An endophytic fungus, Fusarium sp. was isolated from yew bark of eastern Himalaya. Ethyl acetate extract from its fermentation broth displayed considerable antimicrobial activity against three Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis and Staphylococcus epidermidis), three Gram-negative bacteria (Klebsiella pneumoniae, Escherichia coli and Shigella flexneri) and two pathogenic fungi (Candida albicans and Candida tropicalis). The metabolite showed highest inhibition zone against K. pneumoniae (27 mm) and lowest against C. albicans (10 mm). Based on BLAST search analysis of ITS rDNA sequence, the fungus was identified as Fusarium solani (Mart.) Sacc. Phylogenetic trees were generated by four different methods. Phylogenetic tree generated by UPGMA method was used to establish possible phylogenetic relationships of the fungus with other F. solani isolates those exist as endophytes, pathogens and saprotrophs taken from database. The generated tree showed that all F. solani strains have a common endophytic ancestry which gave rise to six clades that radiate into four evolutionary lineages. The possible phylogenetic relationships of F. solani that exist in different lifestyle have been discussed in each clade.

The definition of endophytes ranges from symbiotic to balanced antagonism and/or latent plant pathogens. This indicates a close affiliation between endophytes and pathogens but molecular evidence confirming these relationships are still to be elucidated. We investigated the relationship of endophytic, saprobic and pathogenic Fusarium species as a model genus based on ITS sequences and ITS2 secondary structure analysis. Altogether 212 Fusarium species mostly named in GenBank with ranging lifestyles were used in this study. Species deposited in GenBank were found to be often wrongly named based on sequence comparison as species with the same names were polyphyletic, while differently named species often had the same sequences. Phylogenetic analysis by three different methods (UPGMA, NJ and MP) revealed that similar named species clustered together, but did not form well distinct clades reflective of their lifestyles. Several species were polyphyletic, while several other unrelated species appear to share the same ITS sequences. Since ITS rDNA sequence based phylogeny did not showed distinct relationships, ITS2 consensus secondary structures were used. Endophytes showed close molecular structural affinity with pathogens but structural features of saprobes was very different. Although care must be taken when using sequences from named Fusarium species in GenBank this study provides molecular evidence that endophytes share a close relationship with pathogens and agrees with the assumption that endophytes are latent pathogens.

The occurrence of Fusarium in different lifestyles is quite speculative. Methods to differentiate them morphologically are extremely difficult. Molecular studies available to discriminate this species with varied lifestyles are insufficient. Herein, we investigated affiliation among endophytic, saprophytic and pathogenic Fusarium species considering TEF1 alpha gene sequences and attempted to mark out differences within different groups based on in silico proteomic analyses. The study revealed similar named Fusarium species clustered together based on their lifestyles forming distinct clades, indicating that coding genes could be better used as a phylogenetic marker than non-coding one to differentiate Fusarium species occurring in different forms. Translated proteins showed similarity between endophytic and pathogenic forms in terms of instability and aliphatic indices. Grand average of hydropathicity (GRAVY) exhibited negative values indicating hydrophilic nature of the proteins. The generated consensus RNA secondary structures of different forms revealed distinct structural features supporting the phylogenetic inference. Protein disorders are found to be quite high in all forms of Fusarium species studied implying its complexity and ability to adapt in diverse environments.

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