Endophytic fungi hosted by medicinal plants may potentially exhibit antimicrobial activity

Aboobaker, Zainub
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Endophytes are micro-organisms that colonize plant tissue internally. They are characterized by the feature that they do not cause any harm to the host plant. In recent years, endophytes have generated great interest around the globe as they produce a wide array of functional metabolites. Endophytes have been found in every plant species examined to date and have been recognized as potential sources of novel antimicrobials. The antimicrobial potential of endophytes isolated from South African medicinal plants has, however, been poorly explored, and there is a lack of scientific investigations on the potential of South African endophytes. A large knowledge gap exists in this area of study considering the high floristic diversity in the country. Thus, this study set out to investigate the antimicrobial activity of endophytes isolated from the roots of three popular South African medicinal plant species Pelargonium sidoides DC, Hypoxis hemerocallidea Fisch., C.A.Mey. & Avé-Lall, and Gunnera perpensa L. The study further aimed to isolate and identify secondary metabolites from the endophyte exhibiting promising antimicrobial activity. The hypothesis formulated aims to determine that medicinal plants used as an anti-infective may not only rely on the medicinal plant alone but also on the residing endophytes in combination. Fungal cultures were isolated from root material of P. sidoides, H. hemerocallidea and G. perpensa. Endophyte extracts were prepared and tested independently and in combination with the host plant (aqueous and organic extracts) using the broth micro-dilution technique to determine antimicrobial activity. Minimum inhibitory concentrations (MIC) were determined against four pathogens, Gram-positive (Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 29212) and Gram-negative (Escherichia coli ATCC 8739 and Pseudomonas aeruginosa ATCC 27853) bacteria. The Fractional inhibitory concentration index (FICI) was used to determine if synergistic interactions were apparent during combination studies. Preparatory high performance liquid chromatography-mass spectrometry (prep HPLC-MS) was used to fractionate the bioactive crude extracts, and secondary metabolites were isolated and identified using ultra performance liquid chromatography-mass spectrometry (UPLC-MS) and nuclear magnetic resonance (NMR). Identification of endophytes displaying the most noteworthy antimicrobial activity was undertaken by polymerase chain reaction (PCR) amplification and deoxyribonucleic acid (DNA) sequence analysis. A total of 19 endophytic fungi were isolated from the P. sidoides root. One of these fungal endophyte isolates (PS8) was identified as Penicillium skrjabinii and the ethyl acetate crude extract of this isolate exhibited antimicrobial activity at MIC values 0.03 mg/mL and 0.06 mg/mL (batch 1) and 0.03 mg/mL and 0.13 mg/mL (batch 2) against S. aureus and E. coli respectively. Bioassay-guided isolation was carried out and the major compound, dibutyl phthalate was isolated from the most active fraction. Interactions between P. sidoides endophytes and the host plant displayed higher synergy when compared to H. hemerocallidea and G. perpensa. A total of 144 combinations were tested from P. sidoides with 14.58% displaying synergy and 8.33% displaying antagonism, the remainder 77.03% were either additive/indifferent. One combination PS17 and organic extract displayed synergy (FICI value of 0.09). Six endophytes were isolated from H. hemerocallidea, and one endophyte (H4) displayed noteworthy activity at 0.25 mg/mL against S. aureus. There was a synergistic interaction between the endophyte H1 and aqueous extract (FICI 0.50) while H1 and organic extract displayed synergy at a FIC of 0.38, both these interactions inhibited E. coli at 1.00 mg/mL. From a total of 48 combinations tested, there were no antagonistic interactions. Five fungal endophytes were isolated from G. perpensa. An interesting isolate (GP5) inhibited S. aureus at 0.50 mg/mL when tested independently, and also displayed synergistic activity with the aqueous plant extract against S. aureus and E. coil at 0.25 mg/mL and 0.50 mg/mL respectively. In summary a number of endophyte extracts displayed a synergistic interaction with the host plant extracts, proving the positive influence of endophytes on the antimicrobial activity of plants. This is the first study to identify P. skrjabinii as an endophyte from P. sidoides that produces the compound dibutyl phthalate. It is also the first report documenting endophytes isolated from Pelargonium sidoides, H. hemerocallidea, and Gunnera perpensa root material. Lastly, it is the first report that provides some insight into the combined antimicrobial efficacy of endophytes with the host plant providing evidence of synergy. This study has highlighted the importance of endophytes as a potential source of antimicrobials and has demonstrated their ability to exert a positive effect on plant extracts against pathogenic micro-organisms.
A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Pharmacy, Johannesburg 2018
Aboobaker, Zainub B. (2018) Endophytic fungi hosted by medicinal plants may potentially exhibit antimicrobial activity, University of the Witwatersrand, Johannesburg, <http://hdl.handle.net/10539/27720>