ISSN No. 2321-5488 February 2019 Research Directions Impact Factor-5.7 Special Issue 35 ISOLATION OF ENDOPHYTIC FUNGI FROM AGRICULTURE FIELD OF NAGPUR REGION AND ASSESSMENT OF ISOLATED FUNGI FOR ANTIQUORUM SENSING ACTIVITY Ashok Y. Dawande*1, Vijay N. Charde2, Yogesh S. Banginwar3 1,2 Department of Microbiology, Taywade College, Koradi, Nagpur 3 Department of Microbiology, Arts and Science College, Pulgaon, Wardha Abstract The aim of present study was to isolate the endophytic fungi from agriculture field of Nagpur region and evaluate the isolated fungi for the inhibition of quorum sensing activity. Initially, samples were collected for isolation of fungal endophytes. Fungal endophytes was isolated from the healthy, symptomless leaves, stem and root of various plants such as mustard, Linseed, Bengal gram, safflower, Pigeon pea and Orange. The identification of isolated endophytic fungi was done on the basis of their morphological and microscopic structures. The Slide culture technique was performed using isolated fungi and temporary slides were prepared in Lactophenol cotton blue stain for identification purpose. For quorum sensing inhibitors production, first Liquid- state fermentation and then extraction of inhibitors were carried out. The extract from each endophytic fungal isolate was assayed for the presence of inhibitors using biosensor strains such as Chromobacterium violaceum wild type MTCC 2656. Keywords: Endophytic fungi, quorum sensing inhibitors, Chromobacterium violaceum Introduction: Endophytic fungi reside within host plants exclusive of causing any noticeable symptoms of infection (Pimental et al., 2006).The endophytic fungal hyphae present internal cellular aerial plant parts such as leaf sheaths, sometimes even within the stem and root system also. Association with plants, the endophytic fungi can reduce the damage from the pathogens by inhibiting pathogen infection and propagation within the host. In vitro study showed that endophytic fungi capable of producing various enzymes necessary for colonizing their plant hosts. Enzymes such as amylase, pectinase, xylanase, proteinase, and cellulose have been produced by fungal endophytes. (Rajesh and Rai, 2013). Endophytic fungi have been considered as biofactories of novel bioactive molecules, among the extracted molecules and purified substances derived from the broth or fungal biomass, some have possessed antibacterial activity with moderate to powerful effect when assayed on the bacterial strains resistant to the antibiotics which are currently in use (Radic, N., Strukelj, B., 2012). As endophytes are serving as the richest source of enzymes and secondary metabolites, such metabolites can be used as quorum sensing inhibitors (antimicrobials) directing possible new method for industrially significant substance production and utilization (Rajesh and Rai, 2013). 225 Website- www.researchdirections.org Email- researchdirections2013@gmail.com ISSN No. 2321-5488 February 2019 Research Directions Impact Factor-5.7 Special Issue In the search for these inhibitors, studies have documented that many plants, fungi and even bacteria themselves produce anti-QS substances. Fungal survey on more than 20 years have documented that endophytic asymptomatic infection of land plants by fungi is everwhere. One of the important products produced naturally from endophytic microbes are antibiotics. Pestalotiopsis microspora (a rainforest fungal endophyte) is known to produce many secondary metabolites. Ambuic acid, a secondary metabolite serve as an antifungal agent. Also Colletotric acid, from Collectotricum gloeosporioides, known to show antimicrobial activity. Agriculture field grown plants are supposed to known for presence of various type of fungal endophytes. Such endophytes are capable of producing antiquorum sensing inhibitors. At low cell population, bacteria behave as single cellular organisms, as soon as their population density jumped a threshold level, bacteria may change their behaviour to ‗multicellular‘ type by sensing it. This show kind of cell to cell communication through small signaling molecules responsible expression of genes for certain phenotypes like their virulent behaviour. This is termed as bacterial Quorum Sensing (QS) and the signals molecules are as: Oligopeptides (in Gram-positive bacteria), N-acyl homoserine lactones (AHLs produced by Gram-negative bacteria) , autoinducer Furanosyl borate etc. There are different mechanism of disruption of QS, but among the different possibilities, enzymatic degradation of QS signal molecules (AHLs) has been studied the most (Kalia, V.C, 2013) (Waters, C. M., Bassler, B. L., 2005). The present study mainly focus on isolation and identification of the endophytic fungi and detection of quorum sensing activity inhibitor. Methods: Sample Collection A total of 6 healthy field-grown plants were randomly sampled, the plants collected were mustard, Linseed, safflower (from agriculture field of College of Agriculture, Nagpur), Bengal gram, Pigeon pea and Orange (from nearby agriculture field of Taywade College, Koradi, Nagpur). All samples were utilized immediately after collection. Isolation of endophytic fungi For isolation of endophytic fungi, healthy, symptomless leaves, stem and root were selected from plant species such as mustard, Linseed, Bengal gram, safflower, Pigeon pea and Orange at random, washed several times in running tap water. The samples were fragmented into small pieces and surface-sterilization done by sequentially dipping into 0.1% HgCl2 for 1 min, again cleaned with sterilized distilled water and then placed into distilled water. Then, pieces of each plant were placed on a Potato Dextrose Agar Petri dish supplemented with 0.5 g/l streptomycin sulphate. Plates were incubated at 30 oC and checked for fungal growth and sporulation. Fungi coming out of the plant pieces were then transferred to other PDA plates, and incubated at 30 oC for 7 days. After fungal purity check it is transferred to another PDA medium plate. Endophytic fungal identification was based on morphological characters on PDA plates and microscopic characters of colonies like asexual 226 Website- www.researchdirections.org Email- researchdirections2013@gmail.com ISSN No. 2321-5488 February 2019 Research Directions Impact Factor-5.7 Special Issue or sexual reproductive structures using optical microscopy (Pimentel et al., 2006 and Rajesh & Rai 2013). Quorum sensing inhibitors from endophytic fungi Bacterial strains The bacterial strain used for QS inhibition assays was Chromobacterium violaceum MTCC JCM 1249 (wild type strain). Bacterial strain was kept in 40% glycerol stocks at -20 o C and was cultured in Luria–Bertani (LB) broth prior to use. Fermentation 50 ml of sucrose broth was prepared in a 250 ml Erlenmeyer conical flask. All the isolated endophytic fungi was inoculated and then incubated at 30 0C for 24 hours. After incubation the same culture (2% v/v) transferred into 50 ml sucrose broth and incubated at 30 0 C in a orbital shaker incubator at 140 rpm for 5 days incubation. Extraction of quorum sensing inhibitors After fermentation, the cells were filtered using 1 MM Whatman filter paper and the filtrate was extracted with an equal amount of ethyl acetate. Then, the contration of organic phase was done by evaporation and the residue was dissolved in sterile DMSO and kept in a deep freezer for further use (Ueda et al., 2007). Bioassay The extract from each isolates was assayed for the detection of QS inhibitors. First the LB agar plates were flooded with 1ml of C. violaceum wild type culture to prepare bacterial lawn. One 10 mm diameter well were created in each agar LB plate using a sterile Borer. Then the well of 10 mm was loaded with fungal extract (40µL) and Distilled water and two antibiotic discs namely Piperacillin/Tazobactum (PIT 10mcg) and Aztreonam (AT 30mcg) were used as negative and positive controls, respectively. Then, it was incubated at 28 oC for 24 h. The presence of a clear zone i.e. violet color pigment inhibition around the wells indicates the presence of antiquorum sensing activity. Results and Discussion From Fig. 1, a total of 19 endophytic fungi were isolated from the 53 healthy, symptomless leaves, Stem and root fragments analyzed giving a percentage colonization rate of 48.7% from various plants such as Mustard, Linseed, Bengal gram, Safflower, Pigeon pea and Orange (Table 1). 227 Website- www.researchdirections.org Email- researchdirections2013@gmail.com ISSN No. 2321-5488 February 2019 Research Directions Impact Factor-5.7 Special Issue Table 1 - number of isolates of endophytic fungi obtained from various plants leaf, stem and root fragments Total number of isolates Growth Number of Colonization by organ environment rate *(%) Plant Organ fragments Mustard Root 5 60.00 3 Linseed Root 7 42.85 3 Bengal gram Root 14 7.14 1 Root 10 0 0 Field-grown plants Safflower Pigeon pea Leaf 5 80 4 Stem 4 75 3 Orange Leaf 4 75 3 Stem 4 50 2 Total plants 6 Total fragments 53 taken Percentage colonization rate 48.7 Total isolates 19 *Percentage colonization- the total number of pieces colonized by fungi in relation to the total number of pieces x 100. 228 Website- www.researchdirections.org Email- researchdirections2013@gmail.com ISSN No. 2321-5488 February 2019 Research Directions Impact Factor-5.7 Special Issue Among 19 isolates, only 10 isolates were found to be different from each other and were considered for study (Fig.3). Morphological characteristics of isolated endophytic fungi were shown in Fig.3 Fig. 3- Isolated endophytic fungi cultivated on PDA. Fungal code: F1 to F10 (SPP). Remaining 9 fungi showed morphologically and microscopically similar characteristics with rest of fungi. Endophytic fungi were identified as Pestalotiopsis sp., Colletotrichum sp, 229 Website- www.researchdirections.org Email- researchdirections2013@gmail.com ISSN No. 2321-5488 February 2019 Research Directions Impact Factor-5.7 Special Issue Aspergillus sp1, Penicillium sp, Fusarium sp.1 Phoma sp. Aspergillus sp2 Fusarium sp.2 Trichoderma sp. and Phomopsis sp. Microscopic characters of isolated endophytic fungi were shown in Fig. 2 Quorum sensing inhibitors from endophytic fungi The plant defense machinery uses bioactive compounds produced from the plant or endophytic microorganism. So the production of QS inhibitor from such isolates was detected by bioassay with Chromobacterium violaceum wild type MTCC 2656. Fig. 4 represents the capability of endophytic fungal extract for QS inhibition by inhibiting the violacein pigment production by C. violaceum without inhibiting the bacterial growth. The isolates of Fusarium sp.2 (F9SF) and Phomopsis sp.(F6MP) possess quorum sensing inhibitor by inhibiting the synthesis of violacein pigment. 230 Website- www.researchdirections.org Email- researchdirections2013@gmail.com ISSN No. 2321-5488 February 2019 Research Directions Impact Factor-5.7 Special Issue Table 3- Antiquorum sensing activity of fungal endophytes Inhibition of Isolated endophytic quorum sensing activity Code fungi F1 Aspergillus sp1 F2 Penicillium sp F3(J1) Aspergillus sp2 F4(PP) Pestalotiopsis sp. F5(SPT) Trichoderma sp. F6(MP) Phomopsis sp. + F7(GF) Fusarium sp.1 F8(GC) Colletotrichum sp F9(SF) Fusarium sp.2 + F10(SPP) Phoma sp + Good activity,- No activity Fig.4. Result of anti-QS screening for fungal extract F1 to F10 (SPP) and the controls. Among all, the fungal extracts of Fusarium sp.2 (F9SF) and Phomopsis sp.(F6MP) caused observable inhibition of violacein pigment produced by C. Violaceum wild type as opposed to complete clearing of bacterial growth around the well for the antibiotic control (+ve C) (Aztreonam, AT 30mcg and Piperacillin/Tazobactum, PIT 10mcg). (C) Showing no clearing is for the negative control using sterile distilled water. 231 Website- www.researchdirections.org Email- researchdirections2013@gmail.com ISSN No. 2321-5488 February 2019 Research Directions Impact Factor-5.7 Special Issue With combination to antibiotics, antiquorum sensing molecules could be one of the effective therapeutic tool in future, serving as the alternative to multidrug resistant pathogens. References Bulman, Z., Le, P., Hudson, A. O., Savka M. A., 2011. A novel property of propolis (bee glue): Anti-pathogenic activity by inhibition of N-acyl-homoserine lactone mediated signaling in bacteria. J. Ethanopharm. 138, 788-797. 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