Madsen Sullivan and Brad Geary, Plant and Wildlife Sciences

Introduction

All civilizations have used plants and their byproducts to effectively grow and cultivate crops, as well as manage and treat many ailments. Even today, approximately 25% of prescribed medications are constituted by plants. Many of the active metabolites are produced in such small quantities that mass production by using the plant is unreasonable. However, the endophytes found within plants tend to produce the same metabolites. These fungi and bacteria commonly produce secondary metabolites possessing antibacterial, antifungal or other medicinal properties. By looking to historical medicinal plants, novel endophytes and metabolites have been discovered and integrated into agriculture and medicine. One such plant, Cornus Sericea, has been traditionally used by Native Americans for its relief from colds, fevers and rashes, as well as its analgesic properties. Despite these indicators, endophytes of C. Sericea have not been previously studied.

Methodology

Plant Collection and Endosymbiont Isolation – Newly grown plant tissues of C. Sericea were collected at a location near Provo, UT in the Wasatch Front. All plant samples were placed in plastic bags and immediately transferred onto ice until stored in a 4 °C refrigerator.

Plant material was surface sterilized with 70% Ethanol. Following sterilization, the outer bark was removed and the inner bark and xylum tissues were placed face down on water agar filled Petri dishes. As needed, hyphal tips were collected beginning at three days to several weeks after extraction procedures.

Fermentation – 6 mm plugs of all inhibitory fungi were inoculated in 2L of PDB. Samples were then placed on shakers and incubated for 14 days at room temperature. All broth cultures were then filtered. Filtrates were stored at -20 °C.

Extraction – Culture filtrates were combined and concentrated by lyophilization. Flltrate assays confirmed the presence of target metabolites. Afterwards, the concentrated filtrate was acidified to pH 5.0 with 2 M HCl and extracted with ethyl acetate. The organic layers were then mixed, dried with MgSO4, and evaporated under reduced pressure to dryness.

Antifungal Bioassays – Air dried disks with varying chemical concentrations were applied to agar dishes of ½ PDA. Three disks each were placed 1 cm away from a centered known pathogenic fungus. Inhibition was measured as assessed in fungicidal quantification. Controls included empty air dried disks and 20 μL of pure MeOH.

Chemical Extraction and Purification – All organic extracts of bioactive fungi filtrates were fractioned by column chromatography. TLC will be performed to analyze purity and the homogenous fractions will be combined and renamed. After each isolation, antifungal bioassays will be performed to determine which fractions contained bioactive compounds.

Results

The results of this research suggests the presence of bioactive secondary metabolites being produced by several endophytes of C. Sericea. In primary bioassays there were at least five isolates with significant levels of inhibition to multiple known pathogenic fungi.

Of the inhibiting isolates, isolates 10.A, 12.A, and 13.B proved to be the most potent. 10.A appeared to produce volatile organic metabolites which greatly inhibited the growth of Fusarium, 12.A caused a large zone of inhibition to Penicillium, and 13.B greatly decreased the overall growth of Fusarium.

For primary bioassays, the fungi of interest were inoculated and grown on various corners of a Petri plate of PDA for varying times prior to testing. The test pathogen was placed in the center of the plate. Each pathogen was tested when it was producing new growth. The plate was wrapped with two layers of Parafilm and incubated at approximately 20 °C.

The growth of the test organisms was visually judged on the basis of any new microbial density appearing on the area of the agar that had newly been inoculated. Eventually, the viability of the pathogen was evaluated. The evaluation was performed by removing a sample of the pathogen and placing it on to a PDA Petri plate. Zones of inhibition were measured every three hours for the first 12 hours, every 8 hours for the next 24 hours, and every 24 hours for the next three days. Test cultures were monitored weekly to assess inhibition duration. Prominent zones of inhibition occurred for five of the isolates of which all inhibited multiple fungal pathogens.

Discussion

Further research will be necessary in order to purify the secondary metabolites which have bioactive potential. Additionally, these metabolites will be tested for antibacterial and other medicinal properties.

However, this research provides a simple example of bioactive secondary metabolites in a common Wasatch Front plant. Regionally, in the Intermountain West, there has been very few reported studies of endophytically produced metabolites in poisonous and medicinal plants. This is especially true when compared to rainforests and other regions of ecological diversity. More research should be put into identifying the secondary metabolites of native plants.

Conclusion

While more research is necessary, the current evidence supports the hypothesis that endophytes of Cornus Sericea produce bioactive secondary metabolites with properties similar to those of Cornus Sericea. More research needs to be done on plants native to Utah and the Wasatch Front especially, as these ecologically unique plants have been largely unstudied despite repeated positive results. Endophytes are not a study of the past, but rather hold great potential for treating dangerous and worrisome pathogens of both plant and human.