Isolation, Diversity, and Antimicrobial Activity of Rare Actinobacteria from Medicinal Plants of Tropical Rain Forests in Xishuangbanna, China

Abstract

Endophytic actinobacteria are relatively unexplored as potential sources of novel species and novel natural products for medical and commercial exploitation. Xishuangbanna is recognized throughout the world for its diverse flora, especially the rain forest plants, many of which have indigenous pharmaceutical histories. However, little is known about the endophytic actinobacteria of this tropical area. In this work, we studied the diversity of actinobacteria isolated from medicinal plants collected from tropical rain forests in Xishuangbanna. By the use of different selective isolation media and methods, a total of 2,174 actinobacteria were isolated. Forty-six isolates were selected on the basis of their morphologies on different media and were further characterized by 16S rRNA gene sequencing. The results showed an unexpected level of diversity, with 32 different genera. To our knowledge, this is the first report describing the isolation of Saccharopolyspora, Dietzia, Blastococcus, Dactylosporangium, Promicromonospora, Oerskovia, Actinocorallia, and Jiangella species from endophytic environments. At least 19 isolates are considered novel taxa by our current research. In addition, all 46 isolates were tested for antimicrobial activity and were screened for the presence of genes encoding polyketide synthetases and nonribosomal peptide synthetases. The results confirm that the medicinal plants of Xishuangbanna represent an extremely rich reservoir for the isolation of a significant diversity of actinobacteria, including novel species, that are potential sources for the discovery of biologically active compounds.The class Actinobacteria accounts for a high proportion of soil microbial biomass and contains the most economically significant prokaryotes, producing more than half of the bioactive compounds in a literature survey (46), including antibiotics (6), immunosuppressive agents (55), antitumor agents (18), and enzymes (64). Actinobacteria belonging to the genus Streptomyces, in particular, are excellent producers. The emergence of drug resistance in many bacterial pathogens and the current increase in the number of fungal infections has caused a resurgence of interest in finding new reserves of biologically active compounds (63). As the search for novel natural products continues, it becomes apparent that the rate of discovery of new compounds from soil streptomycetes has decreased, whereas the rate of reisolation of known compounds has increased (28). Recently, evidence has accumulated that rare actinomycete species, which are often very difficult to isolate and cultivate, might represent a unique source of novel biologically active compounds (4). On the other hand, new microbial habitats need to be examined in the search for novel bioactive compounds. One biologically important but relatively overlooked niche is the inner tissues of higher plants. Early studies have demonstrated that some actinobacteria can form intimate associations with plants and colonize their inner tissues. Frankia species and Streptomyce...

Isolation and characterization of culturable endophytic actinobacteria associated with Artemisia annua L.

“…It can be considered that the various isolation methods employed in this study were productive and resulted in the successful isolation of numerous endophytic actinobacteria. The first method used is the simplest and most commonly used (Coombs and Franco 2003;Qin et al 2009). This method was used for selectively isolating fast-growing streptomycetes and other actinobacteria, but there was some interference from contaminating fungi.…”

“…For this method, the optimal time and duration of homogenization needs to be determined according to the properties of the sample. The medium described previously by Qin et al (2009) are useful for A. annua endophytic actinobacteria isolation. Moreover, the addition of a certain amount of salt or plant extracts into the isolation medium is effective as well as using dulcitol or sodium succinate as a carbon sources.…”

“…After drying at room temperature, the tissue segments were surface-sterilized by immersing in 0.1% Tween 20 for 1 min, followed by 5% (available Cl -) NaClO for 4 min (leaf samples) or 6 min (stem and root samples). The samples then were rinsed in 2.5% (w/v) Na 2 S 2 O 3 for 10 min to remove the residual chlorine (Miche and Balandreau 2001;Qin et al 2009) and they were washed three times with sterile H 2 O. The last step was to immerse the samples in 70% (v/v) ethanol for 4 min (leaf samples) or 6 min (stem and root samples), then followed by a minimum of three washes with sterile water.…”

“…Endophytic actinobacteria, as one of the significant residents in plant tissues, have attracted more attention in recent years since there is an ever increasing need for novel organisms and their bioactive products. There have been numerous reports focusing on these useful microorganism, which have been isolated from diverse economic plants such as wheat (Coombs and Franco 2003), rice (Tian et al 2007), potato (Sessitsch and Berg 2004), carrots (Surette et al 2003), tomato, rape (Nejad and Johnson 2000) and citrus (Araujo et al 2002) and various medicinal plants (Taechowisan et al 2003;Zin et al 2007;Li et al 2008;Qin et al 2009). These organisms seem to have important roles in plant development and health because they can promote plant growth by nutrient assimilation or phytohormone production.…”

Endophytic actinomycetes: promising source of novel bioactive compounds

“…collected from 113 plants and they were categorized on the basis of 16S rRNA gene sequences into six families; Streptosporangiaceae (40%), Streptomycetaceae (27%), Thermomonosporaceae (16%), Micromonosporaceae (8%), Pseudonocardiaceae (8%) and Actinosynnemataceae (2%). 11 Notably, the genus Streptomyces in the family Streptomycetaceae accounted for only 26%, meaning that rare actinomycetes (non-Streptomyces) make up 74% of the total and other reports also indicated that rare actinomycetes are majority in plant, [12][13][14] although Kaewkla and Franco reported that Streptomyces strains accounted for 72% of endophytic actinomycetes isolated from Australian native trees. 15 It remains to be seen whether this promising new source of bioactive materials will bear fruit, but the signs are promising.…”