INTERNATIONAL JOURNAL OF INFORMATION AND COMPUTING SCIENCE ISSN NO: 0972-1347

Isolation of Two Endophytes from Glebionis coronaria (L)

Gajendra B. Singh1,*, Gaurav Mudgal1, Mayank Singhal2, Maninder Singh2, Abdul Hafeez2, Rashi Sharma2, Khagendra Singh2 1University Institute of Biotechnology, Chandigarh University, Mohali, Punjab, India 2Department of Biotechnology, Institute of Engineering and Technology, Mangalayatan University, Aligarh, UP, India

Abstract Glebionis coronaria (L), formerly known as Chrysanthemum coronarium (L) is a plant hailing from the daisy family, and belongs to the Mediterranean region. Besides its use as an ornamental flowering plant, its leafy foliage also finds its culinary use as a vegetable in many Asian cuisines, particularly China, Taiwan, Korea and Japan, as also consumed raw by the locals of Crete. Besides, its bioresource potential has been evidenced in literature viz., use as low cost natural alternative for removal of dyes, removal of Cadmium and Cobalt ions from aqueous solution, and for it valued essential oils. The literature also indicates their reserves of bioactive metabolites such as antioxidants, plant growth inhibitors, insect antifeedants, epoxy acids, sesquiterpene lactones, among many others. Endophytes are endosymbiont microorganisms growing inside the plants which have evidenced a plethora of applications viz., medicine, perfumery, secondary metabolites, growth factors to name a few. There are very few reports on isolation of endophytes from Glebionis coronaria. In this work we report preliminary isolation and characterization of two putative endophytes, one a bacterial species and other of fungal origin.

Keywords: Endophytes, Glebionis coronaria (L), Chrysanthemum coronarium (L), MS Media, Bio-resource

Introduction Glebionis coronaria (L), formerly known as Chrysanthemum coronarium (L) is a flowering plant hailing from the daisy family, and belongs to the Mediterranean region. Besides its use as an ornamental flowering plant, its leafy foliage also finds its culinary use as a vegetable in many Asian cuisines, particularly China, Taiwan, Korea and Japan, as also consumed raw by the locals of Crete [1, 2]. Besides, recently its bioresource potential has been evidenced in literature viz., use as low cost natural alternative for removal of dyes, removal of Cadmium and Cobalt ions from aqueous solution, and for it valued essential oils. The literature also indicates their reserves of bioactive metabolites such as antioxidants, plant growth inhibitors, insect antifeedants, epoxy acids, sesquiterpene lactones, among many others. To our knowledge, reports on isolation and identification of endophytes from this plant are negligible [3, 4]. To settle above deficit and tap bioresources from this daisy family member, we have attempted in vitro tissue culture establishment of flowering plant Glebionis coronaria growing within the campus of Mangalaytan University, Aligarh, India.

*Corresponding Author Email ID: [email protected]

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Materials and Methods

Sampling

Whole plant bearing flowers was uprooted from a bush exclusively growing Glebionis coronaria and brought to the biotechnology laboratory of Mangalayatan University. A voucher specimen was sent to the first two authors for help in taxonomic identification of this plant. The sample plants were used for surface sterilization and explant inoculation by the same day of sampling to avoid vitrification [5, 6].

Surface Sterilization and inoculation of explants

Explants included leaves, stems, and parts of flowers, flower buds. Individual explants were excised using a sharp scalpel and processed by initial rinsing in running tap water for 1 hour, under treatment with 2-3 drops of mild detergent (Tween 20) followed by brief (90 seconds) treatment with 0.1% mercuric chloride and a subsequent 3 three washes in 70% ethanol for 3 minutes each. Intermittent to ethanol washes, rinsing with ice cold sterile distilled water was carried out for 2 minutes each wash. The explants were excised at surfaces in order to expose interior to medium [7].

Media and Culture conditions

Explants were placed over 0.8% agar substratum containing basal Murashige and Skoog media (MS) [8]. Plant tissue and endophyte culture media components (MS media, LB=Luria Bertani media, and PD=Potato Dextrose media) were from Himedia and Sigma-Aldreich. Explants were maintained in tissue culture petridishes (J-SilR), test tubes (BorosilR) at 22oC, 16 hours light under fluorescent tubes and 8 hours dark (using a timer setting on the tissue culture rack), 85% humidity. No subculturing was done for 15 days of incubation for any of the replicates [9, 10].

Isolation of endophytes

Any contamination within 2 -3 days post inoculation was ignored and corresponding culture vessels were discarded. For 15 days incubation in culture room any observed microorganismic growth was recorded. Under sterile laminar air flow hood, subculturing for any putative endophytic species was performed into replicates in semisolid MS, LB, media and PD media. Growth characteristics and morphology was recorded for each of the microorganismic species under a microscope or visibly. Glycerol stocks of each isolates were maintained at ultra-deep freezing conditions following standard protocols. Previously established procedures for standard isolation of endophytes from this plant were used [3].

Results and discussion

After about 15 days incubation in culture room, most of the explants exhibited microbial contamination. To surprise our visible observation, contaminating agents included only one each of either a pink coloured bacterium (exhibiting small colonies and slow growing characteristics) (Figure 1 b-i) or a green fungus (Figure 1 b-ii) in almost all explant types. Moreover in none of the 20 replicates did the two contaminating agents grow together on a single explant. This indicates a possible antagonism while also coexistence of two endophytes in the anthers.

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a i) Ii) b

C

d

f e Figure 1. a. Close up of Glebionis coronaria used for explant preparation and surface sterilization. b. i) pink colored bacteria around the stamen explant and individual small colonies which did not overgrow in size even after 15 days of culture on MS-Media. b.ii) Green fungus overgrown from the explant; C. i) and ii) Light Microscopy observations at low and high magnification infer these bacteria (in b.i) as bacilli and morphologically similar to; d) Serratia marcescens [a referenced zoomed image] as per the colony color (in c) and growth delay as recorded; e. streaked fungi on MS agar plate shows mycelia structures extending from along the streaks with possibly sporangial structures erupting over the streaks. Also none of the replicates carrying this bacteria showed coexistence with any other agent; Note that none of the20 replicates carrying this fungus showed coexistence with the pink colored bacteria (in c) or any other microbe; f. Light microscopy observation of the spores (shown) and other morphological details suggest the fungi to be Aspergilus niger. Biochemical tests and molecular analysis underway would confirm the identity of the putative endophytic isolates. Conclusions

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Mercuric chloride is a highly antimicrobial agent, with action against both fungi and bacteria, but frequently also kills the seeds/plant materials [11]. At low concentrations (up to 0.1 %) it is perhaps the most effective disinfective agent for seeds with soil-borne and epiphytic fungi. The results obtained till now infer that most of the contaminants were obtained after 12-15 days of inoculation, indicating that these may be the endogenously evolving microbes, putatively endophytes of Glebionis coronaria (Figure 1a). Usually a pink colored, slow growing and small colony phenotype is shown by Serratia marcescens which is gram negative bacillus belonging to the family Enterobacteriaceae (Figure 1b-i). Though studies are undergoing, growth of only two exclusively either one specific bacterial species or that of fungi from the stamen explants indicates a possible antagonism while also coexistence of two endophytes in the anthers. Serratia marcescens is an infectious bacilli causing hospital-acquired infections (HAIs), particularly catheter- associated bacteremia, urinary tract infections and wound infections [12]. Studies on the antagonistic profiles of the fungi (which preliminarily appears to be Aspergillus niger) isolated from Glebionis coronaria in this study may be useful in isolation of antimicrobial agents for this bacteria and possibly other infectious agents [13-15]. Further studies on identification of the microbial species and their valuable bio-reserves are underway.

Acknowledgment

The authors are thankful to the Department of Biotechnology, UIBT, Chandigarh University for critical review of the manuscript. The views presented here do not reflect in part and/or as a whole an outcome of any mandated research activity(s) of the institution(s) to which the authors are and/or had been affiliated to but in actual are products of authors’ independent observations. GM and GBS are recipients of generous financial assistance from University Committee for Research and Development (UCRD), Chandigarh University. Authors from Mangalayatan University were supported by Institutional Research Grant from Institute of BioMedical Education and Research (IBMER), Mangalayatan University. There exists no conflict of interest amongst the authors. No animal was harmed during the activities to achieve the experimental objectives. Isolation of plants did not involve works in protected areas. Effluents arising from sample processing and sterilization were treated via effluent treatment plants at Mangalayatan University before moving them into water resources. Good Laboratory Practices were exercised throughout the tenure of project.

References

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[10] Ohtsuka, H., & Inaba, Z. (2008). Intergeneric hybridization of marguerite (Argyranthemum frutescens) with annual chrysanthemum (Glebionis carinatum) and crown daisy (G. coronaria) using ovule culture. Plant biotechnology, 25(6), 535-539. [11] Bhojwani, S. S., & Razdan, M. K. (1986). Plant tissue culture: theory and practice (Vol. 5). Elsevier. [12] Hejazi, A., & Falkiner, F. R. (1997). Serratia marcescens. Journal of medical microbiology, 46(11), 903-912. [13] Kang, S. W., Park, Y. S., Lee, J. S., Hong, S. I., & Kim, S. W. (2004). Production of cellulases and hemicellulases by Aspergillus niger KK2 from lignocellulosic biomass. Bioresource technology, 91(2), 153-156. [14] Singh, O. V., Kapur, N., & Singh, R. P. (2005). Evaluation of agro-food byproducts for gluconic acid production by Aspergillus niger ORS-4.410. World Journal of Microbiology and Biotechnology, 21(4), 519-524. [15] Sabu, A., Pandey, A., Daud, M. J., & Szakacs, G. (2005). Tamarind seed powder and palm kernel cake: two novel agro residues for the production of tannase under solid state fermentation by Aspergillus niger ATCC 16620. Bioresource Technology, 96(11), 1223-1228.

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