Bangladesh J. Plant Taxon. 30(1): 111-122, 2023 (June) DOI: https://doi.org/10.3329/bjpt.v30i1.67050 © 2023 Bangladesh Association of Plant Taxonomists MORPHOLOGICAL AND MOLECULAR CHARACTRIZATION OF ENDOPHYTIC FUNGI ISOLATED FROM ANDROGRAPHIS PANICULATA (BURM. F.) WALL. EX NEES AND CENTELLA ASIATICA (L.) URBAN FAZILATUN NESSA, SHAMIM SHAMSI* AND MD. ABDULLAH AL NOMAN Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh Keywords: Morphological identification; ITS sequencing; Medicinal plant; Fungi; Bangladesh. Abstract Fungal endophytes were isolated from the leaves, stems and roots of two widely used medicinal plants viz., Andrographis paniculata (Burm. f.) Wall. ex Nees and Centella asiatica (L.) Urban. A total of 28 endophytic fungi were identified based on morphological and molecular analyses. The identified fungi were: Aspergillus flavus Link, A. fumigatus Fresen., A. niger Tiegh., A. terreus Thom, Cladosporium sp., Colletotrichum sp., Curvularia chonburiensis (ibpromma & K.D. Hyde, C. hominis Da Cunha, Madrid, Gené & Cano, C. lunata (Wakker) Boedijn, C. lycopersici Tandon & Kakkar, Curvularia sp., Fusarium falciforme (Carrión) Summerb. & Schroers, F. phaseoli (Burkh.) T. Aoki & Donnell, F. solani (Mart.) Appel & Wollenw, F. udum (Berk.) Wollenw, Fusarium sp., Lasiodiplodia theobromae (Pat.) Gri. & Maubl., Monodictys paradoxa (Corda) Hug., M. putredinis (Wallr.) Hug., Penicillium commune Thom, P. chrysogenum Thom, P. oxalicum Currie & Thom, Penicillium sp. 1, Penicillium sp. 2, Penicillium sp. 3, Penicillium sp. 4, Scytalidium lignicola Pesante and Talaromyces trachyspermus (Shear) Stolk & Samson. Among them Aspergillus flavus, A. niger, A. terreus, Cladosporium sp., Colletotrichum sp. and Penicillium sp. 1 were isolated from both the plants. Curvularia chonburiensis, C. hominis, C. lycopersici, Fusarium falciforme, F. phaseoli, Monodictys paradoxa, Penicillium commune and Scytalidium lignicola were found to be new records for Bangladesh. Findings of this study will be helpful for better understanding of endophytic fungal diversity and the species richness in those medicinal plants. Introduction The term endophytic fungi refers to the fungi that live within the plant tissues throughout their entire or partial life cycle by establishing a mutually beneficial symbiotic relationship with its host plant without causing any adverse effect or disease (Hyde et al., 2019; Patchett and Newman, 2021). Endophytic fungi have been isolated from many plants, including trees, vegetables, fruits and other crops (Rosenblueth and Martinez-Romero, 2006). Medicinal plants harbor endophytic microflora and they are valuable source of bioprospecting endophytes. Andrographis paniculata (Burm. f.) Wall. Ex Nees and Centella asiatica (L.)Urban are two widely used medicinal plants. The whole plant of Andrographis paniculata has been used for several applications such as anti-dote for snake-bite and poisonous stings of some insects and to treat dyspepsia, influenza, dysentery, malaria and respiratory infections (Chopra, 1980; Jarukamjorn et al., 2010). Aside from healing wounds, C. asiatica is used for the treatment of various skin conditions such as lupus, leprosy, varicose ulcers, eczema, and psoriasis. (Brinkhaus et al., 2000) and also as a blood purifier (Gohil et al., 2010). Endophytic fungi from medicinal plants have significant role in pharmacology and in industries. They can also promote their *Corresponding author, E-mail: prof.shamsi@gmail.com; a part of MS thesis of the first author. https://doi.org/10.3329/bjpt.v30i1.67050 mailto:prof.shamsi@gmail.com; 112 NESSA et al. accumulation of secondary metabolites. In the present study these two important medicinal plants, Andrographis paniculata and Centella asiatica were used for the isolation of endophytic fungi. This study will lead to evaluate the potential bioactive metabolites of the endophytes, relation between endophytes and host plants and also to study the endophytic fungal diversity and the species richness in those medicinal plants. Materials and Methods Centella asiatica and Andrographis paniculata were collected from the Botanical Garden, University of Dhaka and used for the present investigation. Isolation and morphological identification of fungi Endophytes associated with selected samples were isolated by following “Tissue planting method” on Potato Dextrose Agar medium (CAB, 1968). Morphological identities of the fungal isolates were determined following the standard literature (Thom and Raper, 1945; Booth, 1971; Ellis, 1971,1976; Barnett and Hunter, 1972; Sutton, 1980). Molecular characterization of fungi Molecular identification was done by following Amer et al. (2009) with some modifications. DNA extraction Fungi were grown on PDA medium at 28°C for 10 days. Fungal mycelium was harvested by scraping the surface of 10 days old cultures with a sterile spatula from the Petri plates. One gram of fungal mycelium of each isolate was taken in a 1.5 ml sterile Eppendorf tube. The mycelium was immediately ground with a homogenizer with 400μl sterile extraction buffer (200mM Tris- HCI, 250mM NaCl, 25mM EDTA, 0.5% SDS) in each Eppendorf tube. Then 6 μL of 20 mg/ml RNase was added in each Eppendorf. Tubes were stirred with a vortex mixer so that the mixture became homogenous. The tubes were transferred to 65°C preheated water bath for 10 minutes. The samples were taken from the water bath and cooled down to room temperature. 130 μL of 3M sodium acetate, pH 5.2 was added in each tube. Tubes were vortexed for 30 seconds at maximum speed and incubated at -20° C for 10 minutes. The samples were centrifuged at 13,000 rpm for 15 minutes. The supernatants were transferred to fresh tubes and equal volume of chloroform: isoamyl alcohol mixture (24:1) was added and mixed by gentle inversion and then tubes were centrifuged at 12000 rpm for 5 minutes. The supernatant was discarded and the pellet was washed twice with 700 μL of 70% ethanol. The DNA pellets were subsequently air-dried. The resultant DNA pellet was then resuspended in 100 μL of 1 x TE (10 mM Tris- HCI,1 mM EDTA) buffer (pH 8.0). The DNA was allowed to dissolve overnight at 4°C. Then it was stored at-20°C for further analyses. Polymerase chain reaction amplification, sequencing and phylogenetic analysis Molecular identification of the isolates was performed using the internal transcribed spacer (ITS) regions. PCR amplification was conducted using the ITS1 (5'-TCCGTAGGTGA ACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') primers for the ITS regions. The PCR was carried out in 0.2 ml PCR tube with 25 μl reaction volume containing 2.0 μl template DNA, 12.5 μl master mix, 1.0 μl forward primer, 1.0μl reverse primer and 8.5 μl nucleus free H2O. Reaction mixture was vortexed and centrifuged in a microcentrifuge. The PCR was initiated by an initial denaturation step at 94ºC for 5 minutes following 30 cycles of 94, 54 and 72ºC each for 30 sec, with a final extension step of 5 min at 72ºC and ended with 4ºC. PCR amplified products were stored in – 20ºC freezer for analysis by resolving in 1% agarose gel. The gel was prepared using 1.0 g agarose powder containing ethidium bromide. Agarose gel MORPHOLOGICAL AND MOLECULAR CHARACTRIZATION OF ENDOPHYTIC FUNGI 113 electrophoresis was conducted in 1× TAE buffer at 90 Volts and 300 mA for 40 minutes. DNA bands were photographed by a gel documentation system (model: DI-HD, UK).The purified DNA samples were sequenced through automated sequencer in the Centre for Advanced Research in Sciences (CARS), University of Dhaka, Dhaka, 1000. Sequences were aligned with CLUSTAL W alignment using the Molecular Evolutionary Genetics Analysis (MEGA) software version 7.0 (Kumar et al., 2016). The phylogenetic tree was constructed with help of same software using the neighbor-joining method—with relative branch support of 1000 bootstrap replications. Results and Discussion Morphological identification Fungal endophytes were isolated from the leaves, stems and roots of Andrographis paniculata and Centella asiatica, Aspergillus flavus, A. fumigatus, A. niger, A. terreus, Cladosporium sp., Colletotrichum sp., Curvularia sp. 1, Curvularia sp. 2, Curvularia sp. 3, Fusarium sp. 1, Fusarium sp. 2, Fusarium sp. 3, Fusarium sp. 4, Fusarium sp. 5, Lasiodiplodia sp., Monodictys paradoxa, M. putredinis, P. chrysogenum, P. oxalicum, Penicillium sp. 1, Penicillium sp. 2, Penicillium sp. 3, Penicillium sp. 4, Penicillium sp. 5, Scytalidium lignicola, and Talaromyces trachyspermus were identified morphologically. Key morphological features of the isolated fungi Aspergillus flavus Link, Magazin der Gesellschaft Naturforschenden Freunde Berlin 3 (1): 16 (1809) (Fig. 1a) Olive-green colony, flat at their borders while raised in the middle. Conidiophore hyaline, coarsely roughened, up to 1.0 mm in length. Vesicles globose to sub globose, 25-45 µm in diameter. Conidia pale green, globose to sub globose, 3-4 µm in diameter. Specimen examined: Isolated from Centella asiatica and Andrographis paniculata from Curzon Hall Campus botanical garden, University of Dhaka. 23 March, 2021. F. Nessa 1. Aspergillus fumigatus Fresen., Beiträge zur Mykologie3: 81 (1863) (Fig. 1b) Colonies attain a diameter of 3-5 cm within 7 days, consisting of a dense felt of dark green conidiophores intermixed with aerial hyphae bearing conidiophores. Conidiophores short, smooth- walled. Vesicles broadly clavate, 20-30 µm in diameter. Phialides directly borne on the vesicle, often greenish pigmented, 6-8 x 2-3 µm. Conidia globose to subglobose, 2.5-3.0 µ in diameter, green, rough-walled to echinulate. Specimen examined: Isolated from Centella asiatica and Andrographis paniculata from Curzon Hall Campus botanical garden, University of Dhaka. 23 March, 2021. F. Nessa 2. Aspergillus niger Tiegh., Annales des Sciences Naturelles Botanique 8: 240 (1867) (Fig. 1c) Colonies black powdery with conidial production. The reverse is pale yellowish white. Conidiophores arise from long, broad, thick-walled, mostly brownish, sometimes branched foot- cells. Conidia in large, radiating heads, mostly globose, irregularly roughened, 4.0-5.0 µm in diameter, uninucleate. Specimen examined: Isolated from Centella asiatica and Andrographis paniculata from Curzon Hall Campus botanical garden, University of Dhaka. 23 March, 2021. F. Nessa 3. Aspergillus terreus Thom, American Journal of Botany 5 (2): 85 (1918) (Fig. 1d) Colonies yellowish-brown to cinnamon-brown, consisting of a dense felt of conidiophores. Conidiophore stipes smooth-walled, hyaline. Vesicles subspherical, 10-20 µm diameter. Metulae 114 NESSA et al. as long as the phialides. Conidia smooth-walled, striate with SEM, spherical to broadly ellipsoidal, 1.5-2.5 µm, hyaline. Specimen examined: Isolated from Centella asiatica and Andrographis paniculata from Curzon Hall Campus botanical garden, University of Dhaka.16 June, 2021. F. Nessa 14. Cladosporium sp. (Fig. 1e) Colonies raised at the center, umbonate, circular, ash to blackish ash color, thinly hairy. Conidiophore solitary, slightly flexuous, mid brown, smooth 4 – 5.6 µm thick. Conidia arising in simple or branched chains, cylindrical, ellipsoidal, sub-hyaline, smooth, 5 – 6.5 × 2 – 3.5 µm. Specimen examined: Isolated from Centella asiatica and Andrographis paniculata from Curzon Hall Campus botanical garden, University of Dhaka.5 January, 2022. F. Nessa32. Colletotrichum sp. (Fig. 1f) Colony cottony white, fluffy, front side white with light orangish shade, reverse side orangish- white. Conidia hyaline, aseptate, straight to falcate, smooth, thin walled. Conidia length 9.58 µm & width 2.41 µm. Specimen examined: Isolated from Centella asiatica from Curzon Hall Campus botanical garden, University of Dhaka. 11 November, 2021. F. Nessa24. Curvularia hominis Da Cunha, Madrid, Gené& Cano, Persoonia 33: 55 (2014) (Fig. 1g) Colonies on PDA reaching 70-80 mm diam in 1 week, white in color, with moderate aerial mycelium giving the colony a slightly cottony appearance, lobulate; reverse pale to darker luteous towards periphery. Conidia 4–5-celled, slightly curved, ellipsoidal to obovoid, the third cell from the base often larger and unequal sided, end cells subhyaline to pale brown and smooth-walled. Specimen examined: Isolated from fresh and healthy root of Centella asiatica from the botanical garden of Curzon Hall Campus, University of Dhaka. 22 July, 2021. F. Nessa 19. Curvularia lunata (Wakker) Boedijn, Bull. Jard. Bot. Buitenzorg 13 (1): 127 (1933) (Fig. 1h) Colonies on PDA covering the surface of the Petri dish in 1 week, center white to colourless towards periphery; abundant aerial mycelium giving the colony a cottony appearance, lobulate with a fimbriate margin; reverse pale gray. Conidia smooth-walled, pale brown, end cells paler; conidia obovoidal to broadly clavate, curved at the subterminal cell. Specimen examined: Isolated from healthy stem ofCentella asiatica from the Botanical Garden of Curzon Hall Campus, University of Dhaka. 22 July, 2021. F. Nessa 18. Curvularia sp. (Fig. 1i) Colonies on PDA white or pale grey when young, orange to brown. Colonies effuse orangish black, fluffy, cottony, raised. Conidiophore solitary, mostly unbranched, straight or slightlyundulating. Conidia mostly 4-5 septate, brown, slightly curved. Specimen examined: Isolated from Centella asiatica from the botanical garden of Curzon Hall Campus, University of Dhaka. 11 November, 2021. F. Nessa 26. Fusarium falciforme (Carrión) Summerb. & Schroers, Journal of Clinical Microbiology 40 (8): 2872 (2002) (Fig. 2a) Colonies off-white to pale cream, velvety or slightly fluffy, with a slightly raised centre and adpressed margin, growing slowly. Conidia colourless, ellipsoidal to reniform, aseptate or septate. Specimen examined: Isolated from fresh and healthy leaves and root of Centella asiaticafrom MORPHOLOGICAL AND MOLECULAR CHARACTRIZATION OF ENDOPHYTIC FUNGI 115 botanical garden of Curzon Hall Campus, University of Dhaka. 13 April, 2021. F. Nessa 10. Fusarium phaseoli (Burkh.) T. Aoki & O'Donnell, Mycologia 95 (4): 671 (2003) (Fig. 2b) Colony color on PDA white with grayish tint; conidial pustules sometimes present, grayish- green to dark green under fluorescent; macro-conidia septate, 2-4 celled. Specimen examined: Isolated from fresh and healthy stem of Centella asiaticafrom botanical garden of Curzon Hall Campus, University of Dhaka. 22 July, 2021. F. Nessa 19. Fusarium solani (Mart.) Appel & Wollenw., Arbeiten aus der Kaiserlichen Biologischen Anstalt für Land- und Forstwirtschaft 8: 64-78 (1910) (Fig. 2c) Colonies growing rapidly, covering the surface of the Petri dish in 1 week, Aerial mycelium generally abundant, white cottony; Conidiophores arising laterally from aerial hyphae. Monophialides mostly with a rather distinct collarette. Macroconidia produced on shorter, branched conidiophores which soon form sporodochia, usually moderately curved, with short, blunt apical and indistinctly pedicellate basal cells, mostly 3-septate. Microconidia usually abundant, Chlamydospores frequent. Specimen examined: Isolated from fresh and healthy leaves and root of Andrographis apniculata from botanical garden of Curzon Hall Campus, University of Dhaka. 22 July, 2021. F. Nessa 21. Fusarium udum (Berk.) Wollenw., Phytopathology 1: 206 (1913) (Fig. 2d) Colony brownish-black to colorless towards periphery, growth rate medium, raised at center; reverse orange center to white towards periphery with colorless border. Conidia initially produced on simple or verticillately branched conidiophores; variable in size, with a curved apex; there is no clear distinction between microconidia and macroconidia. Specimen examined: Isolated from fresh stem and root of Andrographis apniculata from botanical garden of Curzon Hall Campus, University of Dhaka. 13 April, 2021. F. Nessa 11. Fusarium sp. (Fig. 2e) Colonies growing rapidly, with white to cream-coloured aerial mycelium, reverse usually colourless. Conidiophores arising laterally from aerial hyphae. Macroconidia produced on shorter, branched conidiophores. Microconidia usually abundant, produced on elongate. Chlamydospores frequent, singly or in pairs, terminal, rough-walled. Specimen examined: Isolated from fresh and healthy leaves and root of Andrographis apniculata from botanical garden of Curzon Hall Campus, University of Dhaka. 16 November, 2021. F. Nessa 27. Lasiodiplodia theobromae (Pat.) Griffon & Maubl., Bulletin de la Société Mycologique de France 25: 57 (1909) (Fig. 2f) Colonies on agar greyish sepia to mouse grey to black, fluffy with abundant aerial mycelium; reverse fuscous black to black. Conidiogenous cells hyaline, simple, cylindrical to subobpyriform, holoblastic, annelidic. Conidia initially unicellular, hyaline. Specimen examined: Isolated from fresh and healthy leaves of Andographis paniculata from botanical garden of Curzon Hall Campus, University of Dhaka. 16 June, 2021. F. Nessa16. Monodictys paradoxa (Corda) S. Hughes, Canadian Journal of Botany 36(6): 786 (1958) (Fig. 2g) Colonies white, effuse, dotted with bundles of black conidia, reverse side black and brownish. Conidiophora micronematica. Conidiophore cells inflated. Conidia ellipsoidal, oval, wall-shaped, 116 NESSA et al. Blackish, smooth, often with one or more basal cells, paler than the others, 20-43 x 17-30 µm, black-soot, basal cell subhyaline. Specimen examined: Isolated from fresh and healthy leaf of Andrographis paniculata from Curzon Hall Campus botanical garden, University of Dhaka. 23 June, 2021. F. Nessa 17. Monodictys putredinis (Wallr.) S. Hughes, Canadian Journal of Botany 36 (6): 785 (1958) (Fig. 2h) Colonies effuse, beige in color. Reverse side reddish brown. Mycelium mostly superficial. Stroma none. Setae and hyphopodia absent. Vegetative hyphae hyaline or sooty, 1-2.5 µm wide. Conidiophora micronematic, aggregated. Conidiophore cells not markedly swollen. Conidia individually acrogenous, subglobose or ellipsoid, multicellular, wall-shaped, sometimes slightly constricted at the septa, 20-30 x 15-25 µm, smooth. Specimen examined: Isolated from fresh and healthy root of Centella asiatica from botanical garden of Curzon Hall Campus, University of Dhaka. 12 April, 2021. F. Nessa 8. Penicillium commune Thom, U.S.D.A. Bur. Animal Industr. Bull. 118: 56 (1910) (Fig. 2i) Colony growing rapidly, olive-gray in color, white towards the periphery; reverse white to yellow, Conidiophore stipes rough-walled, penicilli terverticillate. Phialides flask-shaped, tapering into a narrow neck. Conidia spherical to subspherical, smooth-walled. Specimen examined: Isolated from fresh and healthy stem of Centella asiatica from botanical garden of Curzon Hall Campus, University of Dhaka. 1 December, 2021. F. Nessa 29. Penicillium chrysogenum Thom, U.S.D.A. Bur. Animal Industr. Bull. 118: 58 (1910) (Fig. 3a) Colonies bright green with yellow pigmentation in center, velutinous to floccose, exuding a bright yellow pigment into the medium; reverse yellow. Conidiophore stipes smooth-walled, 200-300 µm long; penicilli usually terverticillate. Metulae 8-12 µm long. Phialides flask-shaped, 7-10 µm long. Conidia smooth-walled, ellipsoidal, 2.5-4.0 µm long, blue or bluish-green. Specimen examined: Isolated from Centella asiatica and Andrographis paniculata from Curzon Hall Campus botanical garden, University of Dhaka. 1 December, 2021. F. Nessa 30. Penicillium oxalicum Currie & Thom, J. Biol. Chem. 22: 289 (1915) (Fig. 3b) Colony white, soluble pigment lacking, reverse pale to yellow. Conidial heads irregularly biverticillate. Conidiophores smooth, 200-400 x 3-3,5 µm long. Metulae appressed. Phialides in verticils of 6-10, acerose, 10-15 x 3-3,5 µm. Conidia elliptical, smooth. Specimen examined: Isolated from fresh leaves and root of Andrographis apniculata from botanical garden of Curzon Hall Campus, University of Dhaka. 12 April, 2021. F. Nessa 9. Penicillium sp. 1 (Fig. 3c) Colonies olive in color, with clear white border, moderate growth; Conidiophores arising from the mycelium singly, smooth-walled. Spores minute, globose, white. Specimen examined: Isolated from Andrographis apniculata from botanical garden of Curzon Hall Campus, University of Dhaka. 13 April, 2021. F. Nessa 12. Penicillium sp. 2 (Fig. 3d) Colony yellow in colour with bright orange pigmentation in center, reverse orange. Multiple phialides on each metullae grouped in brush-like clusters (penicilli) at the ends of the conidiophores; conidia unicellular, round. MORPHOLOGICAL AND MOLECULAR CHARACTRIZATION OF ENDOPHYTIC FUNGI 117 Fig 1. Colony on PDA medium and conidia under microscope: a. Aspergillus flavus, b. A. fumigatus, c. A. niger, d. A. terreus, e. Cladosporium sp.,f.Colletotrichum sp., g. Curvularia hominis, h. C. lunata and i. Curvularia sp.(Bar = 50 µm) Fig 2. Colony on PDA medium and conidia under microscope: a. Fusarium falciforme, b. F. phaseoli, c. F. solani, d. F. udum, e. Fusarium sp., f. Lasiodiplodia theobromae, g. Monodictys paradoxa, h. M. putredinis, and i. Penicillium commune. (Bar = 50 µm) Specimen examined: Isolated from Centella asiatica from botanical garden of Curzon Hall Campus, University of Dhaka. 1 December, 2021. F. Nessa 31. 118 NESSA et al. Penicillium sp. 3 (Fig. 3e) Colony yellow in colour with green center, raised at center. No pigmentation present. Conidia round, hyaline, rough walled. Specimen examined: Isolated from fresh and healthy leaves and root of Andrographis apniculata from botanical garden of Curzon Hall Campus, University of Dhaka. 5 January, 2022. F. Nessa 35. Penicillium sp. 4 (Fig. 3f) Colony pale yellow in color, orange in center, reverse orange; reddish orange pigmentation. Conidiophore greenish in colour. phialides grouped in brush-like clusters (penicilli) at the ends of the conidiophores. Conidia round, greenish yellow. Specimen examined: Isolated from Andrographis paniculata from botanical garden of Curzon Hall Campus, University of Dhaka. 5 January, 2022. F. Nessa 35. Scytalidium lignicola Pesante, Annali della Sperimentazione Agaria 11 (suppl.): 265 (1957) (Fig. 3g) Colonies effuse, flat with raised folds, cottony to woolly, initially whitish, finally becoming dark grey to black. Microscopy. Hyphae hyaline at first, later becoming brown. Arthroconidia hyaline, thin-walled, rectangular, about 5-8 x 2 µm. Chlamydospore-like conidia single or in chains, dark brown, thick-walled, swollen up to 7 µm wide. Specimen examined: Isolated from fresh and healthy leaf of Andrographis paniculata from botanical garden of Curzon Hall Campus, University of Dhaka. 23 March, 2021. F. Nessa 4. Fig 3. Colony on PDA medium and conidia under microscope: a. Penicillium chrysogenum, b. Penicillium oxalicum, c. Penicillium sp. 1, d. Penicillium sp. 2, e. Penicillium sp. 3, f. Penicillium sp. 4, g. Scytalidium lignicola and h. Talaromyces trachyspermus. (Bar = 50 µm). Talaromyces trachyspermus (Shear) Stolk & Samson, Stud. Mycol. 2: 32 (1972) (Fig. 3h) The front side of the colony white and reverse side light brown, texture floccose, sporulation moderately dense to dense, and conidia numerous, colonies grew slowly. Phialides lanceolate, MORPHOLOGICAL AND MOLECULAR CHARACTRIZATION OF ENDOPHYTIC FUNGI 119 metulae in small verticils. Conidia ellipsoidal to ovoidal and 5.90~ 7.87 μm in diameter. Specimen examined: Isolated from the fresh and healthy root of Centella asiatica from botanical garden of Curzon Hall Campus, University of Dhaka. 16 June, 2021. F. Nessa 15. Molecular identification Molecular characterization of the fungal species was conducted for proper identification using sequence analysis of ITS region. Ten isolates were identified by analyzing ITS regions sequences using the ITS1 and ITS4 as forward and reverse primers. In order to confirm at the genomic sequence level, PCR amplified bands (~550 bp) from ten samples were subjected to automated sequencing followed by BLAST analysis (Table 1). The endophytic fungi of this study were identified on the basis of sequence similarity of ITS region. PCR amplification of internal transcribed spacer (ITS) regions generated a sharp band of approximately 550 bp in 1% agarose, confirming the presence of the desired region from each of the isolates (Fig 4). Fig 4. Gel electrophoresis of amplified ITS region of the isolated endophytic fungi. (L represents 1kb DNA ladder) Table 1. BLAST analysis of the amplified sequences from the isolated DNA of endophytic fungi. Sample ID Name of Fungi Max score Total score Query coverage (%) E value Identity (%) NCBI Gene Bank Acc. No. F12 Curvularia chonburiensis 704 704 75 0.0 98.47 NR168176 F9 Curvularia hominis 798 798 96 0.0 93.62 MN540244 F10 Curvularia lunata 778 778 96 0.0 92.69 OL699891 F11 Curvularia lycopersici 470 470 100 6E-128 99.23 MT590310 F2 Fusarium falciforme 656 656 84 0.0 91.53 OM372820 F5 Fusarium phaseoli 771 771 92 0.0 94.20 OM839786 F1 Fusarium solani 798 798 94 0.0 94.65 KU939057 F6 Fusarium udum 621 621 84 4e-173 91.01 MW647689 F13 Lasiodiplodia theobromae 753 753 85 0.0 97.56 OL453207 F3 Penicillium commune 714 714 73 0.0 98.53 EU436692 ~550 bp 120 NESSA et al. To confirm identity, the obtained DNA sequences of the isolated endophytic fungi were matched with the already available sequences in National Center for Biotechnology Information database. The obtained DNA sequences showed 98.47% identity with Curvularia chonburiensis, 93.62% identity with Curvularia hominis, 92.69% identity with Curvularia lunata, 99.23% identity with Curvularia lycopersici, 91.53% identity with Fusarium falciforme, 94.20% identity with Fusarium phaseoli, 94.65% identity with Fusarium solani, 91.01% identity with Fusarium udum, 97.56% identity with Lasiodiplodia theobromae, 98.53% identity with Penicillium commune (Table 1). Molecular analysis showed species identification of all the fungal genera studied morphologically (Table 2). Table 2. Comparison between morphological and molecular identification of ten fungal isolates. Isolates No. Morphological identification Molecular identification F9 Curvularia sp. 1 Curvularia hominis F10 Curvularia sp. 2 Curvularia lunata F2 Fusarium sp. 1 Fusarium falciforme F5 Fusarium sp. 2 Fusarium phaseoli F1 Fusarium sp. 3 Fusarium solani F6 Fusarium sp. 4 Fusarium udum F13 Lasiodiplodia sp. Lasiodiplodia theobromae F3 Penicillium sp.5 Penicillium commune F12 Unidentified Curvularia chonburiensis F11 Unidentified Curvularia lycopersici Neighbor-joining tree based on ITS sequences of ten endophytic fungi was also constructed to see the phylogenetic relationship among them (Fig. 5). From this tree, it was demonstrated that fungi belonging to same genera form same cluster. Fig 5. Phylogenetic tree based on ITS sequences of ten endophytic fungi. Scale bar indicates the number of nucleotide substitution per site Curvularia lunata Curvularia lycopersici Curvularia hominis Curvularia chonburiensis Penicillium commune Lasiodiplodia theobromae Fusarium falciforme Fusarium udum Fusarium phaseoli Fusarium solani 100 73 100 90 100 50 50 0.050 MORPHOLOGICAL AND MOLECULAR CHARACTRIZATION OF ENDOPHYTIC FUNGI 121 Among the total 28 fungal endophytes, 18 species were recovered from different parts of the Andrographis paniculata plant. The fungi were Aspergillus flavus, A. niger, A. terreus, Cladosporium sp., Colletotrichum sp., Curvularia chonburiensis, Curvularia lycopersici, Fusarium solani, Fusarium udum, Fusarium sp., Lasiodiplodia theobromae, Monodictys paradoxa, Penicillium chrysogenum, P. oxalicum, Penicillium sp. 2, Penicillium sp. 3, Penicillium sp. 4 and Scytalidium lignicola. Earlier 6 fungal endophytes were isolated from the same plant to study their potential for the production of plant growth promoters and enzymes. (Adhikari Mukhopadhyay, 2022) Sixteen species of endophytic fungi were isolated from the Centella asiatica plant. The fungi were Aspergillus flavus, A. fumigatus, A. niger, A. terreus, Cladosporium sp. Colletotrichum sp., Curvularia hominis, C. lunata, Curvularia sp., Fusarium falsiforme, F. phaseoli, Penicillium commune, Penicillium sp. 1 and Penicillium sp. 2. This work will lead to the study of the endophytic fungal diversity and the species richness in those medicinal plant. Among the isolated fungi, Curvularia chonburiensis, Curvularia hominis, Curvularia lycopersici, Fusarium falciforme, Fusarium phaseoli, Monodictys paradoxa, Penicillium commune and Scytalidium lignicola have been reported as new records for Bangladesh as these were not documented in relevant literature (Siddiqui et al., 2007; Shamsi S, 2017; Nahar et al., 2019; Khatun et al.2022). The present investigation suggests that molecular technique is more accurate and rapid means of fungal identification. ITS-based molecular identification methods might be an important complement to conventional mycological detection by culture. References Adhikari, M. and Mukhopadhyay, M. 2022. Potentials of endophytes of Andrographis paniculata for the production of plant growth promoters, enzymes and antimicrobial compounds. SAARC J. Agric. 19(2): 157–170. Amer, O.E., Mahmoud, M.A., El- Samawaty, A.M.A. and Sayed, S.R.M. 2011. Non liquid nitrogen- basedmethodfor isolation of DNA from filamentous fungi. 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