Phylogenetic reconstruction of endophytic fungal isolates using internal transcribed spacer 2 (ITS2) region

Endophytic fungi are inhabitants of plants, living most part of their lifecycle asymptomatically which mainly confer protection and ecological advantages to the host plant. In this present study, 48 endophytic fungi were isolated from the leaves of three medicinal plants and characterized based on ITS2 sequence – secondary structure analysis. ITS2 secondary structures were elucidated with minimum free energy method (MFOLD version 3.1) and consensus structure of each genus was generated by 4SALE. ProfDistS was used to generate ITS2 sequence structure based phylogenetic tree respectively. Our elucidated isolates were belonging to Ascomycetes family, representing 5 orders and 6 genera. Colletotrichum/Glomerella spp., Diaporthae/Phomopsis spp., and Alternaria spp., were predominantly observed while Cochliobolus sp., Cladosporium sp., and Emericella sp., were represented by singletons. The constructed phylogenetic tree has well resolved monophyletic groups with >50% bootstrap value support. Secondary structures based fungal systematics improves not only the stability; it also increases the precision of phylogenetic inference. Above ITS2 based phylogenetic analysis was performed for our 48 isolates along with sequences of known ex-types taken from GenBank which confirms the efficiency of the proposed method. Further, we propose it as superlative marker for reconstructing phylogenetic relationships at different taxonomic levels due to their lesser length.

approach had strengthened our understanding of fungal evolution and systematics [9]; spurred up proposal for a single identity to an organism with anamorphic and telomorphic stages; uprooted and regrouped many synthetic taxa to erect evolutionarily supported taxon. Internal transcribed region (ITS) rDNA has been the widely accepted standard molecular marker [10,11] for fungal barcoding and features in many scientific literatures of the last two decades than the multilocus approach involving multiple markers such as Cytochrome oxidase c (cox), Tublin (tub), Translation elongation factor 1 subunit alpha (EF1a=tef1) and rpb2 [12,13]. The internal transcribed spacer (ITS) region conventionally includes the entire ITS1, 5.8S gene and ITS2 portion of the nuclear rDNA cistron (Figure 1). ITS based phylogenetic reconstructions provide more clarification at both genus and species level than other gene markers, also it corroborates with the relationship of organism as obtained from mating studies [14]. ITS2 a fast evolving sub-region (<200bp) of internal transcribed spacer; touted as the double edged tool [14] in phylogenetic analysis, has garnered much more attraction [15]. Incorporation of secondary structure data of this region significantly enhances the reliability of sequence alignments, stability of phylogenetic trees and provides finer resolution at both lower and higher taxa levels [16,17,18]. Phylogenetically useful information obtained from ITS2 secondary structure appears highly conserved in pan-eukaryotes [19]. Distinct hallmarks of ITS2 core secondary structure comprises: (1) four helices with (2) helix III as the longest and (3) containing an UGGU motif 5' to the apex (deviations like UGGGU, UGG, or GGU have been described) as well as (4) a U-U mismatch in the second helix. Compensatory base changes (CBCs) were mutations observed at both the nucleotides of a paired site in the helical segments while the pairing itself is maintained. CBCs in the internal transcribed spacer 2 region (ITS2) of the nuclear rRNA cistron have been suggested as a possible marker for distinguishing species. They can be a sufficient but not a necessary criterion to differentiate between distinct species and the result of a CBC analysis may be used to estimate the minimal number of different species present in a multiple alignment [20].
In the present study, endophytes from 3 medicinal plants -Aegle marmelos, Coccinia indica and Moringa oleifera were studied. These three medicinal plants are commonly found in south India. In addition to the well documented knowledge about their utility in traditional medicine and culinary uses, recently novel metabolites of higher therapeutic [21,22] and nutraceutical values were being reported [23,24,25]. This study reports the diversity and phylogenetic relationship of endophytes from three medicinal plants.

Methodology: Collection of Samples
Plant samples of A. marmelos, C. indica and M. oleifera were collected from Chennai, Madurai and Courtallam (Kutralam) of Tamil Nadu, India (Figures 2a & b). Samples were sealed and transported immediately to laboratory; asymptomatic leaves were separately processed within 24 hrs of collection for endophytic fungi isolation [3,26].

Isolation and identification of endophytic fungi from three different medicinal plants
Phylloplane fungal propagules adhering to the surface of the leaves were removed by surface sterilization using the modified method reported [27]: the leaves were washed with running tap water, sterilized with Ethanol (75% V/V) for 1 min and Sodium Hypochlorite (2.5% V/V) for 5 min, then rinsed in sterile water for three times and cut into 1 cm long segments. Plant segments were then transferred to Potato Dextrose Agar (PDA) plates supplemented with Ampicillin (200μg / ml) and Streptomycin (200μg /ml) emerging isolates were sub-cultured on PDA containing plates and incubated at 25ºC for further studies [28].

DNA extraction, amplification of ITS region and sequencing
Genomic DNA was extracted from the endophytic fungus using a modified CTAB method [29].The partial nucleotide internal transcribed spacer (ITS) region was amplified from the genomic DNA using the polymerase chain reaction (PCR) by using the ITS1 forward primer (5' TCC-GTA-GGT-GAA-CCT-GCG-G 3') and ITS4 reverse primers (5'TCC-TCC-GCT-TAT-TGA-TAT-GC 3'). The PCR amplification was performed in an L196GGD Model Peltier Thermal Cycler Version-2.0 with a total 25 μl reaction that comprised of 20 ng of genomic DNA template, 10X buffer with 25mM MgCl2, 10mM DNTP's, 2U of Taq DNA polymerase and 10 pmol of each primer (All molecular chemicals were purchased from Sigma Aldrich). The following reaction conditions were used: 4 min at 94 o C for denaturation, 30 cycles each of 30 seconds at 94 o C for denaturation, 1 min at 58.2 o C for annealing, 2 min at 72 o C for extension followed by the final extension at 72 o C for 7min [30]. The amplified DNA fragments were analyzed by 1% agarose gel electrophoresis with a 100bp ladder purchased from New England Biolabs (Catalogue No. 3231S) and the amplicons were visualized using a gel documentation system (Uvitech). A non-template control was included in each run. PCR products were purified using mini columns (PCR Preps DNA purification System, Sigma) according to the manufacturer's protocol. Further, the amplified products were sequenced by Eurofins Private Limited, Bangalore, India.

ITS2 secondary structure prediction, alignment, phylogenetic analysis
The ITS2 regions were extracted using fungal ITS extractor program. In this study, secondary structures of ITS2 were predicted for 48 query and 28 known isolates (downloaded from genbank NCBI) using Mfold programme (http://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form) with default conditions (linear RNA sequence, folding temperature: 37°C, 1M NaCl (no divalent ions) ionic conditions, 5% sub-optimality, upper bound number of the folding: 50, maximum interior/bulge loop size: 30, maximum asymmetry of an interior/bulge loop: 30, maximum distance between paired bases: no limit). The selected secondary structures were downloaded in Vienna format from Mfold server [31,32]. The consensus structure of each genus was generated using 4SALE [33].

Phylogenetic analysis
ITS sequences of our isolates and control sequences were used for phylogenetic analysis (Neighbor-joining method with 1000 bootstrap replication) using MEGA 5.1. ITS2 sequences and secondary structures were synchronously aligned using 4SALE V 1.7 and resultant alignment was exported to ProfDistS [34] for tree construction.

Figure 3:
Phylogenetic tree inferred using Neighbor-Joining method for query (organism name with GRMPs) and control ITS sequence (organism written in bold and indicated by dark lines).

Results: Isolation of endophytes
A total of 166 isolates were obtained from the above three medicinal plants, among them 48 isolates were characterized based on molecular identification and their ITS sequences were submitted in Genbank (Genbank ID details were listed in Table  1 (see supplementary material). Phylogenetic tree was constructed for our isolates and control isolates using MEGA 5.1 software (Figure 3). Identified isolates belongs to 5 orders (Glomerales, Pleosporales, Diaporthales, Capnodiales and Eurotiales) and 6 genera (Colletotrichum/Glomerella, Diaporthae/Phomopsis, Cochilobolus, Alternaria, Cladosporium, Emericella) of Ascomycota. Colletotrichum/Glomerella genera showed maximum diversity (52%), while Emericella, Cochliobolus and Cladosporium showed minimum diversity (2.0%). Other genera such as Alternaria showed 19% and Diaporthe/Phomopsis had 23% diversity respectively ( Figure 4). C. gloeosporioides and G. cingulata, a telomorph of the former were commonly found in the leaves of all three medicinal plants. C. kusanoi and E. nidulans were present only in C. indica while C. oxysporum was found only from the leaves of M. oleifera. Distribution of other isolates was represented in Table 2

ITS2 secondary structure
The results of the ITS2 extraction for 76 sequences had been summarised in Table 2 & 3 (see supplementary material). ITS2 sequences varied from 157 to 167 base pairs and GC content 48.73 -68.86%. Further they were used to model a consensus structure for each of the respective genera ( Figure 5). In Colletotrichum/Glomerella genus, secondary structures had been modelled for 44 sequences whose minimum free energy (MFE) was -69.51±3.84 (mean ± standard deviation (SD). Similarly, secondary structure predicted for 17 sequences of  Table 4 (Available with authors). To the overall structure we observed a conserved motif like an UGGC sequence preceding the apex of the third helix in all class involved in this study. Further the UGGUUU motif was observed in the loop III of order Capnodiales, Glomerales and Pleosporales, whereas AGGA and CGGA motif was only observed in Diaporthales and Glomerales. Likewise CGGC motif was present in Capnodiales, Eurotiales and Pleosporales.

Phylogenetic reconstruction
Phylogenetic tree was constructed based on ITS2 sequencestructure using our isolates and control sequences (Table 3) have yielded well resolved clades with higher bootstrap support. Monophyletic clades formed were supported with a bootstrap value >50% were represented at their respective nodes ( Figure 6).

Discussion:
A surge in the molecular phylogeny supported research has greatly illuminated the fungal systematics the fungal systematic, ecological and diversity studies as robust computational algorithms with high statistical support and advancements in sequencing technology continue to evolve. ITS2 had been suggested as a standard marker for fungi and integrating ITS2 based phylogenetic analysis with the morphological features of their primary sequences has been the recent trend, which has significantly enhanced the resolution and stability of the clades [35, 36, 37, 38]. ITS2 secondary structure of the sequences analysed in our study were modelled with optimum and sub-optimal free energy in RNAfold program from MFOLD server at default folding conditions [31]. We have employed this approach due to its wide spread use especially in modelling ITS2 secondary structure.
Structures sharing similarities (3-4 helixes) to pan-eukaryotic ITS2 model were chosen from the predicted set of suboptimal structures. On further evaluation of the chosen structures, several conserved motifs were observed. Similar versions of a conserved UGGU motif, preceding the apex of the III helix were observed in our ITS2 structures. A UGGC motif in the 5' side to the apex of the III helix was highly conserved across the investigated genera barring Cochilobolus and Emericella. A profile neighbour joining tree construct based on our sequence-structure alignment resulted in well separated clades. All the investigated isolates belonged to 5 orders and 6 genera of Ascomycota. Distinct clades in Diaporthe and Colletotrichum genera were formed based on the presence of CBCs. Several sub-clades with less bootstrap value were also formed within Diaporthe and Colletotrichum genera that were not supported by CBCs. At species level, several incongruences were observed mainly due to the conventional practices of naming nucleotide sequences. Similar anomalies had been reported earlier [46,47,48] and this mandates revisiting the erroneous Genbank entries and naming after the maximum identical Genbank entries with minimum E-value. Overall resolution of our phylotree at genera level had a bootstrap support >50%. The consensus tree depicted Diaporthales closest to the root and all other genera examined in this studied have to be derived. Glomerales, the other Sorodoriomycetes order studied and Eurotiales of Eurotiomycetes formed sister clades with Dothidiomycetes clade that hosted 2 pleosporales genus (Alternaria and Cochilobolus) and Cladosporium of Capnodiales in subclades, but contradicted with the multigene based phylotree of Ascomycota [49]. The foremost reason for this variation may be due to the random mutation (insertion and deletion) which happens rapidly during the evolution and it depends upon mistakes associated in DNA replication.

Conclusion:
The present result provides novel support from immense analysis of ITS2 sequences and CBC estimation for different endophyte complex. CBC can be used as primary molecular indicator to confirm no genetic exchange between two populations is happened which widens the identification and classification of endophytic species further. The proposed ITS2 based phylogenetics with the fungal isolates from our own study (GRMP) with the reference sequences (ex-types) has clearly distinguish the isolates with greater precision than any other existing methods. This is the first report from India on ITS2 sequence-structure analysis of endophytic fungi from the medicinal plants of A. marmelos, C. indica and M. oleifera. Glomerales (Colletotrichum/Glomerella) a.