Inhibitory Effect of Sambucus ebulus Extracts on Growth of Macrophomina phaseolina and Extraction of their Bioactive

Macrophomina phaseolina is the causal agent of soybean charcoal rot. This disease is one of the most prevalent and destructive of soybean in the north of Iran. This pathogen has broad host range and invades more than 500 plant species of 75 families, such as Caprifoliaceae. Symptoms on stems appear as silver-gray lesions near the base, which eventually decay the stem. Plants show poor seed fill, premature ripening, and undersized heads. Seed yield and oil content are decreased. Numerous tiny black bodies called sclerotia are formed on the decayed tissues giving the stalks a charred appearance. This fungus exists in the soil as sclerotia, a compact mass of hardened mycelia structures, which can remain dormant for many years. As the causal agent, is a soil-borne pathogen, chemical fungicides are not effective for its control. This matter has led to use a large amount of fungicides that are harmful to human health and lead to environmental pollution. There is not any registered fungicide against the charcoal rot pathogen. Some medicinal plants have a potential for controlling various phytopathogenic fungi due to the variety of compounds. Scientists are trying to achieve some plant-derived compounds for diseases control. Natural plant products are biodegradable, show structural diversity and rarely consist halogenated atoms. These can act directly as pesticides or may supply structure lead to pesticidal discovery. The aim of this study was to investigate chemical composition and antifungal activity of alcoholic and aqueous extracts of Sambucus ebulus for control of M. phaseolina and replace the chemical fungicides.
The plant leaves were collected during July - August 2014 in Babol (Mazandaran province- Iran) and washed thoroughly with tap water and then rinsed with distilled water and shade dried at room temperature. The dried plant material was finely powdered using an electric grinder and used for aqueous and organic solvent extraction. The aqueous extract was prepared via maceration method. Extraction with ethyl alcohol lasted 8 hours in a soxhlet extractor. The organic solvent was evaporated by rotary evaporated shaker untile 5 ml of solvent was reached. Then GC-MS analysis was used. For investigation of antifungal effects of Sambucus ebulus extracts, different concentrations of aqueous and alcoholic extracts in PDA (autoclaved) have been prepared. Concentrations of %10, %20 and %30 have been used for aqueous and alcoholic extracts, individually.
The aqueous extract did not show any antifungal effect, but different concentrations (%10,%20,%30) of alcoholic extracts showed significant inhibitory effects. Alcoholic extract at %30 concentration by %100 was the most effective one and at %10 concentration by 39.25 was the least effective one, on inhibiting of mycelial growth and formation of sclerotia of the pathogen. Statistically, there was the significant difference in %1 and %5 levels, between different concentrations of alcoholic extract for control of mycelial growth and formation of sclerotia. Alcoholic leaf extract was more effective than water extract, hence it shows that presence of antifungal agent released in alcohol due to its solubility. The chemical compounds present in the alcoholic extract were identified by using the GC-MS device with non-polar column (HP-5). According to mass spectra library of this device, in extracted organic phase with ethyl alcohol solvent, 27 chemical compounds were identified, which the main components were included as phthalates (%54/3), fatty acids and derivatives (%26/61), terpenoids (%2/65), diterpenes alcohol (%2/09), phenolic derivatives (%1.58), Phytosterols (%3.38) and cycloalkanes (%0.38). The highest abundance of identified chemical compounds was, Mono (2-ethyl hexyl) phthalate (%54.3), palmitic acid (%8.24), α - Linolenic acid (%7.78), Isovaleric acid (%4.33), dihydro Stigmasterol (%3.38), Neophytadiene (%2.65), 1-Butanol (%2.49), Phytol (%2.09), Octadecadienoic acid (%1.97) respectively. Some of these compounds have not previously been reported. Because of the antifungal effects of these constituents, the high potential of antifungal activity for alcoholic extract is justified.
The selected medicinal plant extract used in this experiment are easily and locally available and effective as an antifungal agent, so can be recommended to the farmer after the field test. Leaf extract not only has an adverse effect on the environment but also improve the quality of seed and the soil fertility. Our results showed that S. ebulus has the potential for controlling soybean charcoal rot and can be suitable alternative for chemical toxins. This is the first time that this research studies in Iran.