Diversity of endophytic fungi possessing bioactive compounds isolated from selected medicinal plants

Abstract

Throughout history, mankind has used plants as their primary source of sustainability. This includes their use as sources of food (agricultural commodities), production of clothing and fragrances, fertilizers, the enhancement of flavours, and also to provide shelter. The treatment of infections caused by microorganisms is usually achieved through the administration of antibiotics. However, recent studies have indicated a steady increase in antibiotic resistance among bacteria strains and particularly the detection of multiple antibiotic resistant (MAR) isolates. This therefore presents severe public health challenges to both the medical and veterinary professions and resistance has become an important issue of global concern. Against this background, current research has focused on finding alternative agents that could serve as potential treatment options to address the problems associated with the presence of antimicrobial resistance worldwide. Natural products especially those associated with plants are now regarded as potential agents that could address this concern. Despite the fact that a number of studies have assessed different plant species for potential bioactive compounds, very little emphasis has been placed on investigations that are designed to assess the capabilities of endophytes in producing bioactive compounds. Endophytic fungi are the biggest diverse group of microbes that colonise plants tissues without any damage, infections nor symptoms of infections. Endophytic fungi engage in mutualistic relationships that normally benefits both the fungi and the plant. It has also been reported that plant species that harbour some endophytes have displayed enhanced capability to withstand both abiotic and biotic stress. Fungi are able to produce secondary metabolites that possess bioactive properties as well as pigments that have tremendous benefits to mankind. Despite the fact that South Africa is well known for the use of medicinal plants in both primary health care and ethnomedicine, very little information is documented on bioactive compounds produced by fungi that are harboured by commonly utilized indigenous plants. To our knowledge, this research is the first study on the screening of endophytic fungal diversities present in two medicinal plants Sceletium tortuosum and Pelargonium sidoides that are indigenous to South Africa. This study encompasses the potential of these fungi to produce potent bioactive compounds with broad-spectrum activities against different resistant pathogenic bacterial strains. A total of 193 endophytic fungi were successfully isolated and the dominant isolates belonged to phylum Ascomycota with Fusarium and Aspergillus as the predominant genus. Phylogenetic analysis based on the Internal Transcribed Spacer {ITS) and Transcription Elongation Factor (TEF 1 a) regions was used to establish the relationship and diversities of fungi isolated as well as the evolution time. Cluster analysis produced two-three clusters in each phylogenetic tree and data revealed that three novel fungal isolates that did not clustered with any isolates and were considered to possible new species. Secondary metabolites were extracted from all endophytic fungal isolates and used to assess their potential to inhibit the growth of Gram positive and Gram negative bacteria that comprised environmental strains as well as ATCC strains. Phenotypic antibiotic resistance assays revealed that E. faecium (26%) and E. gallinarum (9%) displayed high levels of susceplibility to the fungal extracts. In addition, E. coli (ATCC 25922) was most often sensitive to a large proportion (70%) of the fungal extracts tested. With the exception of Alternaria, a majority (80%) of the extracts from the fungal species exhibited narrow spectrum activities against the organisms tested. The largest bacterial growth inhibition zone diameter data of 12 mm was produced by an extract obtained from Alternaria. Endophytic fungi were assessed for the ability to produce pigments and a total of thirty-one (16%) isolates produced distinct pigments with varied colours ranging from yellow (26%), red (13%), brown (35%) to black (13%). Based on enzyme activity assays large proportions of the isolates produced amylase (61 %) and lipase (65%). On the contrary, only a small proportion (13%) of the isolates produced the laccase enzyme. The GC-MS based metabolite profiling of selected fungal extracts was used to assess the ability of fungi to produce volatile compounds. A total of 106 different volatile compounds were identified. The chemical characterization indicated that 9, 12-Octadecadienoic acid (Z,Z) and Cyclodecasiloxane were the predominant compounds in extracts that displayed enhanced microbial activities. Greenhouse studies were conducted on maize (Zea mays L.) using nine extracts from these endophytes that were previously selected according to their ability to produce pigments and their antimicrobial properties. An assessment of plant growth parameters between plants in the treatment groups and control group revealed that the extract from Fusarium solani (MHE 55) was the most favourable in enhancing plant growth than the extract from Alternaria (MHE 68) than the control. In conclusion, from 193 isolates only two endophytic fungi namely Alternaria sp. (MHE 68) and F. solani (MHE 55) have shown to have strong antimicrobial activity, pigment production and are growth promoters. Due to time constraint other biological properties such as antifungal, antiviral, antioxidant, cytotoxicity were not assessed and therefore should be investigated. Furthermore, molecular techniques must be used to determine and confirm the novel fungal isolates and the correlation between pigments, flavonoids and phenolic compounds using High performance liquid chromatography (HPLC) should be established.