Physiological and biochemical characters of laboratory induced Fusarium mutants resistant to fludioxonil

Fusarium head blight is a harmful disease of cereals caused by Fusarium fungi. Fusarium graminearum is among the most widespread fungi in the world and is of great economic importance, which leads to grain contamination with the mycotoxins deoxynivalenol (DON) and zearalenone (ZEN) that poses danger to human and animal health. Recently, F. proliferatum , the fumonisins (FUM) producer, has been increasingly discovered in cultivated cereals. Fungicide treatment is the most popular practice for managing pathogens, but also leads to the emergence of fungicide resistance in Fusarium fungi and changes in their features. As a result, the risk of reducing the effectiveness in disease control and increasing a contamination of grain with mycotoxins exists. In this study, the sensitivity of genetically characterized F. graminearum and for the first time F. proliferatum strains isolated from grain, to fludioxonil has been determined, and the laboratory mutants of fungi resistant to this active substance have been generated. Also, for the first time, significant differences between the laboratory mutants and parent strains of F. graminearum and F. proliferatum in terms of growth rate, pathogenicity to wheat and toxin-producing ability have been established. The aim of study was the comparative analysis of physiological and biochemical characters of the parent F. graminearum and F. proliferatum strains with mutants resistant to fludioxonil obtained in the laboratory conditions. The objects of the study were 16 monoconidial Fusarium strains of different geographical origins from the collection of All-Russian Institute of Plant Protection (MFG) (VIZR, St. Petersburg, Russia), previously morphologically identified as F. graminearum and F. proliferatum . For laboratory experiments, the strains were previously grown on potato sucrose agar (PSA) at 25 °C in the dark. The identification of Fusarium strains was confirmed by sequence analysis of the fragment of gene encoding translation elongation factor EF-1a ( tef ) . The fungicide Maxim (Syngenta AG, Switzerland), containing 25 g/l fludioxonil, was diluted in sterile water to a concentration of 5 g/l of the active ingredient. To obtain F. graminearum and F. proliferatum mutants resistant to fludioxonil, the parent Fusarium strains were sequentially cultured on PSA containing increasing concentrations of the fungicide. When determining the sensitivity of fungi to fungicide, the concentrations of fludioxonil in PSA for the parent strains were 5; 0.5; 0.05 and 0.005 mg/l, and for the mutants 250; 50; 5 and 0.5 mg/l. From colonies of the parent strains and mutants grown on PSA, 4 mm discs were cut out and placed on the nutrient medium in the center of a plastic 85 mm Petri dish. In the control, the disk was placed on the surface of PSA without the fungicide. After 3- and 5-day incubation of the F. graminearum and F. proliferatum strains, respectively, the average diameter of the fungal colony was meaasured excluding the diameter of the inoculation disk. The fungicide concentration resulting in 50 % growth inhibition (half-maximal inhibition concentration; IC50) was calculated using the Quest Graph™ IC50 Calculator program. To assess the effect of temperature on the fungal growth rate, the strains were cultured at 25 °C and 35 °C in the dark in Innova 44R incubator (Eppendorf, Germany), and diameter of F. graminearum and F. proliferatum colonies were measured on days 3 and 7, and on days 5 and 7, respectively. The growth rate was determined as the ratio of the colony diameter to the number of days of culture (mm/day). The pathogenicity of the parent strains and mutants was assessed by the detached leaf assay, according to sizes of necrosis caused by fungal strains after inoculation of the wheat (Triticum aestivum L., cv. Wassa). To assess toxin production, fungal strains were grown on autoclaved wheat grain. The determination of DON and ZEN in F. graminearum cultures and FUM in F. proliferatum cultures was performed using the certified commercial enzyme immunoassay test systems (VNIIVSGE, Russia). When cultured on PSA without fungicide, the colony diameter of the parent F. graminearum strains varied from 34 to 52 mm on day 3, and that of the parent F. proliferatum strains were from 48 to 64 mm on day 5. The IC50 concentrations of fludioxonil resulting in half-maximal growth inhibition for F. graminearum and F. proliferatum mutants were 900-60000 and 6-458 times higher than the IC50 values for the parent strains, respectively. Two F. proliferatum strains initially demonstrated high resistance to fludioxonil and produced FUM 6 and 22 times higher than the average for the other parent strains of this species.