I would like to acknowledge the United States Department of Agriculture at the National Center for Natural Products Research for hiring me and allowing me access to all the equipment used in the project. Kumudini Meepagala for her guidance through this project and for giving her the opportunity to learn and do research in her lab since freshman year. Natascha Techen, Jason Martin, Linda Robertson, Amber Reichley, and Robert Johnson for their willingness to teach me various lab techniques and to assist me throughout the project.
A fungus was isolated from Hedera helix (English ivy) leaves experiencing necrosis and identified by molecular techniques as Diaporthe eres. The fungus was grown in potato dextrose broth medium and the culture medium was extracted with ethyl acetate. Bioassay-guided fractionation of ethyl acetate extract of culture filtrate of this fungus led to the isolation of two phytotoxic compounds active against seed germination of the monocot Agrostis stolonifera at 1 mg mL-1.
Structures of these active compounds were determined by spectroscopic methods and were determined to be 8-hydroxy-3,7-dimethylisochroman-1-one and 2-(4-hydroxyphenyl)-ethanol. This is the first finding presented on the phytotoxic effects of ash 8-hydroxy-3,7-dimethylisochroman-1-one.
Introduction
However, even with the discovery of new ways of doing things, there are both benefits and limitations in this research, namely the cost-effectiveness of such studies (Dayan, et al., 2012). The new outlines and research methods conducted in recent studies by Dayan, et al. 2012) and Tresch (2013) can provide ways to combat this financial barrier to the development of natural product herbicides. Following this idea, an observation of necrosis on the leaves of English ivy (Hedera helix) plants on the campus of the University of Mississippi led to an investigation into the isolation and identification of the fungus causing damage to the plants.
It is known to cause small, necrotic lesions on infected plant species (Anagnostakis, 2007, Kaliterna, et al., 2012). However, there is no literature on this species infecting plants in Mississippi or as a pathogen of the plant species Hedera helix. Outside the realm of this particular plant pathogenic fungus, research into the use of phytotoxic metabolites of fungi as potential herbicides is well documented (Martínez-Luis, et al., 2005).
The purpose of this study was to identify and isolate the fungal species that cause necrosis on. English ivy plants and to isolate phytotoxic compounds from the fungal culture medium that could be further investigated for use as potential natural derived herbicides.
Experiemental
- General Experimental Methods
- Fungal material
- Molecular Identification of the fungus
- Extraction and bioassay guided Isolation
- Phytotoxicity bioassays on monocotyledon and dicotyldeon seeds
- Isolation of Phytotoxic Constituents
A leaf with visible necrosis was surface sterilized by immersion in 5% sodium hypochlorite in sterilized deionized water for one minute, then rinsed three times for one minute each by immersion in sterilized deionized water. A strip (approximately 0.5 cm long, 2 mm wide) of leaf material was cut from the infected area of the leaf with visible necrosis with a sterile scalpel and placed on a petri dish containing potato dextrose medium (PDA, Difco). ™) and was. The PDB culture was grown on an orbital shaker at 90 rpm for two weeks at 24 °C under constant light.
This medium was used to inoculate 24 2-liter flasks, each containing 1 L of PDB medium, by adding 3 mL of the fungal culture medium. These culture flasks were allowed to grow on an orbital shaker at 90 rpm for one month at 24°C under constant light. The liquid medium was filtered through miracloth (EMD Millipore, Billerica, MA), followed by vacuum filtration of the medium through semi-cimped, high-speed filter paper (15 cm diameter, Fisherbrand™, Pittsburgh, PA).
The combined ethyl acetate extract was dried over anhydrous sodium sulfate and the solvent was evaporated under reduced pressure at 40 °C to obtain a brown viscous extract of 1.16 g. Thin layer chromatography analysis of the crude ethyl acetate extract eluted in ethyl acetate showed that it contained multiple compounds. The extract was subjected to column chromatography using 0–100% ethyl acetate in hexane for 47 column volumes and 0–10% methanol in ethyl acetate for 21 column volumes.
Fractions obtained by column chromatography on silica gel of the ethyl acetate extract of the fungal culture medium were tested in the seed germination bioassay using Lactuca. Phytotoxic activity was assessed visually, comparing the amount of seed germination in each well with that of the untreated controls after seven days. The qualitative estimate of phytotoxic activity was evaluated using a rating scale of 0-5, where 0 = no effect or 100% germination and 5 = no growth or no germination of the seeds.
Thin layer chromatography of fraction 1, eluted twice in 10% ethyl acetate in hexane, indicated one major compound when examined under UV light (254 nm), but revealed two compounds when exposed to iodine vapor. Preparative thin layer chromatography (20 cm x 20 cm, plate thickness 250 µm) (AnalTech, Newark, DE) of 54 mg fraction 1 eluted twice in 10% ethyl acetate. Further TLC separation of fraction 3 eluted in 50% ethyl acetate in hexane with two drops of acetic acid confirmed this idea.
To isolate the subfractions, 84.2 mg of fraction 3 was subjected to preparative TLC eluted five times in 30% ethyl acetate in hexane with two drops of acetic acid per 10 mL of organic solvent solution. After elution, the four subfractions were identified with 254 nm UV light, scraped from the plates, eluted with 10% methanol in methylene chloride and labeled 3-4 according to increasing polarity (Figure 2b).
Results and Discussion
Identification of Fungal Species
Identification of Phytotoxic Constituents and Results of Bioassay
8-hydroxy-3,7-dimethylisochroman-1-one was isolated as a metabolite from wood rot fungi in a previous study (Alvarenga, et al., 1978). A recent study proposed a reaction that yielded 8-hydroxy-3,7-dimethylisochroman-1-one as the major product using gold catalysis (Hashmi, et al., 2014). 2-(4-hydroxyphenyl)-ethanol, commonly called tyrosol, is a well-known metabolite produced by several phytopathogenic fungi and has been reported as a phytotoxin (Gamboa-Angulo, et al., 2001; Evidente, et al., 2010; Vieira Varejão, et al., 2013).
Conclusions
Diaporthe Eres (Phomopsis Oblonga) as a Pathogen of Butternut (Juglans Cinerea) in Connecticut." Plant Disease: An International Journal of Applied Plant Pathology (2007): AGRIS. Characterization of diaporthe species from grape wood fungus in vineyards of Eastern North America. Plant Disease. Tagetolone and Tagetenolone: Two Phytotoxic Polyketides from Alternaria Tagetica. Journal of Agricultural and Food Chemistry 49.3.
Vine trunk diseases associated with fungi from the Diaporthaceae family in Croatian vineyards” Archives of Industrial Hygiene and Toxicology. Two divergent intragenomic ITS2 rDNA types within a monophletic lineage of the fungus Fusarium are not nortologous.” Molecular Phylogenetic Evolution. Tresch, Stefan “Strategies and future trends for identifying the mode of action of phytotoxic compounds” Plant Science.
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