View of Morphological and Molecular Characterization of Endophytic Fungi isolated from the leaves of Bergenia ciliata

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Annals of R.S.C.B., ISSN:1583-6258, Vol. 25, Issue 4, 2021, Pages. 2239 – 2257 Received 05 March 2021; Accepted 01 April 2021.

Morphological and Molecular Characterization of Endophytic Fungi isolated from the leaves of Bergenia ciliata

Jiwan Raj Prasai¹ S. Sureshkumar¹, P. Rajapriya²C. Gopi³ and M. Pandi^1*

1Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai – 625021, Tamil Nadu, India.

2Department of Zoology, M.S.S. Wakf Board College, Madurai – 625020, Tamil Nadu, India.

3Department of Botany, C.P.A College, Bodi – 625513. Tamil Nadu, India.

*Corresponding Author. Email- [email protected]

Abstract

Endophytic fungi are microorganisms that are present inside the healthy tissue of living plants. Endophytes existence inside the plants tissue enhances the growth and development of the bio-active compounds which increase the quality and quantity of crude drugs. The endophytic fungal assemblages from the medicinal plants are limited around the world. The present study was conducted for the morphological and molecular identification of the endophytic fungi isolated from medicinal plant leaves Bergenia ciliata collected from different mountain areas of Sikkim, India. In this study total of 130 leaves segment was selected for fungal isolation from which 75 fungal colonies were recovered among them 25 different endophytes were isolated and characterized based on the morphological appearance and colony characters. Further all 25 fungi were identified molecular level through Internal Transcribed Spacer (ITS) and ITS2 sequence-secondary structure based analysis. On the basis of morphological and molecular characterization the isolated fungi were belonging to 6 orders i.e. Glomerellales, Trichosphaeriales, Diaporthales, Xylariales, Botryosphaeriales, Pleorotales and 9 genera i.e. Colletotrichum, Nigrospora, Phomopsis/ Diaporthe, Arthrinium, Neofusicoccum, Stagonosporopsis, Phomatodes, Pestalotiopsis, Guignardia of the Ascomycetes class. Among the isolates Colletotrichum sp were dominant. This is the first report of endophytic fungal assemblage from Bergenia ciliata.

Key words: Endophytic Fungi, ITS region, ITS2 secondary structure, Bergenia ciliata.

Introduction

Endophytic fungi typically reside inside the tissues of the plant are unexplored by a community of microorganisms. Their symbiotic relationship protects their host from infectious agents and resists adverse conditions by discharging active metabolites (Petrini, 1991). Currently, 1.5 million fungi species have been identified so far, (Carroll, 1991; Strobel et al.,2004; Gond et al., 2010;

Singh et al., 2011; Blackwell M., 2011; Lahrmann et al., 2013) to this 7% are occupied by endophytic fungi (Chowdhary& Kaushik, 2015). Endophytic fungi are the primary tools for scientific research. They are the origins of new bioactive compounds and have economical significance in various fields such as agriculture, pharmaceutical and industrial (Bills &

Polishook, 1992; Strobel & Daisy, 2003; Arnold et al., 2007). The identification of fungal diversity and ecological analysis was expected to become essential in the field of fungal biology.

Earlier, the fungal taxonomy was carried out by comparing their morphological features (Lodge et al., 1996; Sette et al., 2006; Crous et al., 2007; Zhang et al., 2008). However, it was difficult to distinguish closely related species, identical morphotypes and non-sporulating isolates. This was due to the essence of the media composition, which may impact the morphological characters (Hyde & Soytong,2007). Recently, many molecular techniques are employed for taxonomic classifications because of their sensitivity and specificity to classify species level (Sette et al.

2006). Different molecular markers such as Internal Transcribed Spacers (ITS), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Calmodulin (CAL), Actin (ACT), Glutamine synthetase (GS), ß-tubulin (Tub2), RNA polymerase II second largest subunit (RPB2), Translation

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Elongation Factor 1-alpha (TEF1), etc. have been successfully identified and used in several recent studies (Wang et al. 2005; Arnold &Lutzoni 2007; Ligrone et al., 2007; Silva et al., 2012;

Weir et al., 2012; Sunderasan et al., 2019). The ITS region is located in three sub-regions between the ribosomal large subunit (LSU) and the small subunit (SSU): ITS1, 5.8S and ITS2.

ITS was used primarily as a molecular marker and was proposed as the universal sequence of fungal barcodes (Schoch et al., 2012) (Fig.1).The full length of the region is between 0.45 Kb and 0.75 Kb. Primers ITS1 (forward) and ITS4 (reverse) are widely used to amplify the ITS region using polymerase chain reaction (PCR). The primer will bind to ribosomal regions of the small and large subunit and covers the complete length of the target region. Then the amplified ITS areas is sequenced. The similarity between the collected sequences and those contained in databases is often used to identify the fungal organism (Gardes & Bruns 1993; White et al., 1990)

Fig. 1: Schematic representation of Internal Transcribe Spacer (ITS) region with universal primer ITS1 and ITS4.

In recent times, up to the species level, the ITS2 was used as an effective barcode for fungal classifications and is a mini barcode, simply amplified and sequenced (Keller et al., 2010; Yang et al., 2018). Phylogenetic resolutions in closely related species can be improved by the ITS2 sequence-secondary structure data. Several studies have shown the potential applications of ITS2 for taxonomic classification and phylogenetic reconstruction at both genus and species levels for eukaryotes, including plants, animals and fungi (Yao et al.,2010; Han et al., 2013).More information about variable characters are given in the identification based on the sequence structure, i.e. 5' apex UGGU motif (deviations such as UGGGU, UGG, or GGU have been described), as well as the Helix II-II U-U mismatch, four helix structure, Helix III being the longest compared to others (Schultz & Wolf 2009).Compensatory base change (CBC) has been used as a potential marker for species delimitation (Muller et al., 2007). The aim of this study is to search the fungal endophytes from ethno medicinal plant Bergenia ciliata collected from different mountain regions of the northeastern state of Sikkim, India. Bergenia ciliata is one of the most valuable medicinal plants of the Saxifragaceae family, found mostly in India's high- altitude mountain regions. The plant was used by indigenous people to treat various diseases such as urinary disorders, ear infections, coughs and colds, diarrhea, fevers. Pulmonary disorders, haemorrhoids, asthma, boils, skin disease, ophthalmia, cancer and dissolution of stones in the kidney (Asolkar et al.,1992; Sinha et al.,2001; Ahmad et al., 2018).

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Annals of R.S.C.B., ISSN:1583-6258, Vol. 25, Issue 4, 2021, Pages. 1105 - 1112 Received 05 March 2021; Accepted 01 April 2021.

Materials and Methods Selection of Plant

Healthy leaves of Bergenia ciliata plants were collected from various mountain regions (altitude 5,410ft. Latitude 27.33 " N and longitude 88.60 " E) North Eastern State of Sikkim, India, used for the study. All the collected leaves were sealed and transported to the laboratory using a sterile ziplock polythene bag and stored at 4ºC for further use. Within 24 hours, the leaves are separately processed to extract noble endophytic fungi.

Fig. 2: Location map of sample collection sites of the medicinal plant (Bergenia ciliata) with their corresponding latitude and longitude.

Isolation of Endophytic fungi

The leaves are thoroughly washed by running tape water for five minutes to remove all the imprinted dust and debris on the leaf surface. The cleaned leaves were again washed by sterile distilled water and with the aid of a sterile blade, the washed leaves were cut into tiny pieces ~ 0.5 – 1cm in size. The cut part of the leaves was treated for five seconds with 70 % ethanol and then dipped again for ninety seconds in 4 % sodium hypochlorite, rinsed by autoclaved water for ten seconds and allowed to air dry with the use of sterile tissue papers to remove excess water present on the leaf pieces (Dobranic et al., 1995). The sterile leaf parts were then placed on the Streptomycin (200µg/mL) amended Potato Dextrose Agar (PDA) plate. The inoculated plates were allowed to incubate at 24 ± 1ºC, 12 h light and 12 h dark cycles. To verify the efficacy of surface sterilization, leaf imprinted control plates were also maintained. For the growth of endophytic fungi from the leaf segments, the plates were periodically examined. To obtain a single isolate, the hyphal tips emerging from the inoculated leaf segments were immediately sub- cultured on fresh PDA plates. For further studies, the pure isolates were maintained in PDA slants at 4^ºC.

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Morphological identification of fungi

The isolated endophytic fungi used for the morphological identification were grown on PDA plate at 24^ºC±1^° C. Different morphological characters were used for the study. The major morphological appearance are front and reverse of the colonies' colours on culture plates, colony's surface margin, texture, elevation, growth patterns, hyphae, mycelium, spore shape and size.

DNA Extraction

One-week-old pure fungal culture was selected to isolate the genomic DNA. One gram of fungal mycelia was collected from the culture plate and uniformly ground by mortar and pestle using 4mL of extraction buffer (200mMTrisHCl, 250mMNaCl, 25 mM EDTA, 0.5 percent SDS) at pH 8.5. After that, 600 µL of the crushed sample was taken in centrifuge tubes to this a 300µL of 3M Sodium acetate (pH 5.2) was added and allows for incubation at -20ºC for 10 minutes. After the tubes were centrifuged for 5 minutes at 12,000 rpm. The supernatant was transferred to fresh eppendrof tubes without disrupting the pellet. An equivalent amount of isopropanol was applied to the supernatant and kept at room temperature for 5 minutes for DNA precipitation. The tube was centrifuged again for 2 min at 12,000 rpm. Finally, with 70 % ethanol, the pellet was washed, air-dried and suspended in 50 µL of sterile milliQ water. 0.7 % of agarose gel electrophoresis confirmed the isolated fungal genomic DNA (Cenis 1992).

Amplification of ITS region

The universal primers ITS1 and ITS4 were used by Polymerase chain reaction (PCR) to amplify the fungal ITS region. The Bio-RAD instrument was used for PCR amplification with a total reaction volume of 25 µL comprising 20 ng genomic DNA, 10X PCR buffer with 25mM MgCl2, 10mM dNTP, 2U Taq DNA polymerase and 10 pmol ITS1(forward ) and ITS4 (reverse) primers.

The following parameters were used for thermocycling: initial denaturation at 94ºC for 4 min, followed by 30 cycles, every 30 seconds at 94ºC for denaturation, 1 min at 58.2ºC for annealing, 2 minutes at 72ºC for an extension with a final extension at 72ºC for 7 minutes (Sim et al., 2010).

By using 1% agarose gel electrophoresis with a 100bp DNA marker analyzed the amplified DNA fragments. The amplified products were visualized using gel documentation (Uvitech). A non- template control was included in each run. Then the amplified DNA was sequenced by Eurofins Private Limited Bangalore, Karnataka, India.

Phylogenetic analysis

The EMBOSS merger tool (http://www.bioinformatics.nl/cgi-bin/emboss/merger) was used to get full-length ITS sequences. The fungal ITS sequence was compared using the Basic Local Alignment Search Tools (BLAST) from the National Center for Biotechnology Information (NCBI) database. For the study of phylogenetic relationships, more identical sequences were download and aligned by using the CLUSTAL-W program implemented in MEGA 6. The multiple sequence alignment (MSA) generated was used for phylogenetic tree construction. A neighbor-joining tree was reconstructed using MEGA software version 6 with a bootstrap

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consensus of 1000 replicates to determine the reliability of the generated nodes (Larkin et al., 2007; Tamura et al., 2011).

ITS2 RNA secondary structure analysis

To extract the ITS2 region from the complete length of ITS sequences, the fungal ITS extractor (http://www.emerencia.org/FungalITSextractor.html) was used (Nilsson et al., 2010). Using the Mfold server with a preset temperature of 37^oC, the secondary ITS2 RNA structure of the query sequence and its closest matches in the ITS phylogenetic tree was predicted using the following conditions:- 1M NaCl (no divalent ions) ionic conditions, percentage sub-optimality number 5, upper folding number: 50, maximum interior/bulge loop asymmetry: 30, maximum interior/bulge loop size: 30, maximum interior/bulge loop size. The various parameters, such as structural energy, length, base composition, GC content and standard core-based structure, minimum free energy (MFE) were chosen for the construction of the secondary structure. Structural data were downloaded from the Mfold server in Vienna format (Zuker 2003; Rao & Satish 2016). 4SALE V 1.7 software was used to align and create the consensus structure with the ITS2 sequence- secondary structures. For phylogenetic analysis, the resultant alignment was exported to ProfDistS 0.9.9 (Friedrich et al., 2005).

Result

Isolation and identification of fungi

A total of 75 fungal endophytes were isolated from Bergenia ciliata leaves. They were grouped into 25 morphotypes based on phenotypic features such as colony colour, growth patterns, surface texture (Fig.3) and by using light microscope conidial features, spore dimensions were measured (Fig.4). The morphological features of the fungi have been explained and listed on table - 1. Also, The fungal isolates were also characterized at the molecular level using ITS sequences. All the isolated fungi were examined and arranged on six clades divided into two classes -Sordariomycetes and Dothideomycetes. Sordariomycetes include Colletotrichum (Clade 6), Diaporthe / Phomopsis (Clade 5), Nigrospora, Pestalotiopsis, and Arthrinium (Clade 4).

Dothideomycetes include Guignardia, Neofusicoccum (Clade 2), Stagonosporopsis, Phomatodes (Clade 3), and out-group genera Aureobasidium melanogenum on clade 1 (Clade 1 to 6 counted from lower to upward direction on the phylogenetic tree shown on Fig.5). All the isolates came under six orders (Botryosphaeriales, Diaporthales, Glomerellales, Pleosporales, Xylariales and Trichosphaeriales). All the isolated fungus strain was indicated as JPSK1 to JPSK25. Using ITS based identification, the organism (isolated endophytic fungi) can be identified only at the genus level, which can be further identified up to the species level using ITS2 sequence-structure base identification (Schultz and Wolf 2009).

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Fig. 3. Colony morphology of fungal endophytes on PDA at 24°C. isolated from Bergenia ciliata. Colletotrichum sp. 3, 9, 12, 14, 16, 17,18, 20, 21, 22, 23and 24. Nigrospora sp. 10, 11 and 15.Arthrinium sp. 4 and 8. Neofusicoccum sp. 5 and 6. Diaporthe sp. 13. Stagonosporopsis sp. 1.Phomopsis sp. 2. Phomatodes sp. 7. Pestalotiopsis sp. 19.

Guignardia sp. 25.

Fig. 4. Light Microscopic observation of fungal endophytes isolated from Bergenia ciliata. Colletotrichum sp.

3,9,12,14,16,17,18,20,21,22,23 and 24. Nigrospora sp. 10, 11 and 15. Arthrinium sp. 4 and 8. Neofusicoccum sp. 5 and 6. Diaporthe sp. 13, Stagonosporopsis sp. 1. Phomopsis sp.2. Phomatodes sp.7. Pestalotiopsis sp.19. Guignardia sp. 25.

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Table. 1: Summary of cultural characteristics of the 25 fungal morphotypes from Bergenia ciliata on PDA, at 24ºC.

Morpho - Type Colour (Front) Colour (Reverse) Shape Mycelium Margin Hyphae Spore Shape Spore Size (µm)

JPSK1 White to Dark brown Dark gray Irregular Flat Irregular Septate Cylindrical 4.2 - 8.3 x 2.6 - 3.1

JPSK2 White White to Brown Circular Aerial Entire Septate Cylindrical 8.1 - 15.5 x 1.4 - 2.7

JPSK3 White to pale gray Dark to white Circular Aerial Entire Septate Ellipsoidal 9.4 - 21.3 x 3.1 - 4.1

JPSK4 White brown White to pale yellow Irregular Flat Undulated Septate Cylindrical 6.5 - 12.5 x 3.5 - 4.2

JPSK5 White to grey Pale grey to greenish black Irregular Aerial Irregular Septate Ellipsoidal 9.2 -18.1 x 4.8 - 5.1 JPSK6 White to grey Pale grey to greenish black Irregular Aerial Irregular Septate Ellipsoidal 14.1 - 17.5 x 3.4 -3.5

JPSK7 White to Pale Green Light green Circular Aerial Entire septate Cylindrical 5.1 - 10.0 x 2.1 - 4.0

JPSK8 White brown White to pale yellow Irregular Flat Undulated Septate Spherical 9.8 - 12.2 x 3.1 - 5.4

JPSK9 White or pale gray Dark white & pale gray Circular Aerial Entire Septate Cylindrical 9.1 - 21.1 x 3.3 - 4.1

JPSK10 Gray to Dark Brown Dark gray Irregular Aerial Irregular Aseptate Globose 8.5 - 18.1 x 9.0 -14.9

JPSK11 White White Circular Aerial Entire Aseptate Globose 12.0 - 18.1 x 9.1 - 14.5

JPSK12 White to dark brown Dark blue Circular Aerial Entire Septate Cylindrical 10.1 - 20.5 x 3.5 - 6.2

JPSK13 White to dark gray Dark gray Irregular Aerial undulated Septate Cylindrical 9.5 - 16.2 x 1.4 - 2.5

JPSK14 Grey to light brown yellow to Pale grey Circular Aerial Entire Septate Cylindrical 9.3 - 21.1 x 3.3 - 4.1

JPSK15 White White to light black Circular Aerial Irregular Septate Globose 18.1 - 21.4 x 14.1 -15.3

JPSK16 White Orange yellow to white Circular Aerial Circular Septate Cylindrical 16.5 - 22.5 x 4.5 - 6.5

JPSK17 White to pale yellow White to pale yellow Circular Aerial Entire Septate Cylindrical 13.3 - 15.3 x 4.6 - 5.2 JPSK18 White to light dark White to yellow Circular Aerial Entire Septate Cylindrical 21.1 - 28.5 x 2.1 - 3.2

JPSK19 White White to light yellow Irregular Aerial Irregular Septate Globose 14.8 - 21.2 x 5.4 - 6.6

JPSK20 White Dark blue Circular Aerial Circular Septate Cylindrical 10.5 - 21.3 x 3.0 - 4.3

JPSK21 White to light dark Brick red to pale yellow Circular Aerial Entire Septate Cylindrical 13.5 -15.5 x 4.6 -5.1

JPSK22 Dark gray or black Dark blue Circular Aerial Entire Septate Cylindrical 8.3 - 18.5 x 3.2 - 5.1

JPSK23 White to light gray Light orange to yellow irregular Aerial Entire Septate, Cylindrical 8..3 - 28.5 x 2.4 - 3.3

JPSK24 White to dark gray Gray to black irregular Aerial Irregular Septate Spherical 10.5 - 21 x 3.1 - 4.3

JPSK25 Dark blue to green Dark blue irregular Aerial Entire Septate Cylindrical 6.5 - 11.3 x 5.5 - 6.2

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Fig. 5: Neighbor-Joining (NJ) Phylogenetic tree with ITS rDNA sequences indicating the phylogenetic relationship of endophytic fungi. The endophytic fungi isolated from the selected plant in the current study are labeled in black.

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Nucleotide information and ITS2 RNA secondary structure analysis

The Mfold software was used to predict the ITS2 secondary structures. The ITS2 sequence length ranged from 132 to 162 nucleotide bases, and the GC content ratio was approximately 52.22 – 63.63 (Table 3). Besides, consensus secondary structure was constructed genus-wise.

For Colletotrichum 20 sequences whose minimum free energy (MFE) was -67.85 kcal/mol (mean value). Likewise, the Arthrinium, Diaporthe/Phomopsis, Nigrospora and Neofusicoccum secondary structure was constructed with 4 sequences whose MEF -68.97, - 73.00, -61.12 and -73.08 respectively. The next series 2 sequences of Pestalotiopsis, Guignardia, Phomatodes and Stagonosporopsis had -53.48,-76.83,-61.58 and -62.06 MFE respectively. The secondary structure of ITS2 containing two forms they are (i) 4-helix domain (ii) 3 helix domains with the most extended third helix and the fourth helix not always present. In the present study, a genus called Arthrinium, Diaporthe / Phomopsis, Nigrospora, and Neofusicoccum contain four helixes with a more extended third helix. While Colletotrichum, Pestalotiopsis, Phomatodes, Stagonosporopsis and Guignardia having three helixes with the longest third helix was found in all genera.

Fig. 6: Consensus ITS2 secondary structure of endophytic fungal genera (Arthrinium, Colletotrichum, Phomopsis / Diaporthe, Guignardia Neofusicoccum, Nigrospora, Pestalotiopsis, Phomatodes and Stagonosporopsis) and their Minimum Free Energy (MEF) value.

CBC analysis

By using 4SALE tools the compensatory base change (CBC) was observed in a pair of structures. Observing one or more CBCs between a pair of secondary structures signifies the two structures under comparison to being distinct belong to different species (Fig.7). The black mark region indicates a lack of CBC between the pair of sequences (Fig.7). It may belong to the same or different species. In this study Colletotrichum horii, and Colletotrichum fructicola were distinguished from Colletotrichum gloeosporioides, Colletotrichum brevisporum, Colletotrichum sydowii because of presence of CBC in the isolates. Whereas

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Nigrospora oryzae, N. chinensis and N. sphaerica show no CBC but the nucleotide variation found between their sequences (Details of CBC were mentioned in Table.3).

Phylogenetic Analysis

For the phylogenetic analysis, Sequence-structure based alignment performed in 4SALE was subsequently exported to ProfDistS. The phylogenetic clade formation was separated. Based on the analysis, 15 different species were identified, which included Stagonosporopsis cucurbitacearum, Phomatodes aubrietiae, Arthrinium hydei, Neofusicoccum mangifera, Pestalotiopsis microspora, Guignardia mangiferae, Nigrospora oryzae, N. chinensis, N.

sphaerica, Colletotrichum brevisporum, C. horii, C. fructicola, C. sydowii, C. boninense, C.

gloeosporioides. Seven isolates were classified up to the genus level that is Arthrinium sp. (1), Neofusicoccum sp (1), Phomopsis / Diaporthe sp (2), Colletotrichum sp (3). In this study, Colletotrichum was most dominant among the twenty-five isolates. They are Colletotrichum sp. (3) Colletotrichum sydowii (2), Colletotrichum boninense (2), Colletotrichum gloeosporioides (2), Colletotrichum brevisporum (1), Colletotrichum horii. (1), and Colletotrichum fructicola (1). The remaining isolates are Nigrospora oryzae, N. chinensis, N.

sphaerica, Stagonosporopsis cucurbitacearum, Phomatodes aubrietiae, Arthrinium hydei, Neofusicoccum mangiferae, Pestalotiopsis microspora, Guignardia mangiferae. All the isolates of the present study belong to nine genera and six orders of Ascomycetes (Table 2).

All these identified fungal rDNA-ITS sequences were submitted to GenBank (www.ncbi.nlm.nih.gov), and their accession numbers are listed in (Table 3).

Table. 2. Number of fungal endophytes identified from Bergenia ciliata.

Sl. no. Number of Genus Number of species Number of strains

1

Colletotrichum sp.

Colletotrichum sp. 3

Colletotrichum sydowii. 2

Colletotrichum boninense. 2 Colletotrichum gloeosporioides. 2 Colletotrichum brevisporum. 1

Colletotrichum horii. 1

Colletotrichum fructicola. 1 2

Nigrospora sp.

Nigrospora oryzae. 1

Nigrospora chinensis. 1

Nigrospora sphaerica. 1

3 Phomopsis sp./Diaporthe sp. Phomopsis sp./ Diaporthe sp. 2

4 Arthrinium sp. Arthrinium hydei. 1

Arthrinium sp. 1

5 Neofusicoccum sp. Neofusicoccum sp. 1

Neofusicoccum mangiferae 1

6 Stagonosporopsis sp. Stagonosporopsis cucurbitacearum. 1

7 Phomatodes sp. Phomatodes aubrietiae. 1

8 Pestalotiopsis sp. Pestalotiopsis microspora. 1

9 Guignardia sp. Guignardia mangiferae. 1

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Fig.7. Compensatory base change (CBC) analysis of the isolated fungi and reference sequences. The black mark region indicates lack of CBC and remains are presence of CBC.

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Fig. 8: Secondary structure-based (ITS2) phylogenetic analysis of query (organisms name ranging from JPSK1 to JPSK25 suggesting black mark) reference sequence by neighbor joining process. Constructing the phylogenetic tree by ProDistS software.

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Table. 3. ITS region nucleotide information a- maximum length of ITS sequences, b-ITS2 sequence and nucleotide compositions (A, U/T, G and C), percentage of secondary structure GC content and minimum free energy (MFE). 1 to 25 is our isolates (JPSK) and 26 to 52 reference sequences from NCBI.

The last number 51 & 52 are out group ref. sequence from NCBI.

Sl. no. Name of the Organism Accession No aTotal

length

bITS2 region

A’s U/T’s G’s C’s % GC

Free Energy

(kcal / mol)

1 Stagonosporopsis cucurbitacearum. JPSK1 MT555754 485 314 -470 32 43 37 45 52.22 -62.58

2 Phomopsis sp. JPSK2 MT560320 550 349-508 33 28 47 51 61.63 -84.34

3 Colletotrichum brevisporum. JPSK3 MT560321 612 354-510 33 29 39 56 60.50 -80.32

4 Arthrinium hydei. JPSK4 MT672781 582 386-539 32 47 36 39 48.70 -69.48 5 Neofusicoccum mangiferae. JPSK5 MT798866 533 402 -533 23 25 37 47 63. 63 -66.70

6 Neofusicoccum sp. JPSK6 MT560383 642 399-554 28 35 42 51 59.61 -76.86

7 Phomatodes aubrietiae. JPSK7 MT560384 506 312-467 32 45 37 42 50.64 -62.33

8 Arthrinium sp. JPSK8 MT560587 587 388-541 32 47 36 39 48.70 -69.48

9 Colletotrichum sp. JPSK9 MT560588 573 358-515 31 37 41 49 56.96 -74.03

10 Nigrospora oryzae. JPSK10 MT561421 526 327-481 30 44 35 46 52.25 -64.68

11 Nigrospora chinensis. JPSK11 MT561422 520 320-472 30 48 35 40 49.01 -60.79

12 Colletotrichum fructicola. JPSK12 MT708144 728 401-558 31 37 41 49 56.96 -65.84

13 Diaporthe sp. JPSK13 MT672790 573 352-513 35 35 43 50 57.05 -73.30

14 Colletotrichum sydowii. JPSK14 MT561432 516 310-467 32 36 41 49 56.96 -67.25

15 Nigrospora sphaerica. JPSK15 MT561433 559 344-498 30 45 36 44 51.61 -57.89

16 Colletotrichum horii. JPSK16 MT568591 578 362-518 31 37 41 48 56.68 -65.94

17 Colletotrichum boninense. JPSK17 MT568592 581 379-536 30 37 42 49 57.59 -67.43 18 Colletotrichum gloeosporioides. JPSK18 MT568594 595 386-542 29 38 42 48 57.32 -63.42 19 Pestalotiopsis microspora. JPSK19 MT568595 524 319-480 35 58 33 36 42.59 -53.48

20 Colletotrichum sp. JPSK20 MT568596 554 349-505 31 37 41 48 56.68 -65.94

21 Colletotrichum boninense. JPSK21 MT568597 596 376-533 30 37 42 49 57.59 -67.43 22 Colletotrichum sydowii. JPSK22 MT568598 519 305-462 32 36 41 49 56.96 -67.25 23 Colletotrichum gloeosporioides. JPSK23 MT568599 682 370-526 29 38 42 48 57.32 -63.42

24 Colletotrichum sp. JPSK24 MT568600 554 345-501 31 37 41 48 56.68 -65.94

25 Guignardia mangiferae. JPSK25 MT568601 641 421-583 30 38 47 48 58.28 -76.83

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Table 3 Continued

Sl. no. Name Organism Accession No aTotal

length

bITS2

region A’s U/T’s G’s C’s % GC

Free Energy (kcal /mol)

26 Stagonosporopsis cucurbitacearum isolate PIIND-03 MF446620.1 539 333-489 32 43 37 45 52.22 -61.55

27 Phomopsis sp. YE3250 KF733183.1 558 339-498 33 29 47 51 61.25 -84.35 28 Colletotrichum brevisporum strain. CBS129958 MG600763.1 558 344-500 33 29 39 56 60.50 -81-90 29 Arthrinium hydei culture CBS 114990. KF144890.1 699 469-622 32 47 36 39 48.70 -69.40 30 Neofusicoccum mangiferae isolate MBA02. KY052909.1 514 348-502 27 34 43 51 60.64 -69.95 31 Neofusicoccum sp. SFC-2017b strain CERC3497 KX278053.1 562 356-514 29 36 42 52 59.11 -78.82

32 Phomatodes aubrietiae. CBS627.97 NR136003.1 485 305-459 32 46 36 41 49.67 -60.83

33 Arthrinium sp. isolate SFC-R851 MH498543.1 591 393-547 32 48 36 39 48.38 -67.46

34 Colletotrichum sp. MH156052.1 568 353-510 31 37 41 49 56.96 -74.03

35 Nigrospora oryzae. strain APBSMLF76. MG733724.1 555 342-496 30 44 35 46 48.33 -64.68

36 Nigrospora chinensis. strain LC4433 KX986013.1 522 321- 473 30 48 35 40 49.01 -60.79

37 Colletotrichum fructicola. strain HJH-11. MH370509.1 566 360-517 31 37 41 49 56.96 -65.84 38 Diaporthe sp. Ina CC F-2310. AB899787.1 606 387-548 36 33 42 51 57.40 -72.70 39 Colletotrichum sydowii strain CBS 132889. KY263784.1 530 335-492 32 36 41 49 56.96 -67.25 40 Nigrospora sphaerica strain H2. KU878079.1 558 345-499 30 45 36 44 51.61 -57.89 41 Colletotrichum horii isolate DZ-10. MK429844.1 538 362-518 31 37 41 48 56.68 -72.70 42 Colletotrichum boninense isolate LD3-8-1. KX343044.1 599 381-538 30 37 42 49 57.59 -67.43 43 Colletotrichum gloeosporioides. EU847425.1 594 379-535 29 38 42 48 57.32 -63.42 44 Pestalotiopsis microspora isolate. MK801280.1 551 332-493 35 58 33 36 42.59 -53.48 45 Colletotrichum sp. JQ658860.1 554 350-506 31 37 41 48 56.68 -65.94

46 Colletotrichum boninense isolate LD3-8-1 KX343044.1 599 381-538 30 37 42 49 57.59 -67.43

47 Colletotrichum sydowii strain CBS 132889. KY263784.1 530 335-492 32 36 41 49 56.96 -67.25 48 Colletotrichum gloeosporioides. EU847425.1 594 379-535 29 38 42 48 57.32 -63.42 49 Colletotrichum sp. BDS-2011 strain GS 04. JN248671.1 579 373- 529 31 37 41 48 56.68 -65.94 50 Guignardia mangiferae strain 01 27. AY277711.1 630 416 -578 30 37 47 49 58.89 -76.83 51 Aureobasidium melanogenum CBS 105.22 NR 159598.1 588 379 - 533 34 42 41 38 50.96 -69.47

52 Aureobasidium melanogenum strain CBS 123.37 MH855849.1 589 378 -534 34 44 41 38 50.31 -70.39

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Discussion

Fungi are a group of species with the largest variety of organisms and are the world's leading components of the tropical ecosystem. To understand the role of endophytic fungal species and diversity in the ecosystem, more focus should be given to fungal research. Endophytic fungi stand as an important genetic resource in the search for novel bioactive compounds (Guimaraes et al. 2008). The endophytic fungi isolated from ethno medicinal plant Bergenia ciliate collected from a different region of high altitude mountains of north-eastern hilly state Sikkim, India were used in this study. The plant was selected due to its multiple ethno medicinal uses to dealing and cures various forms of human infection such as urinary disorders, ear infections, coughs and colds, diarrhea, fevers, pulmonary disorders, hemorrhoids, asthma, boils, skin disease, ophthalmia, cancer, and dissolution of stones in the kidney (Asolkar et al.,1992; Sinha et al.,2001; Ahmad et al., 2018). On the other side, endophytic fungi play a major role in plant tissue physiology and protect plants from various pathogens (insects, pests, nematodes, etc.) and also provide resistance to various environmental factors causing the disease (Clay & Holah, 1999., Omacini et al., 2001, Brundrett., 2006). In recent years, endophytic fungal research has been used up on cataloging the species to clarify the endophytes' existence with specific emphases in researching the novel compound residing in medicinal plants. Mostly endophytic fungi that multiply when temperature and humidity occur in a favorable condition have been documented and more different bioactive compounds are produced, particularly those isolated from medicinal plants that grow in such type of healthy climatic condition (Blackwell M., 2011;

Mishra et.al., 2012). The colonization of fungal endophytes may provide additional benefits to their host by generating many substances that provide defense and increase the hosts' fitness (Redman et al.,2002, Arnold et al., 2003; Tejesvi et al., 2007). The most significant steps in the isolation process are choosing healthy plant components and removing dust debris and epiphytes that adhere to the plant's parts. Their ecological and medicinal importance does not distinguish fungal endophytes (Sette et al. 2006., Tao et al. 2008). Therefore, it is very essential to classify and characterized the fungal endophytes to know their phylogenetic as well as molecular relationship. The endophytes that exhibited characteristic colony morphology and microscopic features such as colonies, shape, size, margin, the colour of the fungal culture, hyphal and spore arrangement were putatively identified at genus level but morphological features only were difficult to identify up to the species level (Larone, 2002., G. S. Barseghyan and S. P. Wasser, 2010). The endophytes which were difficult to identify morphologically were subjected to molecular based on sequencing the ITS1 - 5.8S - ITS2 region as well as the relation of these strain to their close related isolates using neighboring joining plot. Differences between Candida and Penicillium species have previously been used to identify using the amplified ITS1 - 5.8S - ITS2 region (Skouboe et al. 1999; Korabecna et al. 2003; Morakotkam et al. 2007). The ITS region has been frequently used to classify fungi as a phylogenetic marker (Schoch et al., 2012;

Sun & Guo 2012). Through molecular characterization, many studies have been done on the identification of endophytic fungi isolates from several different medicinal plants (Chen et al., 2011; Yoo & Eom, 2012; Bhagat et al., 2012). For the delimitation of species levels between closely related organisms, ITS2 sequence-secondary structure-based analysis is now used as a third-dimensional approach to characterizing fungi and other eukaryotic organisms, including plants, animals, insects, etc. (Zhang et al. 2015).

Sequence-secondary structure-based study of ITS2 is more descriptive than structural evidence can provide additional support for organism characterization, as the secondary RNA structure was stronger and more detailed, and well suggested in phylogenetic reconstruction (Coleman

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2003, 2009). In particular, ITS data has enhanced molecular-based identification since 2010 in comparison with other approaches (Raja et al., 2017). This has recently helped to classify fungi by providing additional information (GokulRaj et al., 2014; Sundaresan et al., 2019). Another additional supporting technique for distinguishing structural pairs in the series of CBC (Compensatory Base Change). The existence of CBC among pairs of the structure shows that the two distinct species and their likelihood were ~93%. If that there is no CBC in the gap between the structural pairs in that case, with a likelihood of ~ 76 percent, which indicates that they may belong to the same or different species. (Muller et al., 2007; Wolf et al., 2013). Due to its very shorter length with a lower GC gap, the ITS sequence - secondary structure is used as a suitable for fungal species level recognition, and the structural characters have greater phylogenetic utility.

ITS2 sequences and neighboring joining method further analyzed structural details that differed from other strategies based on our sequence-structure-based alignment, resulting in more accurately segregated clades. Based on these sequences, the phylogenetic tree was built along with aligned reference sequences NCBI databases. The dendrogram generated 25 isolates that were arranged on six clades which include nine genera and six orders. The generated isolates were divided into two classes i.e. Sordariomycetes and Dothideomycetes belonging to the diverse group of Ascomycota. Among all, Colletotrichum was the highly dominant genera.

Conclusion:

On the study of morphological and molecular information, the 25 fungal morphotypes were isolated from Bergenia ciliata. The 25 isolates were arranged on six different orders they are Botryosphaeriales, Diaporthales, Glomerellales, Pleosporales, Xylariales, and Trichosphaeriales and nine genera are Stagonosporopsis, Phomopsis / Diaporthe, Guignardia ,Phomatodes, Pestalotiopsis, Neofusicoccum, Arthrinium, Nigrospora and Colletotrichum. Among the 25 morphotypes, 15 isolates were identified to species level and the remaining seven were at the genus level. Of all the 25 isolates, Colletotrichum was found more dominant in this study. The ITS2 sequence secondary structure could be the most essential and useful tool for distinguishing between closely related species. Further investigation is now being focused on extract, and purifies the bioactive compounds for their antimicrobial, antioxidant, and anticancer activities.

Acknowledgement

The authors thank to DST- PURSE, Madurai Kamaraj University, Madurai, Tamil Nadu for providing laboratory and instrumentations facilities. One of the authors (Mr. Jiwan Raj Prasai) thanks to Government of Sikkim, India for providing the fellowship (Post Metric Scholarship) for pursuing the research work.

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