INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY ISSN Print: 1560–8530; ISSN Online: 1814–9596 16–339/2017/19–2–219–225 DOI: 10.17957/IJAB/15.0234 http://www.fspublishers.org Full Length Article Evaluation of Antagonistic Activity of Actinobacteria in Saxicolous Lichens Dongsheng Wang* and Haike Ren College of Life Science, Shanxi Normal University, Linfen, Shanxi 041000, China *For corresponding author: [email protected] Abstract Actinobacteria are important producers of novel bioactive compounds. New sources need to be explored for isolating previously unknown bioactive compound-producing actinobacteria. We evaluated the potential of saxicolous lichens as a natural source of novel bioactive actinobacterial species. Saxicolous lichen samples were collected from three climatic belts at different elevations (1600–3400 m) on Qin Mountain, Shaanxi Province, China. Actinobacteria were cultivated, enumerated, and isolated using serial dilution and spread-plate techniques under normal nutrient and oligotrophic conditions. Antimicrobial activity of actinobacterial isolates was analyzed using an agar block method against fifteen typical bacterial and fungal species and plant pathogens. The dominant isolates and isolates with broad-spectrum antagonistic activity were identified by 16S rRNA-based sequence analysis. Results showed that colony counts, total number of species, and the number of bioactive species of actinobacteria in saxicolous lichens were higher in elevation <3000 m than in elevation >3000 m. The dominant isolates were classified into 42 species of 10 genera. Antagonistic activities were detected in approximately 66% of the actinobacteria isolates. Of these, 42 isolates (28%) showed broad-spectrum antagonistic activity against ≥5 microorganisms tested. In conclusion, the saxicolous lichen possesses a high diversity of actinobacteria and serves as a natural source of bioactive compound-producing actinobacteria. © 2017 Friends Science Publishers Keywords: 16S rRNA; Gause’s synthetic agar; Modified humid acid agar; Oligotrophic media; Qin Mountain Introduction might be involved in microbe-microbe and microbe-host communication (Yim et al., 2007; Yoon and Nodwell, 2014). Actinobacteria are the main source of antibiotics which have Lichens are symbiotic associations consisting of fungi and produced 100–120 out of 150 clinical and agricultural photosynthetic partners like green algae and cyanobacteria. antibiotics (Bérdy, 2005). Isolation of novel bioactive They are abundant as epiphytes on plant, bare rock and compound-producing actinobacteria is a fundamental work exposed soil surfaces in various environments including for research and development of new drugs. Actinobacteria some of the most extreme environments on Earth, such as are widely distributed in natural ecosystems. Extensive high mountains, hot deserts and arctic tundra. Saxicolous studies have explored soils, sediments and water bodies lichens could adapt more harsh conditions than those from which numerous bioactive actinobacterial species colonizing on other media because bare rock surface is were isolated. Representative genera are Streptomyces extraordinarily oligotrophic, dry in most time of the year and Micromonospora (Bérdy, 2005). In recent decades, and irradiated strongly by ultraviolet. Although a number of it has become increasingly difficult in finding novel individual bioactive isolates or species in lichens have been bioactive actinobacteria in the above-mentioned natural described (Singh et al., 1997; Davies et al., 2005; ecosystems. Exploring new sources is urgent. In this Motohashi et al., 2010; Brana et al., 2015), little is known context, alternative ecosystems have attracted about the diversity of the microbial community especially substantial attention of researchers for actinobacteria actinobacteria inhabiting saxicolous lichens. Recently, screening, including extreme environments (Okoro et al., González et al. (2005) have isolated a wide diversity of 2009), animal feces (Jiang et al., 2013), beehives actinobacteria from saxicolous and arboricolous lichens, of (Promnuan et al., 2009), wasp and swallow mud nest which many isolates possessed biosynthetic genes. (Kumar et al., 2012), termite guts (Watanabe et al., 2003), Knowledge remains lacking regarding diversity and the internal and external environments of plants (Verma et antagonistic activities against plant pathogenic al., 2009) and microbial symbioses (Suzuki et al., 2016). microorganisms of actinobacteria in saxicolous lichens at It is hypothesized that microbial bioactive compounds different elevations. To cite this paper: Wang, D. and H. Ren, 2017. Evaluation of antagonistic activity of actinobacteriain in saxicolous lichens. Int. J. Agric. Biol., 19: 219‒225 Wang and Ren / Int. J. Agric. Biol., Vol. 19, No. 2, 2017 To this end, the present study investigated counted. Data are reported as colony-forming-unit (CFU)/g actinobacterial populations residing in saxicolous lichens at stove-dry lichen. The colony numbers were compared different elevations under normal and oligotrophic between different elevations by t-test in SAS 9.0 statistical conditions. We evaluated the potential for isolating novel software (SAS Institute Inc., Cary, NC, USA). bioactive compound-producing actinobacteria from the special ecosystem of saxicolous lichens by colony counts Antimicrobial Activity Assay and species numbers of actinobacteria, and evaluation of their antagonistic activity followed by identification of Antimicrobial activity of actinobacteria isolates was dominant and valued actinobacterial species. analyzed using an agar block method against four bacterial species and eleven fungal species provided by the Materials and Methods Microbiology Laboratory in Shanxi Normal University. The bacterial species included Escherichia coli E1, Bark Sampling Staphylococcus aureus S4, and two pathogens of konjac soft rot, Serratia sp. H1 and Dickeya dadantii subsp. Dadantii This study was carried out in the north Qin Mountain D3; the fungal species included Penicillium sp. P1, Candida (33°57′–34°58′N, 107°45′–107°53′E), Shaanxi Province, tropicalis C1, and nine plant pathogens, Verticillium dahliae China. Ten saxicolous lichen samples were collected from 3 V2, Fusarium oxysporum FO1, F. solani (Mart.) Sacc FSS1, climatic belts: 5 from alpine cold temperate zone (elevations F. sulphureum FS1, F. oxysporum f. sp. cucumerinum FOC1, of 1500‒3000 m), 3 from alpine subfrigid zone (elevations F. oxysporum f. sp. niveum FON1, F. solani FS3, F. of 3000‒3350 m) and 2 from alpine frigid zone (elevations oxysporum f. sp. vasinfectum FOV1, and Didymella above 3350 m). The samples were collected with sterile bryoniae DB1. blades, individually sealed in sterile polyethylene bags, transported to the laboratory within 7 h, and stored in the Antagonistic Potentiality Assay of Lichen Actinobacteria dark at 4°C until use. Antagonistic potentiality of lichen actinobacteria (APLA) Actinobacteria Isolation was calculated by considering the number and antimicrobial spectrum of actinobacterial isolates in the lichen ecosystem Saxicolous lichen samples were grinded using sterilized using the following equation (Zhu et al., 2011): pestles and mortals. Serial dilution and spread-plate n m n techniques (Williams and Davies, 1965) were used to APLA(%) Tn / Tn 100 1 1 1 isolate actinobacteria. Serial dilutions were prepared by adding the 3.0 g of grinded lichen to 27.0 mL of sterile Where m and n are the numbers of tested lichen distilled water (10-1) in a conical flask, followed by samples and actinobacterial isolates with antagonistic oscillation at 160rpm for 10 min and further dilution to activity, respectively; Tn is the number of target 10-3. The dilutions (0.05 mL aliquots) were inoculated to microorganisms to which the actinobacterial isolate is the agar media by spread plating. Four agar media were antagonistic. tested: Gause’s synthetic agar (soluble starch 20.0 g; KNO3 1.0 g; K2HPO4 0.5 g; MgSO4·7H2O 0.5 g; NaCl Identification of Actinobacteria Isolates 0.5 g; FeSO4 0.01 g; agar 10.0 g; distilled water 1000 mL), modified humic acid agar (humic acid 10.0 g; The dominant actinobacteria isolates and isolates with Na2HPO4 0.5 g; KCl 1.0 g; MgSO4·7H2O 0.05 g; CaCl2 broad-spectrum antagonistic activity were identified by 16S 1.0 g; agar 10.0 g; distilled water: 1000 mL), oligotrophic rRNA-based sequence analysis. Actinobacteria DNA was Gause’s synthetic agar (Gause’s synthetic agar at one extracted from pure isolates using the method described fifty the recommended concentration) and water agar by Saito and Miura (1963). Partial 16S rRNA gene (agar 10.0 g; distilled water: 1000 mL). All media were fragments were amplified by polymerase chain reaction supplemented with 80 mg/L potassium dichromate to inhibit (PCR) using the bacterial primers 27F: 5'- the growth of bacteria and fungi. After inoculation, all plates AGAGTTTGATCCTGGCTCAG-3' and 1541R: 5'- were incubated at 28°C for 15 days. Actinobacterial AAGGAGGTGATCCAGCCGCA-3'. Amplification was colonies were identified by visual examination of the carried out in a DNA Engine thermal cycler (BIO-RAD, cultural and morphological characteristics; microscopic USA), using a 50µL reaction mixture containing 4µL examination was performed if needed. Morphologically Taq DNA polymerase (2.5 U/µL, Genscript, Nanjing), 5 distinct colonies were transferred onto Gause’s synthetic µL 10× buffer (Transgene, Beijing), 1 µL 20 mM agar slants separately, incubated at 28°C for 7 days, and deoxynucleoside