Bioprospecting of endophytic bacterial plethora from ...

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PLANT SCIENCES FEED ORIGINAL ARTICLE

2013

ISSN : 2231 - 1971 http://psf.lifescifeed.com

BIOPROSPECTING OF ENDOPHYTIC BACTERIAL PLETHORA FROM MEDICINAL PLANTS

SYED BAKER AND SREEDHARAMURTHY SATISH* Plant and microbe interaction has fostered the researcher towards harnessing the valuable potentials bearing pharmaceutical importance. One such area gaining impetus importance is endophytic plethora

which remains largely untapped reservoir. Hence the present study evaluates in bioprospecting of

bacterial endophytes from five medicinal plants viz, Annona squamosa L., Coffea arabica L., Tridax procumbens L., Euphorbia hirta L. and Mimosa pudica L. upon evaluation the preliminary investigation resulted in isolation of three hundred and thirty two endophytes which will be forged towards isolation of bioactive compounds for antimicrobial activity in future studies.

KEYWORDS: Biodiversity, Plants, endophytes, bacterial endophytes.

Cite this article as: Syed Baker and Sreedharamurthy Satish. Bioprospecting of endophytic bacterial plethora from medicinal plants. Plant Sciences Feed, 2013; 3 (3): 42-45 AUTHOR AFFILIATIONS: EMAIL :

Herbal Drug Technological Laboratory, Department of Studies in Microbiology, University of Mysore, Manasagangotri, Mysore 570 006 Karnataka (India) [email protected]

Isolation of microorganism inhabiting plants has a wide chasm, upon closer inspection, their interaction realms with hosts gives knit towards the beneficial aspects for host and its counter partner. Endophytes are microorganisms that reside asymptomatically in the tissues of higher plants and reported to be promising source of bioactive compounds. Bioactive compounds discovery is a multidisciplinary endeavor that includes the search for new pharmaceuticals from various sources. Endophytes secrete structurally diversified bioactive compounds as secondary metabolite via fermentation process and can be inexhaustible and sustainable resource. Perusal of studies reported so far envisioned that endophytes forms ware house of biologically active compounds. Modern technologies have opened new avenue on endophytic research for highly sustainable and economically feasible novel natural products which are presumed to push forward the frontiers of drug discovery [3, 4]. The spawning scientific literatures have promoted us to execute in isolating endophytes from various significant plants whose reports have been briefly summarized in the present investigation. Selection of plants was inspired based on the records of their scientific

Annona squamosa L. genus name “Annona” is from the Latin pertaining yearly produce, Annona squamosa Linn. It is commonly known as custard apple, is a woody, semi deciduous tree grown throughout India. It is a native species of West Indies and was introduced to Taiwan during the 17th century. This plant is reputed to possess several medicinal properties which has been conferred by various studies and evaluated against cardiac disease, diabetes hyperthyroidism and cancer. The root is considered as a drastic purgative [5,6]. Coffea arabica L. is the second-largest worldwide commodity and undoubtedly coffee has been one of the most popular beverages across the globe due to the fact that its main active constituents are overwhelmed with medicinal properties. Coffee and its plant have perused to have a rich medical history and the therapeutic benefits which are being supported by a rapidly growing and significant level of scientific validation [7, 8]. Euphorbia hirta L. belongs to the plant family Euphorbiaceae and genus Euphorbia. Euphorbia hirta is used in the treatment of gastrointestinal disorders (diarrhea, dysentery, intestinal parasitosis, etc.), bronchial and respiratory diseases (asthma, bronchitis, hay fever, etc.), and in conjunctivitis. Hypotensive and tonic properties are also reported in E. hirta. The aqueous extract exhibits anxiolytic, analgesic, antipyretic, and anti-inflammatory activities. The stem sap is used in the treatment of eyelid styes and a leaf poultice is used on swelling and boils. Extracts of E. hirta have been found to show anticancer activity. The root exudates exhibit nematicidal activity against juveniles of meloidogyne incognita [9, 10]. Mimosa pudica L. is a small and short-lived shrub, native to South America, but has become a pan-tropical weed. It is often grown for its curiosity value: When touched, it quickly folds its leaflets and pinnae and wilt downward at the petiole attachment. Whole plant is evaluated to treat headache, alopecia (Panama), diarrhea, dysentery (Haiti), insomnia (Panama, Trinidad), as antidote to poison (Venezuela), sedative (Guatemala, Java). Some Chinese medicine preparations containing M. pudica are also tested and used treating schizophrenia, pervigilium, arresting bleeding, skin diseases, hyperosteogeny, arthralgia, hyperosteogeny, AIDS [11, 12]. Tridox procumbens L. is a common medicinal herb used by ethno medical practitioners, belonging to the family asteraceae. It is best known as a widespread weed and pest plant. Leaf extracts are used to treat infectious skin diseases in folk medicines. The scientific literatures on this medicinal herb are enormous and have reported to confer activity against gastritis, heart burn, antioxidant, anti diabetic, antimicrobial, antiseptic, insecticidal and antiparasitic [13,14].

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Bioprospecting also referred as “biodiversity prospecting” which seeks to capture useful aspects of the world’s biological diversity in order to apply them in industrial, medicine, agriculture or other endeavours towards welfare of the society. It is most reasonable to carry out bioprospecting efforts in communities where the desired activities have been demonstrated or probably occurring. Microorganism and their interactions between other living creatures represent a large swath which have shaped biosphere and its inhabitants since life arose from our comparatively simple ancestor’s records. Yet, we have just begun to appreciate the extent of these invisible wonders of life and their influences. The microbial activities has unanticipated biochemical feats and fruitful new endeavours with groundbreaking research have upsurge new leads and challenges towards basic science. Moving towards their interactions, plants in natural environments establish multiple interactions with myriad microorganisms throughout their lifetime in the form of epiphytes and endophytes [1]. Despite their potentially paramount importance for plants, these extremely complex microbial communities have remained largely uncharacterized. As the research on plant-microbe interaction has upsurge, chemical diversity bearing pharmaceutical potential reached beyond the plant kingdom for exuberant microbial source, discovering such plethora of microbes bearing pharmaceutical potentials and the study have forced researchers to broaden their thoughts about significant, limitations and their activities of these invisible wonders of world which in turn highlights the wealth to gain tantalizing glimpses of its promising roles on the biosphere [2].

significance and availability hence with perturbation of isolating potent endophytic bacteria following plants were screened and results were compelled towards a fruitful endeavor in isolating antimicrobial potentials.

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1. INTRODUCTION

Plant Sciences Feed Vol. 3 Issue 3

Plant materials such as leaves, stem and bark were randomly collected from four healthy plants from Mysore Southern India. Selection of plant species was attributed based on their traditional scientific records and the based on the perusal of endophytes by far. Hence in the present investigation the four medicinal plants were evaluated viz, Annona squamosa L., Coffee arabica L., Euphorbia hirta L., Mimosa pudica L., Tridox procumbens L., b. Sample collection:

The materials were collected in a sterilized polybags and transported to laboratory within two hours before processing. Collected plants materials were thoroughly washed under running tap water then were immersed in a double distilled water containing 50 µg/ml of cycloheximide for 60 mins (minutes) to suppress the growth of fungal endophytes [3]. c. Surface sterilization protocol:

Stem and roots were subjected to surface sterilization under aseptic washed thoroughly with tap water followed by distilled water to remove adhering soil and debris Later the surface sterilization was carried by sequential steps initially by immersing in 3.15% sodium hypochlorite for 5 mins and then followed by ethanol 70% for 30 seconds. In every step of the surface sterilization procedures the plant materials were washed in sterile double distilled. To confirm that the surface disinfestations process was successful and to verify that no biological contamination from the surface of stem and roots, sterility checks were carried out for each sample to monitor the effectiveness by impressions and 0.1 ml from the final rinse was plated out on nutrient agar as control plate [4]. d. Isolation of bacterial endophytes:

After proper drying, the surface sterilized plant material outer tissue of surface sterilized plant material were removed with sterilized scissor scalpel, later carefully excise into 0.5-1.0 cm tissue blocks of plant material and placed on surface of nutrient agar supplemented with 250 μg/ml of cycloheximide and incubated for 48hrs (hours) to observe bacterial colonies [4]. 3. RESULTS

Mining the medicinal plants towards haunting for potent endophytes in the present investigation claimed successful isolation of three hundred and thirty two isolates upon screening five significant plants which has been cited in table-1. During surface sterilization protocol addition of cycloheximide resulted in suppression of fungal endophytes. Sterility checks showed no growth which claimed the successful sterilization procedure. Upon screening Annona


4. DISCUSSION

When Annona squamosa L., Coffee arabica L., Euphorbia hirta L., Mimosa pudica L., Tridox procumbens L., were cited for any early reports of endophytes during which much isolates are being reported attributing the fungal endophytes very few of bacterial endophytic plethora are reported. Xylaria sp. A23 isolated from Annona squamosa L. resulted in secreting cytochalasin H2, a new cytochalasin compounds bearing cytotoxic activity [15]. Similarly Xylarialean sp. A45 was isolated from another study which represented cytotoxic activity but none of the reports represented isolation of bacterial endophytes from Annona squamosa L. [16]. which prompted us to screen bacterial endophytes from this plant. In Coffee arabica L. reports of endophytes displayed eighty-seven culturable endophytic bacterial isolates in 19 genera were obtained from coffee plants collected in Colombia [17]. Burkholderia sp. has been reported as endophytes from root nodules of Mimosa pudica L. [18]. But no reports were available pertaining to Euphorbia hirta L., Tridox procumbens L. 5. CONCLUSION

Potential of endophytes have received tremendous attention due to the fact that, Isolation of endophytes might present bioactive compounds bearing pharmaceutical importance as these plethora are largely underexplored. In

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a. Selection of plant:

squamosa L. fostered towards isolation of eight four endophytic bacteria among which forty one isolates were from procured from surface sterilized stem and forty three isolates were obtained from leaves. The obtained isolates were given accession number AS01 to AS84 until further use. Similarly Coffee arabica L. revealed thirty two isolates among which eleven isolated were from stem and twenty one segments were procured from surface sterilization leaves which were maintained with accession number CA01 to CA32. When Euphorbia hirta L. was evaluated, forty six isolates were obtained among which twelve isolates were obtained from stem segments, eighteen isolates from leaves and sixteen isolates from root segments which were maintained with accession number from EH01 to EH46 until further use. Mimosa pudica L. revealed highest numbers of isolates with one forty one endophytes as this herb is very shy which folds itself upon touch hence large segments of stems and roots were evaluated in isolation of potent endophytes among the total isolates of Mimosa pudica L. fifty seven isolates were obtained from stem and eighty four were obtained from roots. All the isolates were given accession number from MP01 to MP 141 and were well maintained until further use. Lastly Medicinal plant Tridax procumbens L. upon evaluation resulted in isolation fourteen isolates from surface sterilized stem, six isolates from leaves and nine isolates from roots respectively. All the obtained isolates were provided with accession number from T01 to T29.

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2. MATERIALS AND METHODS

Authors are pleased to thank UGC for their financial assistance and are also expresses their gratitude towards Department of Studies in Microbiology, University of Mysore for providing infra structure.

7. REFERENCES [1] Baker S and Satish S. Endophytes: Toward a Vision in Synthesis of Nanoparticle for Future Therapeutic Agents. Int J Bio-Inorg Hybd Nanomat 2012; 2: 6777. [2] Baker S and Satish S. Endophytes: Natural warehouse of bioactive compounds. Drug Inven Today 2012 ; 11: 548-553. [3] Baker S, Santhosh P, Rakshith D and Satish, S. Screening of bacterial endophytes inhabiting Mimosa pudica L. Sci J Microbiol 2012; 5:136-140. [4] Baker S, Sahana S, Rakshith D, Kavitha HU, Kavitha KS and Satish S. Biodecaffeination by endophytic Pseudomonas sp. isolated from Coffee arabica L. J. Pharm. Res 2012; 7: 3654-3657. [5] Yushau M, Taura DW, Bello B and Abdullahi N. Screening of Annona squamosa extracts for antibacterial activity against some respiratory tract Isolates. Int Res Pharma Pharmaco 2011; 9: 237-241 [6] Vanitha V, Umadevi KJ and Vijayalakshmi K. Determination of Bioactive Components of Annona squamosa L Leaf by GC- MS Analysis. Int J Pharma Sci Drug Res 2011; 4: 309-312. [7] Bisht S and Sisodia SS. Coffea arabica: A Wonder gift to medical science. J Natural Pharma 2010; 1: 58-65. [8] Farah A. Coffee: Emerging Health Effects and Disease Prevention. In: Coffee Constituents. John Wiley & Sons, In Press, 2011. [9] Upadhyay B, Singh KP and Ashwani Kumar. Pharmacognostical and antibacterial studies of different extracts of euphorbia hirta L. J Phytol 2010; 2: 55-60. [10] Huang L, Chen S and Yang M. Euphorbia hirta (Feiyangcao): A review on its ethnopharmacology, phytochemistry and pharmacology. J Medicin Plants Res 2012; 39: 5176-5185. [11] Varnika S, Ashish S and Imran A. A review on ethnomedical and traditional uses of mimosa pudica (Chui-Mui). Int Res J pharma 2012; 2: 41-44. [12] Kaur P, Kumar N, Shivananda TN and Kaur G. Phytochemical screening and antimicrobial activity of the plant extracts of Mimosa pudica L. against

[14] [15] [16] [17] [18]

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6. ACKNOWLEDGEMENTS

[13]

selected microbes. J Medicin Plants Res 2011 22; 5356-5359. Nazeruddin GM, Shirish S, Pingale, Samir S and Shaikh. Pharmacological review of Tridax procumbens L. Der Pharmacia Sinica 2011; 4: 172175. Kuldeep G and Pathak AK. Pharmacognostic and Phytochemical Evaluation of Tridax Procumbens Linn. J Pharmaco Phytochem. 1; 5: 2012 Li Y, Lu C, Huang Y, Li Y and Shen Y. Cytochalasin H2, a New Cytochalasin, Isolated from the Endophytic Fungus Xylaria sp. A23. Rec Nat Prod 2012; 2: 121126. Lin T, Lin X, Lu CH, and Shen YM. Three New Triterpenes from Xylarialean sp. A45, an Endophytic Fungus from Annona squamosa L. Helv Chimi Acta 2011; 94: 301-305. Vega F, pavaripoll M, Posada F and Buyer JS Endophytic bacteria in Coffea arabica L. J Basic Microbiol 2005; 45: 371–380. Pandey P, Kang SC and Maheshwari DK. Isolation of endophytic plant growth promoting Burkholderia sp. MSSP from root nodules of Mimosa pudica L. Curr Sci 2005; 89: 177-180.

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order to harness these plethora of microorganism much studies are to be awaited across the globe which could exert huge impact on life expectancy. Hence in the present investigation preliminary work has been carried out towards isolating endophytic bacterial plethora for further antimicrobial assay.
