Biodiversity , Antimicrobial Potential , and Phylogenetic Placement of an Endophytic Fusarium oxysporum NFX 06 Isolated from Nothapodytes foetida

Biodiversity of endophytic fungi associated with the medicinal plant Nothapodytes foetida of Agumbe forest was determined and evaluated for its microbial activity. A total of 170 endophytic isolates were obtained from leaf, stem, seed, and fruit tissues of Nothapodytes foetida. The dominant endophytic fungi belong to genera Fusarium, Penicillium, Aspergillus, and Colletotrichum. Maximum endophytic isolates were obtained from leaves segments followed by fruit, stem, and seed tissues. Hyphomycetes were the dominant group found with 75.29% over other fungal groups. Shannon-Weiner and Simpson indexes showed rich diversity of endophytic fungi suggesting even and uniform occurrence of various species. 88.57%, 74.28%, 62.85%, and 65.71% of isolates have shown activity against Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922), and Candida albicans (ATCC 69548), respectively. One of the isolate NFX 06 isolated from leaf has showed considerable antimicrobial activity against all the test pathogens. It was identified asFusariumoxysporum by ITS sequence analysis; the nucleotide sequence was submitted in the GenBank with an accession number KC914432. Phylogenetic relationship confirmed that the strain F. oxysporum NFX 06 has evolved from an endophytic ancestor.


Introduction
Endophytes are microbes that colonize living, internal tissues of plants without causing any immediate, overt negative effect [1].Endophytic fungi represent an important and quantifiable component of fungal diversity, with an estimate of at least 1 million species [2][3][4].They are found in nearly all plant families including bryophytes [5], pteridophytes [6], gymnosperms [7], and both monocotyledonous [8] and dicotyledonous angiosperms [9,10].Endophytic fungal community has been found in plants living in a unique ecosystem [11], which can be a source for a variety of bioactive metabolites such as antibiotics, antimycotics, immunosuppressants, and anticancer compounds [12].
The discovery of novel antimicrobial metabolites from endophytes is an important alternative to overcome the increasing levels of drug resistance by plant and human pathogens [13][14][15].The antimicrobial compounds can be used as drugs and also as food preservatives in the control of food spoilage and foodborne diseases [16].Thus there is a huge interest across the world to determine the biodiversity of endophytic mycoflora within the host for its novel and undescribed species [17,18].In this study an attempt has been made to isolate the endophytic mycoflora of N. foetida, to screen for their antimicrobial activity against selected human pathogens and to determine the phylogenetic relationship of Fusarium sp. 2 NFX06 with other endophytic and pathogenic isolates of different plant species.

Materials and Method
2.1.Study Area and Collection of Plant Material.N. foetida, commonly known as "Amrutha" is a medicinal plant that belongs to the family Icacinaceae and it was selected for the study because of its well known pharmacological properties.The plant samples were collected in the month of August from Agumbe forest located at 13 ∘ 30  N and 75 ∘ 2  E in the Western Ghats, Dakshina Karnataka, India (Figure 1).The plant material was identified and authenticated by an experienced botanist.Fresh and healthy parts of the plant like leaves, stem, seed, and fruits were cut with a sterile scalpel and stored at 4 ∘ C in a sterile polythene bag prior to use.

Isolation and Identification of Endophytic Fungi.
Isolation was carried out as described by Wang et al. [19], with little modifications.Plant samples were surface sterilized with 75% ethanol (1 minute) and 2.5% sodium hypochlorite (3 minutes) followed by washing with sterile distilled water (2 minutes).Each plant sample was cut aseptically into 0.5 cm long segment and placed on petri dishes containing potato dextrose agar (PDA) supplemented with chloramphenicol (50 g/mL, Sigma) and streptomycin sulphate (250 g/mL Sigma).The plates were sealed using Parafilm and incubated at 25 ∘ C ± 1 ∘ C in a light chamber with 12 hours of light followed by 12 hours of dark cycles [20].The petri dishes were monitored every day to check the growth of endophytic fungal colonies from the plant segments.As and when the hyphal tips emerged out from plant segments they were isolated and subcultured and brought to pure culture by serial subculturing.Identification of fungal endophytes was carried out based on their microscopic and macroscopic characteristics.Lactophenol cotton blue staining method was used for staining the fungal cultures [21] and visualized under microscope (MOTIC BA 400).

Determination of Species Diversity.
Colonization rate (CR) was calculated as the total number of plant tissue segments infected by fungi divided by the total number of segments incubated [22].Isolation rate (IR) was determined as the number of isolates obtained from plant segments divided by the total number of segments incubated.Colonization frequency (CF %) was calculated as the number of plant segments colonized by a single endophyte divided by the total number of segments observed × 100.Isolation frequency (IF %) was calculated as the total number of isolates of one species divided by the total number of isolates in that sample × 100.Simpson dominance index and Shannon-Wiener's diversity index were calculated for fungal diversity [23,24].
Simpson's index of diversity was calculated using formula 1- as given in the following equation: where  is the total number of organisms of a particular species and  is the total number of organisms of all species.Shannon-Wiener diversity index was calculated using the following equation: where   is the symbol for the diversity in a sample of  species or kinds,  is the number of species in the sample,   is the relative abundance of th species or kinds measures = /,  is the total number of individuals of all kinds,   is the number of individuals of th species, and ln is the log to base 2. The concentrations were confirmed via spectrophotometer readings at 580 and 626 nm, respectively.Yeast samples were inoculated on Sabouraud agar (1% peptone, 4% dextrose, and 2% agar), and the bacteria were plated on Brain Heart Infusion agar (Difco) using a swab.

Fermentation.
The fungal fermentation was carried out using 100 mL of potato dextrose broth (PDB) in 250 mL of Erlenmeyer flask inoculated with (1 cm × 1 cm) mycelial agar plug taken from an actively growing colony on potato dextrose agar plate.It was then incubated at a temperature of 25 ∘ ± 1 ∘ C and at 120 rpm under light and dark conditions of 12 hours.After 14 days of fermentation the biomass was separated from the broth by filtration using sterilized preweighed filter cloth.

Antimicrobial Screening of Fungal Endophytes.
The antimicrobial activity of the fermentation broth was carried out for the 35 endophytic fungal isolates.The fermented broth was sterilized by filtration with 0.22 m Millipore syringe filters and 50 L of the broth were tested by modified agar well diffusion assay as described by NCCLS [25].The antimicrobial activity was assessed by the diameter (mm) of inhibition zones relative to those of positive and negative The PCR products were examined by electrophoresis in 1% (w/v) agarose gel with ethidium bromide (10 mg/mL) with a 100-base-pair ladder and checked for size and purity.The purified PCR product was sequenced using the forward and reverse primer in Applied Biosystems 3130 Genetic Analyser.The consensus sequence obtained was subjected to BLAST search to assign putative identity, designation of operational taxonomic units based on sequence similarity measures, and phylogenetic inference.It was then aligned with other sequences of endophytic and pathogenic F. oxysporum downloaded from GenBank to determine their possible evolutionary relationship using MEGA program [27].

Results and Discussion
Endophytic fungi are known to be ubiquitous in nature and every plant species examined to date has been found colonized with fungal endophytes.It has been found that a single plant species may harbour hundreds of endophytes and may inhabit all available tissues, including leaves, petioles, stems, twigs, bark, xylem, roots, fruit, flowers, and seeds [28][29][30].

Species Diversity.
In the present study a total of 170 isolates of endophytic fungi were recovered from 250 samples of leaf, stem, seed, and fruit of ten N. foetida trees.The population of Zygomycetes and basidiomycetes were totally absent in this study and usually they are isolated in very low numbers in plant endophyte research as reported by Suryanarayanan et al. [31].Colonization rates (CR %) of leaf, stem, seed, and fruit were found to be (88.0%,36.0%,24.0%, and 72.0%) and isolation rates (IR) of leaf, stem, seed, and fruit were found to be (1.1, 0.48, 0.46, and 0.92), respectively (Table 1).It indicates a wide variation between the segments of plants selected.Endophytic fungi were more prevalent on leaf tissue (39.45%) than on stem (16.32%), seed (14.96%), and fruit tissues (29.25%) which comprises 6.47% ascomycetes, 34.70% coelomycetes, 75.29% hyphomycetes, and 1.17% sterile fungi, respectively.Colonization frequency (Table 2) indicates that Colletotrichum sp. 1, Pestalotiopsis sp., Aspergillus flavus, Alternaria sp., Fusarium sp. 1, Fusarium sp. 2, Fusarium sp. 3, and Penicillium sp. 2 were the common isolates found in leaf, stem, seed, and fruit whereas Chaetomium sp., Drechslera sp., and Mycelia sterilia were restricted to the leaf tissues.
As shown in Table 3, this difference in prevalence may be tissue specific which may be due to their anatomical structure [32,33].In the comparative study of twig and leafassociated endophytes in Quercus ilex [34], the commonest endophytes were found only in leaves, whereas the remainder were found in both leaves and twigs, but no twig-specific taxa were recovered.Similarly no bark specific taxa were found in Kigelia pinnata by Maheswari and Rajagopal [18].This interestingly coincides with the present study, as there were no stem, seed, or fruit-specific taxa in the overall isolates.

Effect of Secondary Metabolites of Endophytic Fungi on
Selected Pathogens.The endophytic fungi were evaluated for their antimicrobial activity against some clinically significant human pathogens.The fermentation broths of 35 endophytic fungi were screened for antimicrobial activity against three pathogenic bacterial strains, S. aureus, E. coli, and P. aeruginosa and one yeast C. albicans.88.57%, 74.28%, 62.85%, and 65.71% of isolates have shown activity against S. aureus, P. aeruginosa, E. coli, and C. albicans, respectively.
NFX 06-Fusarium sp. 2, NFX 23-Fusarium sp. 5, and NFB 04-Penicillium sp. 3 isolated from leaves and seeds, respectively, were found to exhibit very good activity against all the pathogenic strains with a zone of inhibition of more than 15 mm.NFX 13-Penicillium sp. 1 and NFX 20-Phomopsis sp. 2 isolated from leaf, NFS 02-Xylaria sp. 4 isolated from stem, and NFB 02-Emericella isolated from seeds have shown a zone of inhibition of about 10 to 15 mm against the bacterial pathogens and more than 15 mm against C. albicans.[35], the endophytic fungi that showed low antimicrobial activity might possess the active compound in a lesser concentration.And the concentration of these compounds could be enhanced by considering some factors like mode of fermentation, concentration of inoculum, period of incubation, solvents used for extraction, and most importantly purification process [36].
NFX 15-Xylaria sp. 2, NFX 17-Drechslera sp. 1, NFS 01-Xylaria sp. 3, and NFT 05-Lasiodiplodia sp. 1 isolated from leaf, stem, and fruit, respectively, were found to exhibit no activity against the selected human pathogens.Among all the isolates, NFX 06-Fusarium sp. 2 (Figure 2) has shown maximum zone of inhibition, as shown in Table 4, of about 17.7 mm hence further studies were carried out with this isolate. of F. oxysporum [37] and genetic diversity within pathogenic and nonpathogenic isolates with endophytic isolates from various plants [38,39] and also with other isolates that exist as pathogens, saprophytes, and endophytes [40] have been reported.In this study, an attempt was made to place an endophytic F. oxysporum isolated from N. foetida leaves with other endophytic and pathogenic F. oxysporum of different plant species (Table 5).Phylogenetic tree was generated using maximum parsimony method.The generated tree showed twelve clades and one independent lineage which have descended from a pathogenic F. oxysporum ancestor (Figure 3).The evolutionary relationship of 40 taxa revealed consistency index (0.229865), retention index (0.534473), and composite index (0.122857), respectively, with a tree length of 6295.Clade I showed two clusters with an endophytic F. oxysporum and in Clade II all the isolates were pathogenic F. oxysporum whereas both the clades shared common endophytic ancestor.Clade III has all endophytic descendants with a common endophytic ancestor.Clades IV and VI have two coevolved endophytic isolates with two major clusters giving rise to one pathogenic descendant.Clades VIII and XI have two clusters with all pathogenic isolates.Clades VII and XII have two endophytic isolates and one pathogenic isolate which depicts the possible evolution of a pathogenic strain from an endophytic isolate.Clades IX and X have two clusters each with an endophytic and pathogenic isolate coexisting together which shares a common pathogenic ancestor.

Phylogenetic Placement of F. oxysporum. Molecular methods to determine the genetic diversity within the species
Based on the phylogenetic analysis we hypothesize that endophytic strains have given rise to pathogenic forms.This hypothesis is supported by the fact that there is a great deal of genetic relatedness between pathogenic and nonpathogenic F. oxysporum isolates, which has led some researchers to conclude that some particular pathogenic isolates might have evolved from nonpathogenic strains by mutations involving a few loci [41,42].

Conclusions
However, this is perhaps the first report on endophytic fungal diversity of the endangered medicinal plant N. foetida of Agumbe forest.This study contributes to the understanding of species richness of endophytic fungi in different tissues of the plant like leaf, stem, seed, and fruit.More number of endophytic isolates in leaf tissue may be due to the fact that the sampling was done in a wet season and in many instances leaves sampled during the wet season harboured more endophytes than those screened during the dry season [43,44].The rich diversity of Fusarium species as endophytes in different tissues of both the plants may be due to germination of more number of spores of this fungus due to favourable environmental condition.Most of the species isolated comprise mostly Fusarium, Penicillium, Colletotrichum, Pestalotiopsis, Aspergillus, and Alternaria which was in agreement with the results of Jeewon et al. [45] from Antidesma madagascariense.The result of antimicrobial activity indicated that most of the isolates showed varying degree of activity against the selected human test pathogens.Hence the endophytic fungi existing in the plant are potential sources of antimicrobial substances as reported by several researchers [46][47][48][49].Among the 35 selected endophytes Fusarium sp. 2 has shown very good activity which was further identified based on ITS sequencing to be F. oxysporum strain NFX06 and submitted in the GenBank with the accession number KC914432.Phylogenetic analysis of the endophytic strain F. oxysporum NFX06 with the other endophytic and pathogenic F. oxysporum indicated that there is a strong relationship between the pathogenic and nonpathogenic F. oxysporum and from the results it was concluded that F. oxysporum NFX06 has evolved from an endophytic ancestor.

Figure 3 :
Figure 3: Tree showing phylogenetic relationship of 40 F. oxysporum strains.The evolutionary history was inferred using the maximum parsimony method.Tree number 1 out of the 280 most parsimonious trees (length = 6295) is shown.The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches.* denotes the endophytic fungus used in the present study.

Table 1 :
Colonization and isolation rate of endophytic fungi from different plant tissues of N. foetida collected from Agumbe forest.
[26]ly the ITS region of the fungal genome.The fungal ITS regions, including the intervening 5.8 S gene and the flanking ITS1 and ITS2, were amplified with universal primers ITS 4 (5  TCCTCCGCTTATTGATATGC3  ) and ITS 5 (5  GGAAGTAAAAGTAACAAGG3  ) designed by White et al.[26].Amplification was performed in a 50 L reaction mixture containing 28 L MilliQ Water, 5 L of 10X Mg free PCR buffer, 2 L of 25 mM MgCl 2 , 5 L of 2.5 mM deoxyribonucleotide triphosphates (dNTPs), 2 L each of 10 M primers (ITS4 and ITS5), 2 L of DNA template, 2 L of 2.5 units of Taq DNA polymerase, and 2 L of DMSO.The thermal cycle consisted of 3-minute initial denaturation at 94 ∘ C, followed by 35 cycles of 30-second denaturation at 94 ∘ C, 30-second primer annealing at 54 ∘ C, 1-minute extension at 72 ∘ C, and a final 10-minute extension at 72 ∘ C.

Table 2 :
Endophytic fungi isolated from different parts of N. foetida.

Table 3 :
Species diversity in terms of dominance, richness and evenness of endophytic fungal assemblages in different tissues of N. foetida.Figure 2: Colony morphology and conidia of endophytic fungus Fusarium oxysporum NFX06.

Table 4 :
Antimicrobial activity of the fermentation broth of endophytic fungi isolated from different parts of N. foetida.Pestalotiopsis sp. 2, and NFX 21-Phomopsis sp. 3 were found to exhibit low activity of less than 10 mm zone of inhibition.According to Fabry et al.

Table 5 :
List of F. oxysporum strains used in this study and their GenBank accession numbers.