C. 770
Gange. 2009. Endophytic Actinomycetes from Azadirachta indica A. Juss: 771
Isolation, Diversity, and Anti-microbial Activity. Microbiol. Ecol. 57: 749-756. 772
84. Vickers, J. C., S. T. Williams, and G. W. Ross. 1984. A taxonomic approach to 773
selective isolation of streptomycetes from soil. In Biological, Biochemical and 774
Biomedical Aspects of Actinomycetes, pp. 553–561. Edited by L. Ortiz-Ortiz, L. F. 775
Bojalil & V. Yakoleff. Orlando: Academic Press. 776
85. Wang, H. X., Z. L. Geng, Y. Zeng, and Y. M. Shen. 2008. Enrichment plant 777
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2684-2691. 779
86. Wayne, L. G., D. J. Brenner, R. R. Colwell, P. A. D. Grimont, O. Kandler, M. 780
I. Krichevsky, L. H. Moore, W. E. C. Moore, R. G. E. Murray, E. 781
Stackebrandt, M. P. Starr, and H. G. Truper. 1987. Report of the ad hoc 782
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Evol. Microbiol. 37: 463–464. 784
87. Zhao, P. J., H. X. Wang, G. H. Li, H. D. Li, J. Liu, and Y. M. Shen. 2007. 785
Secondary Metabolites from Endophytic Streptomyces sp. Lz531. Chemistry & 786
Biodiversity. 4: 899-904. 787
88. Zheng, Z., Z. L. Feng, M. Cao, Z. F. Li, and J. H. Zhang. 2006. Forest 788
Structure and Biomass of a Tropical Seasonal Rain Forest in Xishuangbanna, 789
Southwest China. Biotropic. 38: 318–327. 790
89. Zin, N. M., N. I. M. Sarmin, N. Ghadin, D. F. Basri, N. M. Sidik, W. M. Hess, 791
and G. A. Strobel. 2007. Bioactive endophytic streptomycetes from the Malay 792
Peninsula. FEMS Microbiol. Lett. 274: 83–88. 793
794
795
796
797
798
799
800
801
802
803
804
805
806
807
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Table 1. Compositions of the 11 different media for isolation of endophytic 808
actinobacteria from plant samples. 809
810
Media Compositions References
M1 TWYE 21
M2 modified TWYE supplemented with plant extract This study
M3 glycerol-asparagine agar (ISP 5) 73
M4 HV agar 40
M5 inorganic salts-starch agar (ISP 4) 73
M6 YIM 38 medium (4.0 g malt extract, 4.0 g yeast extract, 4.0 g glucose, vitamin
mixture and agar 15.0g
This study
M7 raffinose–histidine agar 84
M8 sodium propionate agar (sodium propionate 1.0g, L-asparagine 0.2 g, KH2PO4 0.9
g, K2HPO4 0.6 g, MgSO4·7H2O 0.1 g, CaCl2·2H2O 0.2 g, agar 15.0g)
This study
M9
cellulose-proline agar (cellulose, 2.5g, sodium pyruvate 2.0g, KNO3 0.25g, proline
1.0g, MgSO4˙7H2O 0.2g, K2HPO4 0.2g, CaCl2 0.5g, FeSO4˙7H2O 10mg and agar
15.0g)
This study
M10 trehalose-proline medium (fucose 5.0 g, proline1.0 g, (NH4)2SO4 1.0 g, NaCl 1.0
g, CaCl2 2.0 g, K2HPO4 1.0 g, MgSO4·7H2O 1.0 g, agar 15.0g)
This study
M11 xylan-arginine agar (xylan 2.5g, arginine1.0g, (NH4)2SO4 1.0g, CaCl2 2.0g,
K2HPO4 1.0g, MgSO4·7H2O 0.2g, FeSO4˙7H2O 10mg and agar 15.0g)
This study
811
812
813
814
815
816
817
818
819
820
821
822
823
824
825
826
827
828
829
830
831
832
833
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Table 2. Polymerase chain reaction (PCR) primers used in this study. 834
835
Primers
name
Sequences (5’-3’) Target
genes
Length of the target
gene fragments (bp)
Reference
PA 5′-AGAGTTTGATCCTGGCTCAG-3′ 16S rRNA 1400-1500 22
PB 5′-AAGGAGGTGATCCAGCCGCA-3′
K1F 5'-TSAAGTCSAACATCGGBCA-3' PKS-І 1200-1400 4
M6R 5'-CGCAGGTTSCSGTACCAGTA-3'
KSα 5'-TSGCSTGCTTGGAYGCSATC-3' PKS-Ⅱ 600 45
KSβ 5'-TGG AANCCG CCGAABCCTCT-3'
A3F 5'-GCSTACSYSATSTACACSTCSGG-3' NRPS 700-800
A7R 5'-SASGTCVCCSGTSCGGTAS-3' 4
836
837
838
839
840
841
842
843
844
845
846
847
848
849
850
851
852
853
854
855
856
Table 3. Actinobacteria isolated from different organs of host plants using three 857
different methods, with taxonomic status of plants and similarity values of 16S rRNA 858
gene sequences. 859
860
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Isolates
(GenBank accession no.)
Host plants (species) Taxonomic status
(family, genus) of host plants
Organs Closest cultivated species Similarity
(%)
Isolation
methods
YIM 61095 (EU814512) Maytenus austroyunnanensis Celastraceae, Maytenus Root Saccharopolyspora flava AS 4.1520T (AF154128) 97.7 1
a
YIM 65359 (FJ214364) Tripterygium hypoglaucum Celastraceae, Tripterygium Stem Saccharopolyspora flava AS 4.1520T (AF154128) 98.4 1
YIM 60513 (EU005371) Gloriosa superba Liliaceae, Gloriosa Stem Saccharopolyspora gregorii NCIB 12823T (X76962) 99.1 1
YIM 65085 (FJ214357) Osyris wightiana Santalaceae, Osyris Root Amycolatopsis pretoriensis NRRL B-24133 T
(AY183356) 98.0 3
YIM 65117 (FJ214358) Ficus tikoua Moraceae, Ficus Root Lentzea albida IFO 16102 T
(AB006176) 99.1 1
YIM 61043 (FJ214340) Maytenus austroyunnanensis Celastraceae, Maytenus Root Pseudonocardia zijingensis JCM 11117T (AF325725) 98.6 3
YIM 56051 (EU200680) Callicarpa longifolia Verbenaceae, Callicarpa Leaf Pseudonocardia alni IMSNU 20049T (AJ252823) 97.5 1
YIM 56035 (DQ887489) Lobelia clavata Campanulaceae, Lobelia Leaf Pseudonocardia kongjuensis DSM 44525T (AJ252833) 98.4 1
YIM 65003 (EU325542) Cercidiphyllum japonicum Cercidiphyllaceae, Cercidiphyllum Leaf Rhodococcus fascians DSM 20669T (X79186) 99.6 1
YIM 61441 (FJ214349) Maytenus austroyunnanensis Celastraceae, Maytenus Root Nocardia carnea DSM 46071T (AF430037) 98.3 3
YIM 61394 (FJ214346) Maytenus austroyunnanensis Celastraceae, Maytenus Stem Tsukamurella tyrosinosolvens DSM 44234T (AY238514) 99.9 2
b
YIM 65220 (FJ214359) Paris yunnanensis Liliaceae, Paris Leaf Mycobacterium monacense B9-21-178T (AF107039) 97.2 3
c
YIM 65001 (EU375845) Schima sp. Theaceae, Schima Stem Dietzia maris DSM 43672T (X79290) 99.8 1
YIM 65002 (EU375846) Cercidiphyllum japonicum Cercidiphyllaceae, Cercidiphyllum Root Dietzia natronolimnaea 15LN1T (X92157) 99.5 1
YIM 61527 (FJ214354) Maytenus austroyunnanensis Celastraceae, Maytenus Stem Gordonia sputi DSM 43896T (X80634) 99.0 3
YIM 61401 (FJ214347) Maytenus austroyunnanensis Celastraceae, Maytenus Stem Gordonia polyisoprenivorans W8130T (DQ385609) 97.9 3
YIM 60475 (EU200683) Maytenus austroyunnanensis Celastraceae, Maytenus Root Streptomyces hainanensis YIM 47672T (AM398645) 99.2 1
YIM 65188 (EU925562) Sedum sp. Crassulaceae, Sedum Root Streptomyces special GW 41-1564T (AM934703) 97.5 1
YIM 65287 (FJ214361) Tripterygium wilfordii Celastraceae, Tripterygium Root Blastococcus aggregatus DSM 4725T (AJ430193) 99.6 1
YIM 61359 (FJ214343) Maytenus austroyunnanensis Celastraceae, Maytenus Root Polymorphospora rubra DSM 44947T (AB223089) 97.6 2
YIM 65312 (FJ214363) Tripterygium wilfordii Celastraceae, Tripterygium Root Dactylosporangium aurantiacum DSM 43157T (X93191) 98.0 2
YIM 65268 (FJ214360) Berberis chingii Berberidaceae, Berberis Stem Catellatospora chokoriensis 2-25(1)T (AB200231) 99.0 1
YIM 61481 (FJ214350) Maytenus austroyunnanensis Celastraceae, Maytenus Leaf Micromonospora narashino DSM 43172T (X92602) 99.3 3
YIM 61363 (FJ214344) Maytenus austroyunnanensis Celastraceae, Maytenus Leaf Micromonospora peucetia DSM 43363 T
(X92603) 98.0 1
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YIM 65293 (FJ214362) Tripterygium wilfordii Celastraceae, Tripterygium Stem Kineosporia aurantiaca IFO 14067T (D86937) 98.2 1
YIM 65377 (FJ214365) Tripterygium wilfordii Celastraceae, Tripterygium Stem Kineococcus aurantiacus IFO 15268T (X77958) 98.0 2
YIM 61515 (FJ214352) Maytenus austroyunnanensis Celastraceae, Maytenus Leaf Promicromonospora aerolata IFO 16526T (AJ487303) 99.0 3
YIM 56864 (EU200679) Cymbopogon citratus Poaceae, Cymbopogon Root Promicromonospora sukumoe DSM 44121T (AJ272024) 99.4 2
YIM 60535 (EU200684) Ginkgo sp. Ginkgoaceae, Ginkgo Leaf Oerskovia jenensis DSM 46097T (AJ314850) 99.3 1
YIM 61525 (FJ214353) Maytenus austroyunnanensis Celastraceae, Maytenus Root Janibacter melonis KCTC 9987T (AY522568) 99.8 1
YIM 61410 (FJ214348) Maytenus austroyunnanensis Celastraceae, Maytenus Stem Microbacterium paraoxydans H110bT (EF204432) 100 3
YIM 61510 (FJ214351) Maytenus austroyunnanensis Celastraceae, Maytenus Leaf Arthrobacter mysorens LMG 16219T (AJ639831) 98.2 3
YIM 65004 (FJ214355) Polyspora axillaris Theaceae, Polyspora Root Micrococcus flavus CGMCC 1.5361T (DQ491453) 99.7 1
YIM 56238 (EU005372) Aquilaria sinensis Thymelaeaceae, Aquilaria Root Micrococcus flavus CGMCC 1.5361T (DQ491453) 99.0 1
YIM 61341 (FJ214342) Maytenus austroyunnanensis Celastraceae, Maytenus Stem Nocardiopsis dassonvillei JPL-3T (AY030320) 98.4 1
YIM 61333 (FJ214341) Maytenus austroyunnanensis Celastraceae, Maytenus Root Nocardiopsis dassonvillei JPL-3T (AY030320) 98.2 3
YIM 56155 (FJ214339) Duranta repens Linn Verbenaceae, Duranta Root Actinocorallia aurantiaca JCM 8201T (AF134066) 99.1 2
YIM 60542 (EU005370) Millettia reticulata Papilionaceae, Millettia Leaf Actinocorallia herbida IFO 15485T (D85473) 99.1 2
YIM 61435 (FJ157185) Maytenus austroyunnanensis Celastraceae, Maytenus Leaf Actinomadura atramentaria DSM 43919T (AJ420138) 97.4 2
YIM 61375 (FJ214345) Maytenus austroyunnanensis Celastraceae, Maytenus Stem Streptosporangium vulgare DSM 44112T (X89957) 97.4 2
YIM 61105 (FJ157184) Maytenus austroyunnanensis Celastraceae, Maytenus Leaf Nonomuraea candida DSM 45086T (DQ285421) 98.2 1
YIM 65070 (FJ214356) Osyris wightiana Santalaceae, Osyris Stem Herbidospora cretacea IFO 15474T (D85485) 99.5 2
YIM 61503 (FJ157186) Maytenus austroyunnanensis Celastraceae, Maytenus Stem Jiangella alkaliphila DSM 45079T (AM422451) 98.8 2
YIM 56134 (EU200681) Carex baccans Nees Cyperaceae, Carex Root Glycomyces algeriensis NRRL B-16327T (AY462044) 98.9 1
YIM 56256 (EU200682) Scoparia dulcis Scrophulariaceae, Scoparia Root Glycomyces sambucus DSM 45047T (DQ460469) 99.7 1
YIM 61331 (EU814511) Maytenus austroyunnanensis Celastraceae, Maytenus Root Glycomyces sambucus DSM 45047T (DQ460469) 98.7 1
a Samples were aseptically crumbled into small fragments and directly placed on the selective media and incubated at 28 ºC for 2-8 weeks. 861
b Combined calcium carbonate enrichment, rehydration and centrifugation (RC) 862
c Combined enzymatic hydrolysis and differential centrifugation method. 863
864
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Table 4. Statistical analyses on the relationships between taxonomic status of the 46 865
endophytic actinobacterial strains associated with particular plant organs and taxa of 866
plant samples. 867
Taxonomic status of isolates Plants Organs Isolates
no. Suborder Family Genus
Roots
7
Pseudonocardineae
Streptomycineae
Micrococcineae
Streptosporangineae
Glycomycineae
Pseudonocardiaceae
Streptomycetaceae
Promicromonosporaceae
Thermomonosporaceae
Glycomycetaceae
Amycolatopsis,
Lentze, Glycomyces
Pseudonocardia
Streptomyces
Promicromonospora
Actinocorallia
Stems 2 Pseudonocardineae
Streptosporangineae
Pseudonocardiaceae
Streptosporangiaceae
Saccharopolyspora
Herbidospora
Leaves 3 Pseudonocardineae
Corynebacterineae
Pseudonocardiaceae
Mycobacteriaceae
Pseudonocardia
Mycobacterium
Herbaceous
Total no. 12 6 7 10
Roots
13
Pseudonocardineae
Streptomycineae
Corynebacterineae
Frankineae
Micromonosporineae
Micrococcineae
Streptosporangineae
Glycomycineae
Pseudonocardiaceae
Streptomycetaceae
Dietziaceae
Nocardiaceae
Geodermatophilaceae
Micromonosporaceae
Intrasporangiaceae
Micrococcaceae
Nocardiopsaceae
Glycomycetaceae
Saccharopolyspora
Pseudonocardia,
Dietzia, Glycomyces
Streptomyces
Nocardi, Janibacter
Blastococcus
Dactylosporangium
Micrococcus
Nocardiopsis
‘Plantactinospora’
Stems
12
Pseudonocardineae
Corynebacterineae
Micromonosporineae
Kineosporiineae
Micrococcineae
Streptosporangineae
Propionibacterineae
Pseudonocardiaceae
Tsukamurellaceae
Dietziaceae
Nocardiaceae
Micromonosporaceae
Kineosporiaceae
Microbacteriaceae
Nocardiopsaceae
Streptosporangiaceae
Nocardioidaceae
Saccharopolyspora
Tsukamurella
Dietzia, Jiangella
Gordonia
Catellatospora
Kineosporia
Kineococcus
Microbacterium
Nocardiopsis
Streptosporangium
Leaves
9
Corynebacterineae
Streptosporangineae
Micrococcineae
Micromonosporineae
Nocardiaceae
Streptosporangiaceae
Thermomonosporaceae
Cellulomonadaceae
Promicromonosporaceae
Micromonosporaceae
Micrococcaceae
Rhodococcus
Nonomuraea
Actinomadura
Actinocorallia
Oerskovia
Promicromonospora
Micromonospora
Arthrobacter
Woody
Total no. 34 10 18 28
868
869
870
871
872
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Table 5. Antimicrobial activity and PKS/NRPS genes of culturable actinobacteria 873
from medicinal plants. 874
875
Isolate no. Candida
albicans
Staphylococcus
aureus
Bacillus
subtilis
Pseudomonas
aeruginosa
PKS-I PKS-Ⅱ NRPS
YIM 61095 - - - - - - +
YIM 65359 - - - - - - -
YIM 60513 - - - + + - -
YIM 65085 - - - - - - -
YIM 65117 - ++ ++ - - - +
YIM 61043 - + - - - - +
YIM 56051 - ++ - - - + -
YIM 56035 - - - - - + -
YIM 65003 - - - - - - +
YIM 61441 - - - - + - +
YIM 61394 - - - - - - -
YIM 65220 - + + - - + -
YIM 65001 - - - - - - -
YIM 65002 - - - - - - -
YIM 61527 + - + - - - -
YIM 61401 - - - - - - -
YIM 60475 - - ++ - - - -
YIM 65188 - - - - - - -
YIM 65287 - - - - - - -
YIM 61359 - ++ - ++ - - -
YIM 65312 - ++ - - - - -
YIM 65268 - - ++ - - + -
YIM 61481 ++ - - - - - +
YIM 61363 ++ - - - + - -
YIM 65293 - - + - - + +
YIM 65377 - - - - - + +
YIM 61515 - + ++ ++ - - -
YIM 56864 - - - ++ - - -
YIM 60535 - - - - - - -
YIM 61525 - - - - - - -
YIM 61410 + - - - - - -
YIM 61510 - - - - - - -
YIM 65004 - - - - - - -
YIM 56238 - - - - - - -
YIM 61341 +++ - ++ - - - +
YIM 61333 +++ - ++ +++ - - +
YIM 56155 - + - - - - -
YIM 60542 - - - - - - -
YIM 61435 - - - - - - -
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YIM 61375 ++ - - - + + +
YIM 61105 +++ - - - - - -
YIM 65070 - - ++ - - - +
YIM 61503 - - ++ - - - -
YIM 56134 - +++ + - + - -
YIM 56256 - - - - - - -
YIM 61331 - - - - - - -
The antimicrobial activity was estimated by measuring the diameter of the clear zone of growth 876
inhibition. Symbles: +, ++ and +++ indicate presence, moderate and strong activity, respectively. 877
878
879
880
881
882
883
884
885
886
887
888
889
890
891
892
893
894
895
896
897
898
899
900
901
902
903
904
905
906
907
908
909
910
911
912
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