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PLANT SCIENCE TODAY, 2021 Vol 8(3): 501–508 HORIZON E-Publishing Group ISSN 2348-1900 (Online) PLANT SCIENCE TODAY, 2021 Vol 8(3): 501–508 HORIZON https://doi.org/10.14719/pst.2021.8.3.1104 e-Publishing Group ISSN 2348-1900 (online) RESEARCH ARTICLE Study of antibacterial, anti-proliferative and pro-apoptotic potential of the cell extracts of endophytic fungi and bacteria isolated from Pajanelia longifolia (Willd.) K. Schuman Gowthami G A1, Subhankar Das1, 2, Yalpi Karthik1 & Manjula Ishwara Kalyani1* 1Department of Studies and Research in Microbiology, Mangalore University, Jnana Kaveri, P G Centre, Chikka Aluvara, Kodagu 571 232, Karnataka, India 2Biotechnology Unit, Mangalore University, Mangalagangotri 574 199, Mangalore, Karnataka, India *Email: [email protected] ARTICLE HISTORY ABSTRACT Received: 25 January 2021 Endophytes contribute to the synthesis of significant metabolites in symbiotic association with their Accepted: 11 May 2021 host plants. On considering the medicinal importance of the prominent tree species Pajanelia Available online: 01 July 2021 longifolia (Willd.) K. Schuman, the study was conducted to isolate and identify the endophytic bacteria and fungi for their bioactivity. The isolation of endophytic bacteria and fungi were performed by surface sterilisation of the stem and leaf samples of P. longifolia. The obtained bacterial and fungal KEYWORDS endophytic isolates were maintained in nutrient agar and Potato Dextrose Agar (PDA) media and were Pajanelia longifolia examined for colony morphology and microscopic appearances with varied biochemical Bacteria characterisations. Furthermore, both the fungal and bacterial isolates were subjected to solvent Fungi extractions to evaluate antibacterial activity. Also, anti-proliferative effects due to apoptotic induction Antiproliferation activity by the endophytic fungal extracts were checked against proliferative yeast cells. Moreover, endophytic Pro-apoptotic Antibacterial activity bacteria belonging to Enterococcaceae had shown antibacterial activity against Salmonella species. In the present study, fungal species belonging to Cladosporium predominantly found to inhabit as endophytic fungi in the plant samples. Also, this particular fungus among other selected endophytic fungi attributed to causing effective anti-proliferative activity. The endophytic bacteria belonging to Enterococcus and Micrococcus genera showed significant antimicrobial activity against Salmonella typhimurium (ATCC 23564). Introduction deciduous tree belonging to the family of Bignoniaceae, is considered as one of the important Plant-Microbe interaction is known to have crucial plant species among Western Ghat diversity in biological activities for both the species participating Karnataka due to its antibacterial and antioxidant in the association. Endophytic microbe play a vital properties. The pharmacological importance of this role in stabilising physiological and biochemical status tree is reported in the treatment of skin disorders, the within the host attributing to the defensive role bark extract is used for treating eczema by local tribes against invading plant pathogens (1). The role of in certain regions of Dakshina Kannada district in endophytes and their associations with the medicinal Karnataka. The scientific interventions have reported plant contributes significantly to the medicinal antioxidant, hepatoprotective and cytotoxic activity of properties of the particular plant (2, 3). Endophytic the plant (8, 9). Many important metabolites from the microbes have also been isolated from diverse P. longifolia, have been isolated and studied over a medicinal plants and have showed significant long time for medicinal properties such as contributions to the treatment for many ailments by antibacterial, antifungal, anti-tumour, metabolite synthesis and the host biosynthesis (4, 5). immunosuppressant, anti-parasitic and antiviral, anti- According to the literature, the endophytic microbes oxidant and anti-inflammatory (10, 11). protect their host from infectious agents and against any adverse conditions by secreting bioactive The present study focus on isolation of secondary metabolites (6, 7). endophytic bacteria and fungi from leaf and stem of P. longifolia. The isolated endophytes were examined for Since time immemorial medicinal plants have the colony morphology, microscopic staining and played an important role in the treatment of various biochemical characterisations were also determined. diseases. Pajanelia longifolia (Willd.) K. Schuman, a © Gowthami et al (2021). This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited (https://creativecommons.org/licenses/by/4.0/). 502 GOWTHAMI ET AL The Intracellular metabolites were further extracted biochemical characterisations for IMViC tests, to check their pharmacological properties such as carbohydrate utilisation test, hydrogen sulphide test, antimicrobial, anti-proliferative and pro-apoptotic catalase test and gelatin hydrolysis tests (12, 13). activities. The Ioslates fungal strains were subjected Identification of Endophytic Fungi to apoptosis induction as a measure of cell death due Endophytic fungal cultures were examined for their to cytotoxicity was analysed in the mutagen characteristics such as growth pattern, size, the transformed cell model after treating with colouration of the mycelium and other colony intracellular extracts from endophytic fungi. morphological features. The morphological Furthermore the isolated bacterial endophytic strains observation of the fungal isolates were observed by showed prominent antibacterial activity against lactophenol cotton blue staining for spore Salmonella typhimurium (ATCC 23564). arrangement and seen under Phase contrast Microscope (Lawrence and Mayo LM-52-3501) (14, Materials and Methods 15). Plant collection The isolation rate (IR) was calculated using the formula: IR = (Ni/Nt), Ni: the number of segments Pajanelia longifolia was collected from yielding the fungal isolates, Nt: the total number of Honnahanakodu region of Karnataka (12°37'02.1"N segments incubated. The colonisation rate (%) was 75°51'36.6"E). The healthy mature leaves and stem calculated using the formula: CR = (Ni/Nt) × 100 (16). samples were collected and transferred to the laboratory in sterile bags. Intracellular extraction from endophytic bacteria Isolation of Endophytes from Pajanelia longifolia To obtain bacterial biomass, each of the bacterial isolates was grown by inoculating onto Nutrient The collected plant parts (as shown in Fig. 1) were broth pH 7.0 and incubated at 37˚C for 48 hours in a washed, dried and cut into small segments measuring shaker incubator (ROTEK Incubator shaker ROSI-2). about 1 cm. The leaf and stem segments were initially The bacterial biomass was collected and their surface sterilised using sterile distilled water, intracellular fractions were extracted using subsequently rinsed with 70% ethanol and 4% phosphate buffer pH 7.2 by crushing the biomass in sodium hypochlorite. Successive washing were mortar pestle. The buffer extracted fractions were carried out thrice using sterilised distilled water and further centrifuged at 10000 rpm (Remi CRP 30 Plus dried aseptically. Surface sterilised segments were VCEF-6571/450 LAL) and the culture-free extract was placed on water agar media containing obtained. Protein content was estimated by Lowry’s chloramphenicol (0.1 mg), Nutrient agar media and method using BSA as standard (17) where as the (Potato Dextrose Agar) PDA media. Each plate was Carbohydrate was estimated by DNS method using placed with ten segments in equidistance, followed glucose as standard (18). by setting standard bacterial and fungal growth conditions. Biomass extraction of endophytic fungal isolates Identification of Endophytic Bacteria using Endophytic fungal isolates were cultured in PDB Biochemical Characterisation medium for ten days at room temperature on a rotary shaker Remi (Model no. RIS-768) (19, 20). After The colony morphology and staining characteristics incubation period the broth was filtered using were observed for each of the Endophytic isolates Whatmann filter paper No. 1 the fungal biomass was and documented. The isolates were subjected for then collected and homogenized using mortar and Fig. 1. Leaf and stem of Pajanelia longifolia plant. PLANT SCIENCE TODAY 503 pestle in aseptic conditions in biosafety cabinet (ESCO stained with trypan blue dye. Cell viability counts AC2-4EB class II bio-safety cabinet) by adding equal were performed for the different time interval of amount of 0.1M phosphate buffer pH 7.2. The incubation. The treated and untreated, proliferative intracellular fractions were centrifuged using Remi yeast cells were subjected to staining with Giemsa to CRP 30 Plus VCEF-6571/450 LAL at 4000 rpm for 10 analyse morphological changes of a cell undergoing min and the supernatant was collected. The culture death (24). The cells were then fixed on to slide using free extract was obtained for further analysis. methanol and glacial acetic acid solvent mixture; the Antibacterial activity of the intracellular extract slides were immersed in Giemsa staining solution for of endophytic bacterial and fungal isolates 30–40 min, subsequently, washed with distilled water, air-dried and observed under 40X objective The obtained endophytic bacterial and fungal using a phase contrast microscope (Lawrence and extracts were tested for growth inhibition against
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