Citotoxic and mutagen action of secondary metabolites Phomopsis Helianthi M

Abstract

Infection diseases can cause 70-100 % deaths of valuable single- and perennial crops, identifying and studying their phytotoxins is an important scientific problem. Phytotoxins of pathogenic microorganisms (fungi and bacteria) play an important role in pathogenesis processes and manifest themselves at level of the body, tissue or cells. Sunflower is an important herbivorous crop grown in Ukraine. The field conditions of southern Ukraine sunflower plants are affected by various harmful pathogens, but the most dangerous is the pathogen of disease phomopsis – Phomopsis helianthi M. Losses of crop damage by phomopsis can reach up to 90%, therefore the comprehensive study of cytotoxic and mutagenic action of secondary metabolites Phomopsis helianthi is an important task.

The purpose of research was to study effects of secondary metabolites Phomopsis helianthi on the nuclear apparatus and the processes of plant cell division.

In order to isolate Phomopsis helianthi in a pure culture, stems of sunflower were removed from the phomopsis symptoms, cut into fragments of 5-10 mm, sterilized in 96 ° alcohol and placed in Petri dishes on the surface of potato-glucose agar. Subsequently, the prepared material was incubated in a thermostat for two weeks. Isolated pure culture of the fungus was grown on liquid potato glucose surface at 25 °C for 7, 14, 17 and 21 days, then the culture fluid was filtered and used in further studies. The total determination of phenolic compounds was carried out using the Folin-Chicol'tou method in the Singleton and Rossi modification. The concentration of phenolic antioxidants in the extracts was determined spectrophotometrically by Brand Williams method. The biological activity of extracts was determined by their effect on cell division in the apical meristems of Allium cepa L. of the variety "Stuttgart" (Allium-test). DNA was detected in cells by cold hydrolysis and painting by Reagent of Schiff (Merck) by Folgene. The mitotic activity of the cells and the number of anomalies in core structure and in process of cell division on a Nikon Eclipse E-200 microscope were calculated.

Photo-coding and processing of digital images were performed in specialized Image-Pro Premier 9.0 program. Regression analysis and selection of mathematical models were performed using the SigmaPlot 12.0 program. Statistical data processing in Statistica 7.0.

In Phomopsis helianthi cultivation process, the fungus biomass and its exometabolites are accumulated. It has been established that the accumulation of secondary metabolism products, including mycotoxins, is intensified by the second week of cultivation. The maximum concentration of aromatic compounds in the culture fluid was detected on 17 day, accumulation of phenolic compounds in the culture fluid is accelerated by 8–9 day and reaches a maximum on 15–17 days. With prolonged cultivation of mycelium, the amount of phenolic compounds begins to gradually decrease.

It was determined that Phomopsis helianthi secondary metabolites contribute to changes in the processes of cell division of Allium cepa: in apical meristem cells of roots increase the diameter of nuclei was observed, compared to the control  plants by 16 %, and nucleoli diameter increased by 32%. The acceleration of cell division processes indicates a high biological activity and ability of exometabolites Phomopsis helianthi to penetrate into plant cells.

Finding out the mechanisms for stimulating mitoses and accelerating processes of nucleation reconstruction by products of secondary synthesis P. helianthi in low concentrations deserves special attention and requires further research.

Keywords: Phomopsis helianthi, culture fluid, secondary metabolites, Allium-test, nucleus, DNA