Rare actinobacteria from medicinal plants of tropic al rainforests

31 Pages·2009·0.39 MB·English

AEM Accepts, published online ahead of print on 31 July 2009
Appl. Environ. Microbiol. doi:10.1128/AEM.01034-09
Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
1 Rare actinobacteria from medicinal plants of tropical rainforests,
2 Xishuangbanna: isolation, diversity and antimicrobial activity
3 Sheng Qin1, Jie Li1, Hua-Hong Chen1, 2, Guo-Zhen Zhao1, Wen-Yong Zhu1,
4 Cheng-Lin Jiang 1, Li-Hua Xu1, Wen-Jun Li1*
5 1. The Key Laboratory for Microbial Resources of the Ministry of Education, P. R.
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6 China, and Laboratory for Conservation and Utilization of Bio-Resources, Yunnan w
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7 Institute of Microbiology, Yunnan University, Kunming, 650091, P. R. China a
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8 2. Department of Chemistry, Chuxiong Normal College, Chuxiong, Yunnan, 675000, P. f
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9 R. China h
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11 *Author for correspondence: Wen-Jun Li .
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12 Tel & Fax: [phone] .
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13 E-Mail: [email protected] ; [email protected] o
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16 Running title: Rare actinobacteria from medicinal plants of tropical rainforests 1
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23 Abstract
24 Endophytic actinobacteria are relatively unexplored as potential sources of novel
25 species and novel natural products for medical and commercial exploitation.
26 Xishuangbanna is well recognized in the world for its diverse flora, especially the
27 rainforest plants, many of which have indigenous pharmaceutical history. However,
28 little is known about the endophytic actinobacteria from this tropical area. In this
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29 work, we studied the diversity of actinobacteria isolated from medicinal plants
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30 collected from tropical rainforests in Xishuangbanna. Due to the use of different n
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31 selective isolation media and methods, a total of 2174 actinobacteria were isolated. 46 d
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32 isolates were selected on the basis of their morphology on different media and were
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33 further characterized by 16S rRNA gene sequencing. The results showed that they m

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34 represent an unexpected variety of 32 different genera. To our knowledge, this is the tt
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35 first report describing isolation of Saccharopolyspora, Dietzia, Blastococcus, /a
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36 Dactylosporangium, Promicromonospora, Oerskovia, Actinocorallia and Jiangella
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37 species from the endophytic environments. At least 19 isolates are considered as novel m
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38 taxa by our current research. In addition, all of the 46 isolates were tested for r
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39 antimicrobial activity and screened for the presence of genes encoding polyketide o
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40 synthetases (PKS) and nonribosomal peptide synthetases (NRPS). These results p
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41 confirm that medicinal plants from Xishuangbanna represent an extremely rich il 1
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42 reservoir for the isolation of significant diversity of actinobacteria and novel species 2
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43 which are potential sources for discovery of biologically active compounds. 9

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44 g
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45 Keywords: Actinobacteria, Endophytic, Tropical rainforests, Medicinal plants, s
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46 Diversity
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51 Introduction
52 The class Actinobacteria present a high proportion of soil microbial biomass and
53 contain the most economically significant prokaryotes, producing over half of the
54 bioactive compounds in the Antibiotic Literature Database (47), notably antibiotics
55 (7), immunosuppressive agents (57), antitumor agents (20), and enzymes (65),
56 especially those belonging to the genus Streptomyces, which are the excellent
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57 producers. The advent of drug resistance in many bacterial pathogens and the current
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58 increase in the number of fungal infections has caused a resurgence of interest in n
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59 finding other reserves of biologically active compounds (64). As the search for novel d
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60 natural products continues, it becomes apparent that the rate of discovery of new
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61 compounds from soil streptomycetes has decreased, whereas the rate of re-isolation of m

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62 known compounds has increased (29). Recent evidence is being accumulated that rare tt
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63 actinomycete species, which are often very difficult to isolate and cultivate, might /a
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64 represent a unique source of novel biologically active compounds (5). On the other
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65 hand, new microbial habitats need to be examined, in the search for novel bioactive m
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66 compounds. One biologically important but relatively overlooked niche is the inner r
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67 tissues of higher plants. Early studies have demonstrated that some actinobacteria can o
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68 form intimate associations with plants and colonize their inner tissues. Frankia p
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69 species and Streptomyces scabies can penetrate their host and establish either il 1
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70 pathogenic or endophytic associations (6, 25). The actinomycete bacteria that reside 2
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71 in the tissue of living plants and do not visibly harm the plants are known as 9

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72 endophytic actinobacteria (37). These actinobacteria are relatively unstudied and are y

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73 potential sources of novel natural products for exploitation in medicine, agriculture, e
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74 and industry (74).
75 Endophytic actinobacteria have attracted attention in recent years, with increasing
76 reports of isolates from a range of plant types, including crop plants, cereals, such as
77 wheat, rice, potato, carrots, tomato and citrus (3, 18, 63, 72, 75, 81) and medicinal
78 plants (76, 89) The culturable endophytic actinobacteria from these plants were found
79 to fall within a narrow species distribution, with Streptomyces spp. as the most
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80 predominant species and Microbispora, Micromonospora, Nocardioides, Nocardia
81 and Streptosporangium as the common genera. Endophytic actinobacteria have been
82 demonstrated to improve and promote growth of host plants as well as to reduce
83 disease symptoms caused by plant pathogens through various mechanisms, including
84 the production of secondary metabolites, which are used in direct antagonism against
85 pests and diseases (10, 11, 13), changes in host physiology (42) and induction of
86 systemic acquired resistance in plants (17). Another significant function of these D
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87 actinobacteria was found to exhibit antibiotic activity, suggesting that endophytic w
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88 actinobacteria can be an interesting source for bioprospecting. New antibiotics a
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89 Alnumycin, Munumbacin A-D and Coronamycin have been reported from endophytic d

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90 Streptomyces spp. (8, 12). Recently, two novel antitumor anthraquinones, lupinacidins o
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91 A and B were isolated from a new endophytic Micromonospora sp. (43). Moreover, h
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92 new species of endophytic actinobacteria have been increasingly reported (26, 35). :/
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93 Thus, endophytic actinobacteria are expected to be potential sources of new species m
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94 and new bioactive agents. s
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95 Of the myriad of ecosystems on earth, those having the greatest general .o
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96 biodiversity of life seem to be the ones also having the greatest number and most /
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97 diverse endophytes (74). Tropical and temperate rainforests are the most biologically A
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98 diverse terrestrial ecosystems on earth that hold the greatest possible resource for il
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99 acquiring novel microorganisms and their products (74). One of the areas of ,

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100 enormous plant biodiversity is Xishuangbanna, located in P. R. China at the boarder 1
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101 to Myanmar, at the ecotone between the Asian tropics and subtropics, and is b
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102 dominated by tropical seasonal rain forests (88). It contains over 5000 species of u
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103 vascular plants, comprising 16 percent of China’s total plant diversity, with more than t
104 3000 being endemic species (54, 61), many of which have ethnobotanical history.
105 Until this time, only few researches were carried out on the isolation of endophytic
106 actinobacteria and their secondary metabolites from Xishuangbanna (36, 87). As our
107 long term study on endophytic actinobacterial diversity and bioactive metabolites
108 from tropical rainforest medicinal plants in Xishuangbanna, many bioactive
109 endophytic Streptomyces have been isolated (50). However, this is insufficient to
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110 provide a general understanding of tropical rainforest endophytic actinobacteria
111 regarding to their diversity, distribution, and ecology, as well as for further
112 exploitation of diverse functions of this novel microbial source.
113 In the present study, the diversity of rare endophytic actinobacteria associated
114 with medicinal plants from tropical rainforest in Xishuangbanna was investigated by
115 combined special culturing techniques. The selected isolates were also identified by
116 16S rRNA gene analysis. The overall aims of this study were (i) to analyze D
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117 actinobacterial community and reveal whether the investigated rainforest in w
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118 Xishuangbanna represents a valuable source for abundant endophytic actinobacteria a
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119 and new species, (ii) to evaluate their antimicrobial activities and biosynthetic d

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120 potential of related secondary metabolites, and (iii) to study the relationships between o
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121 taxa of these endophytic actinobacteria and applied isolation methods. h
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122 :/
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123 Materials and methods m
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124 Sample collection m
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125 An extensive collection of various plant materials was set up in the tropical r
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126 seasonal rainforest in Xishuangbanna for two years (from November 2005 to May o
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127 2006 and from September 2006 to June 2007) in the Menglun Nature Reserve p
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128 (21◦56’N, 101◦11’S) and Jinghong Nature Reserve (22◦01’-22◦19’N, 100◦47’ il 1
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129 -100◦57’S ). Each plant selected for endophyte isolation was based on its local , 2
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130 ethnobotanical properties including its properties to antibacterial, insecticidal, 9

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131 antitumour, heal wounds and others. No history indicates that the plants had ever been y

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132 previously studied for endophytic actinobacteria. Healthy root, stem and leaf samples e
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133 of each plant were placed in sterile plastic bags and taken to the laboratory, which
134 were subjected to isolation within 96 h. Some representative plants samples of the
135 nearly 90 selected for study are as follows: Phyllanthus urinaria, Kadsura heteroclite,
136 Maesa indica, Rauvolfia verticillata, Paris yunnanensis, Maytenus austroyunnanensis,
137 Gloriosa superba, Scoparia dulcis, Tadehagi triquetrum, Goniothalamus sp.,
138 Cephalotaxus sp., and Azadirachta sp.
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140 Selective isolation producers and media
141 Samples were air dried for 48 h at room temperature and then washed with an
142 ultrasonic step (160W, 15mim) to remove the surface soils and adhered epiphytes
143 thoroughly. After drying, the samples were subjected to a five-step surface
144 sterilization procedure: a 4 to 10-min wash in 5% NaOCl, followed by a 10-min wash
145 in 2.5% Na2S2O3, a 5-min wash in 75% ethanol, then wash in sterile water, and a final D
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146 rinse in 10% NaHCO for 10-min. After thoroughly drying in sterile conditions, the w
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147 surface-sterilized tissues were continuously subjected to drying at 100 ºC for 15 a
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148 minutes. Surface treated tissues were then pretreated by a variety of methods d

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149 described below. o
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150 Method 1. Most of the samples were aseptically crumbled into small fragments and h
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151 directly placed on the selective media and incubated at 28 ºC for 2-8 weeks. :/
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152 Method 2. Some samples (1.0 g) treated as above were mixed in a mortar with 0.5 g m
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153 of sterile powdered calcium carbonate and placed in a Petri dish. 2 ml of sterilized tap s
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154 water was added to the samples to give a moisture environment. The Petri dish was .o
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155 maintained at 28 ºC for 2 weeks and then air-dried at room temperature to a constant /
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156 weight. Parts of the samples were directly diluted to 10-4 with sterile water and A
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157 spread-plated onto media. The other mixtured samples were continuously enriched il
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158 according to Otoguro et al. (66). Samples were placed in a glass vessel and flooded ,

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159 with 50 ml of 10 mmol-1 phosphate buffer (pH 7.0) containing 10% plant extract or 01
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160 soil extract at 28 ºC for 5 h to liberate actinomycete spores. A portion (10 ml) of the b
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161 flooding mixture was transferred into a centrifuge tube and centrifuged at 1500×g for u
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162 30 min. After settling for 30 min, a portion of the supernatant enriched with spores t
163 was serially diluted with sterile tap water and plated onto media.
164 Method 3. Some plants, e.g. Maytenus austroyunnanensis, were aseptically
165 crumbled into small fragments and isolated using the combined enzymatic hydrolysis
166 and differential centrifugation method (44). The homogenate was centrifuged at 200 g
167 for 20 min at 4 ºC, and the supernatant was subsequently centrifuged at 3000 g for 30
168 min(4 ºC) to collect the sediments, which will be diluted to 10-2 with sterile water and
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169 spread-plated onto media.
170 200 µl each of the samples pretreated dilutions was spread over the surface of solid
171 media that were designed for cultivation of actinobacteria (Table 1). The inoculated
172 plates were incubated at 28 ºC for 2–4 weeks. The pH of all media was adjusted to
173 7.0-7.4. All media were amended with nalidixic acid (50 mg l-1) and nystatin (100 mg
174 l-1) to supress the Gram-negative bacterial and fungal gowth. As the colonies appeared
175 from the plates, they were observed and selected carefully according to their D
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176 characteristics. Special attention was given to the non-mycelium forming w
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177 actinobacteria. They were inoculated onto yeast extract-malt extract agar (ISP 2) a
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178 slants and incubated at 28 ºC for 2–3 weeks. d

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179 o
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180 Effectiveness of surface sterilization h
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181 Two experiments were carried out to check the validation of the sterilization :/
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182 procedures. Firstly, the surface-sterilized tissue was imprinted onto the ISP 2 agar, m
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183 incubated at 28 ºC, then check for microbial growth. Secondly, the surface sterilized s
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184 samples were washed in sterile distilled water thrice and then soaked in 5ml sterile .o
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185 water and stirred for 1min. An aliquot of 0.2ml suspension was then inoculated on to /
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186 ISP 2 agar plates, incubated at 28 ºC, and observed for microbial growth. If no A
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187 microbial growth occurs on the media surface, the sterilization is considered il
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188 complete. ,

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189 1
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190 Preliminary identification of actinobacteria b
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191 Isolates were tentatively grouped and dereplicated by observing the morphological u
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192 and cultural characteristics, including the characteristics of colonies on the plates and t
193 slants, the presence of aerial mycelium and substrate mycelium, spora mass colour,
194 distinctive reverse colony colour, diffusible pigment, and sporophore and spore chain
195 morphology. Many colonies that were similar in colour, shape and size, were
196 observed for hyphal length and structure with light microscopes, which allowed them
197 to be segregated into distinct isolates. Cell wall type was also determined on the base
198 of the occurrence of isomers of diaminopimelic acid to exclude the Streptomycetes
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199 from other spore-forming actinomycetes as whole-organism hydrolysates of
200 Streptomyces strains contain the LL-isomer and the latter the meso- diaminopimelic
201 acid. The diagnostic sugars of the representatives of each group were also detected
202 (38). Based on the preliminary grouping, 46 isolates were selected for further
203 research.
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205 DNA extraction, sequencing and analysis D
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206 The 46 selected isolates were subjected to 16S rRNA gene sequence analysis for w
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207 precise genera and species identification. The identity of the organisms was a
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208 determined based on partial or nearly full length of 16S rRNA gene sequence analysis. d

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209 Genomic DNA of each isolate was extracted using a method of Li et al. (53). The 16S o
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210 rRNA genes from pure cultures were amplified using the primer pair PA and PB h
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211 (Table 2). Polymerase chain reaction was carried out under the following conditions: :/
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212 initial denaturation at 94°C for 4 min, followed by 30 cycles of 94°C for 1 min, 55°C m
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213 for 1 min and 72°C for 2 min, with a final extension 72°C for 10 min. The reaction s
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214 mixture (50 µl) contained dNTPs (0.25 mM each), 1×reaction buffer (20 mM Tris pH .o
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215 8.4, KCl 50 mM), MgCl2 (3 mM), primers (1 µM each), Taq DNA polymerase (1.25 o/
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216 units) and 1 ng of template DNA (all PCR reagents were purchased from TaKaRa, A
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217 Dalian, China). The PCR products were separated by agarose gel electrophoresis and il
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218 purified using the QIA quick Gel Extraction Kits (Qiagen, Hilden, Germany) and ,

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219 sequenced on the ABI PRISM 3730 sequencer. 1
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220 The determined 16S rRNA gene sequences were compared with the b
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221 GenBank/EMBL/DDBJ databases by using the BLASTN (1) search program. A u
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222 phylogenetic tree was constructed with the neighbour-joining (71) method using the t
223 software package MEGA 3.1 (46) after pairwise alignments using the CLUSTAL_X
224 1.8 program (80). The stability of relationships was assessed by performing bootstrap
225 analyses (28) of the neighbour-joining data based on 1000 resamplings. DNA
226 sequences were deposited to GenBank and the Accession numbers were shown in
227 Table 3.
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228 Detection of PKS-I, PKS-ⅡⅡⅡⅡand NRPS sequences
229 Three sets of degenerate primers for the amplification of genes encoding polyketide
230 synthases (PKS-І, PKS-Ⅱ) and nonribosomal peptide synthetase (NRPS) from the
231 tested isolates were carried out as recommended by Ayuso-Sacido and Genilloud (4)
232 and Ketela et al. (45) (Table 2). The reaction mixture contained 2.5 units of Taq DNA
233 polymerase, 1 mM MgCl , 0.4 mM dNTPs, 2 µM of each primer and 5% DMSO in a
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234 50 µl reaction volume. Control reactions, without the actinobacterial DNA template. D
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235 Thermocycling conditions consisted of one denaturation step of 94°C for 5 min w
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236 followed by 30 amplification cycles of 94°C for 1 min, 57°C (for K1F/M6R, a
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237 A3F/A7R) or 58°C (for KSα/ KSβ) for 1 min, 72°C for 2 min followed by a final d

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238 extension at 72°C for 5 min. o
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239 h
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240 Fermentation, extraction and evaluation of the antimicrobial activity :/
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241 Each isolate was cultured in soybean mannitol liquid medium [soybean flour 12 g m
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242 and mannitol 20 g in 1000 ml tap water (pH 7.2–7.4)] at 28°C and shaked at 180 s
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243 rev/min). After 7-12 days of cultivation, the fermentation broth was extracted with .o
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244 ethanol. Each organic solvent extract was then evaporated under reduced pressure to /
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245 yield an ethanol extract. The ethanol extracts were then used for the antimicrobial A
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246 activity screening. The inhibitory effect of the extract obtained from endophytic il
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247 actinobacteria was tested using the paper disk (7 mm diameter) assay method (59). ,

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248 25µl of ethanol extract suspension were pipetted onto each disc and incubated at 37°C, 1
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249 followed by measurement of the diameters of the inhibition zone after 24 h. 25µl b
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250 ethanol was used as the control. Pathogenic bacterial organisms Staphylococcus u
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251 aureus, Bacillus subtilis, Pseudomonas aeruginosa and yeast Candida albicans were t
252 used as indicator organisms to determine the antimicrobial activity. The pathogenic
253 microorganisms were deposited in the Yunnan institute of microbiology, Yunnan
254 University.
255 Results and Discussion
256 Evaluation of surface sterilization protocol
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257 Surface sterilization is important for studying endophytes. Imprinted agar of ISP 2
258 plate from each surface-sterilized sample showed no microbial growth after 15 days
259 incubation at 28°C. In addition, ISP 2 agar plates spread with the last washing water
260 from plant samples also did not emerge colonies of microorganisms after two weeks
261 incubation. This indicated that the five steps surface sterilization protocol was
262 effective to kill the epiphytic microorganisms. Thus the subsequent isolates can be
263 considered as true endophytic actinobacteria. D
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264 A 2.5% sodium thiosulfate solution was used instead of sterilized water rinses in w
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265 this study in order to remove the chlorine cover left on the plants surface by a
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266 hypochlorite disinfection. This was based on the consideration that even though plant d

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267 tissues were rinsed thoroughly with sterilized water after treated with NaOCl, there o
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268 were still toxic compounds present on their surfaces, which may kill endophytes or at h
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269 least stressed them so much that they were unable to form colonies on the plates. By :/
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270 our tentative experiments, the increase in the number of CFU was indeed obvious m
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271 compared to the control (supplementary Table S1). This was consistent with the result s
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272 of the effects of rice seed surface sterilization with 2.0% sodium thiosulfate instead of .o
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273 water rinses (60), and the fact that thiosulfate can suppress the detrimental effects of /
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274 hypochlorite on skin (34). It is also known that the alkaline environment favors the A
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275 growth of actinomycetes but not for endophytic fungi. The growth of fungal il
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276 endophyte was effectively inhibited by soaking the plant tissues in 10% NaHCO3 ,
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277 solution in our study so that the actinobacterial endophytes could grow out of tissues 1
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278 before fungi overgrow the samples and mask the actinobacteria. The present surface b
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279 sterilization method was effective to help us acquire endophytic actinobacteria. u
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280 t
281 Selective isolation of culturable endophytic actinobacteria from medicinal plants
282 On the basis of characteristic colonial morphology, notably the ability to form
283 aerial hyphae and substrate mycelia, organisms putatively identified as actinobacteria
284 were selected. In total, 2174 actinobacterial strains were isolated from nearly 90
285 selected plant samples. According to the preliminary morphological identification, the
286 most abundant genus was Streptomyces (87%), which was consistent with the other
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