Complementation of the cpxAR mutation almost restored expression of all the tested genes

ical analysis From each of the F. culmorum FcUK99 and Fc233B wild-type strains, respectively, single-spore FcStuA deletion mutants and an ectopic transformant were chosen for morphological characterisation. To evaluate mycelium hydrophobicity, a droplet of sterile H2O was deposited on the surface of colonies grown on PDA for 5 days. The time elapsed to absorb the drop by FcStuA deletion mutants and by ectopic transformants was compared to the respective wildtype strain. Hyphal structures, sporodochial development, conidiogenesis, as well as the time and extent of spore germination were TL32711 chemical information observed by a light microscope after growth in CMC liquid medium or SNA solid substrate. These experiments were replicated three times. Briefly, three mycelium plugs were inoculated into 5070 mL of liquid CMC medium in the dark at room temperature and 120 rpm shaking for 5 days. The spore suspension was filtered with a MiraclothH membrane and then washed twice with sterile water. The filtrate was centrifuged to collect conidia and subsequently the concentration was estimated using a Burker Turk hemacytometer. The growth experiment was done on SNA at 25 uC with a photoperiod of 24 hours for 18 days to check sporodochial production. To determine the timing of spore germination, 1 mL of spore suspension was inoculated into Erlenmeyer flasks containing 30 mL of CzapekDox broth. The evaluation was made after 0, 2, 4,and 8 h of incubation at 25 uC in the dark with slow shaking. Pathogenicity tests FcStuA deletion mutants confirmed by Southern blot analysis were tested on durum wheat seedlings to evaluate the role of FcStuA in the pathogenic process by the soil-borne fungus F. culmorum. Mycelium plugs bearing one seed of durum wheat were placed into a plastic sowing pot and covered by sterile soil. Pathogenicity tests were conducted according to in a greenhouse at 25u C and three weeks after inoculation the severity of disease was assessed using the McKinney index. Head Blight symptoms were evaluated on Triticum durum cv Simeto plants inoculated according to the procedure described in. Plants were observed every 3 days and final evaluation was carried out at 14 days after inoculation. Score indices ranged from 0 to 10, which is equivalent to the number of spikelets infected above the inoculation point. 3 FcStuA Gene Characterisation in Fusarium culmorum Oxidative stress effects To evaluate the ability of FcStuA mutants to grow in oxidative stress conditions, strains were cultured for 5 days at room temperature on CM plates amended with 3 mM H2O2, or with 50 mM potassium persulphate or with 10 mM methyl viologen. In addition, vegetative growth and pigment production were evaluated on PDA. Ten mL of a spore suspension were spotted in the middle of each plate. After 5 days of incubation at room temperature the radial development was measured. The effect of oxidative stress on the mutants was measured 1700309 by comparing the ratios of growth of each strain to the respective wild type strain. A series of hydrogen peroxide concentrations were used to determine if the mutant and wild type fungal colonies differed in their sensitivity to high oxidative 2837278 stress. To analyse catalase activity in macroconidia, spores of FcUK99. Fc233B and DFgStuA S12 and S19 mutants were suspended in 3.5 mM H2O2. The absorbance at 240 nm was measured every minute for 80 minutes with a Lambda 35 spectrophotometer. The linear part of the degradation curve was compared and the resulting rate of degrad