A MULTIPHASIC PHYLOGENETIC AND GENOMIC CHARACTERIZATION OF THE FUNGAL ENDOPHYTE STEMPHYLIUM STRAIN PNW2016-02 AND A SEMI-AUTOMATED BIOINFORMATIC APPROACH TO INVESTIGATING THE STRAIN SECRETOME
As part of development of a human pathogen suppressing in plantae model system within Spinacia oleracea, a known pathogen transmitting produce plants, the secretome of the Stemphylium strain PNW2016-02, an endophytic fungal isolate of spinach plants, was studied. This strain was previously isolated in Purdue University Northwest laboratories in the Biological Sciences Department. As the secondary metabolite secretions of PNW2016-02 were shown to have antibiotic properties against a broad range of bacteria, we sought to improve our understanding of these properties and other characteristics of PNW2016-02 by sequencing and annotating the genome of this strain. Chemical compound characterization was achieved using HPLC-MS, which provided the ability to identify and quantify chemical compounds contained within the PNW2016-02 secretome. Through multi-gene phylogenetic analysis and comparative genomics, we found that PNW2016-02 clusters with a clade representing Stemphylium vesicarium; however, genome annotation also uncovered several genes unique to PNW2016-02. To assist in more fully understanding the secondary metabolites PNW2016-02, a semi-automated bioinformatic pipeline was developed in the R statistical environment in order to reconstruct and characterize the metabolic pathways, especially those pertinent to antibiotic production. Analysis using the bioinformatic pipeline revealed the presence of a number of antimicrobial metabolites, such as flavonoids, and suggests these compounds are produced and transformed in pathways, such as the phenylpropanoid synthesis pathway. These findings also suggest an important vital role of the shikimate pathway for antimicrobial metabolite production within PNW2016-02. Overall, the findings presented here have implications for understanding the antimicrobial strategies employed by Stemphylium PNW2016-02 and its potential for use in the above-mentioned model system.