缺氮处理禾谷镰刀菌在氮素恢复后的致病力变化研究.

To clarify the effects of nitrogen recovery on the pathogenicity variation of Fusarium graminearum (F. graminearum) suffered nitrogen starvation, an off-line simulation method was used to test the growth, spore germination, toxicity and infectivity of F. graminearum. The MM-N media containing 0, 25%, 50%, 75% and 100% nitrogen were set as five treatments, among which 100% nitrogen level treatment contained 1 g/L ammonium chloride. Hyphae growing in Potato Dextrose Agar liquid media (PDA) for 72 h were then shifted to MM-N media for the nitrogen starvation. After cultured in different nitrogen levels media for 168 h, hyphae were shifted to MM total nitrogen media for the nitrogen recovery. Cultures were sampled at 0, 2, 24, 48 and 72 h for further analysis. Five millilitres of culture were sampled and centrifuged at 10 000 rpm for 10 min to separate the mycelium from the media; then the weights were recorded for charactering the growth of F. graminearum. The spores of F. graminearum were calculated and then transferred to MM total nitrogen media; after cultured for 48 h, the germination of spores in 40 μL cultures was observed under a microscope and the germination rate was calculated. Toxin Deoxynivalenol (DON) was extracted using specific immunoaffinity columns; the samples were evaporated under a low nitrogen stream at 50°C and resuspended in the mobile phase (methanol: water=18:82, v/v). The determination of DON was performed using high-performance liquid chromatography with Shimadzu SPD-M20A Infinity II Diode Array Detector (DAD); A 150 mm×4.6 mm C18 column with a particle size of 5 μm from Phenomenex was used; The mobile phase comprised methanol and water (18:82, v/v) and was set at a flow rate of 0.8 mL/min; The column temperature was 35°C, the injection volume was 20 μL, and the total run time was 15 min for mycotoxin analyses. The infection lengths of wheat seedlings were used to test the infectivity of F. graminearum, and the lesion length was measured with a digital calliper. Significant differences were not found for the growth rate of F. graminearum treated by different nitrogen levels deficiency after nitrogen recovery for 24 h (P<0.05). The biomass of total nitrogen deficiency treatment was significantly higher than the other treatments (P<0.05), reaching 0.354 g/mL liquid media. The biomass of each treatment was not significantly different after 48 h culture. However, after 72 h of nitrogen recovery, the biomass of nitrogen treatment (25%) was significantly higher than that of the other treatments (P<0.05). The treatment of total nitrogen deficiency only produced 8.2×105 spores/mL, but it had the highest spore germination rate, reaching 46.00%. The DON production of F. graminearum of total nitrogen deficiency treatment was always low after the nitrogen recovery. The nitrogen treatment (25%) for 24 h and the nitrogen proportion 50%, 75% and 100% treatments for 72 h had the higher toxin production compared to the other treatments at the same time (P<0.05), the toxin content were 1.361, 0.515,1.409 and 0.540 μg/g. After the nitrogen recovery, the pathogenicity of F. graminearum showed a trend of decreasing firstly and then increasing. At the initial stage of nitrogen recovery, the infection length of nitrogen proportion 100% treatment reached 13.28 mm. Then, the infection length was decreased significantly from 2 h to 48 h, and increased to 11.96 mm at 72 h. The infection lengths of the three treatments (nitrogen proportion 25%, 50% and 75%), reached the lowest at 24 h, and then increased to the maximum at 72 h after nitrogen recovery. Cultured for 72 h, the infection length of the treatment with total nitrogen starvation was 7.73 mm, significantly lower than the treatments of nitrogen proportion of 25%, 50% and 100% (P<0.05). The results would provide valuable information for establishing rational strategy of fertilizer application after straw incorporation into soils. To clarify the effects of nitrogen recovery on the pathogenicity variation of Fusarium graminearum (F. graminearum) suffered nitrogen starvation, an off-line simulation method was used to test the growth, spore germination, toxicity and infectivity of F. graminearum. The MM-N media containing 0, 25%, 50%, 75% and 100% nitrogen were set as five treatments, among which 100% nitrogen level treatment contained 1 g/L ammonium chloride. Hyphae growing in Potato Dextrose Agar liquid media (PDA) for 72 h were then shifted to MM-N media for the nitrogen starvation. After cultured in different nitrogen levels media for 168 h, hyphae were shifted to MM total nitrogen media for the nitrogen recovery. Cultures were sampled at 0, 2, 24, 48 and 72 h for further analysis. Five millilitres of culture were sampled and centrifuged at 10 000 rpm for 10 min to separate the mycelium from the media; then the weights were recorded for charactering the growth of F. graminearum. The spores of F. graminearum were calculated and then transferred to MM total nitrogen media; after cultured for 48 h, the germination of spores in 40 μL cultures was observed under a microscope and the germination rate was calculated. Toxin Deoxynivalenol (DON) was extracted using specific immunoaffinity columns; the samples were evaporated under a low nitrogen stream at 50°C and resuspended in the mobile phase (methanol: water=18:82, v/v). The determination of DON was performed using high-performance liquid chromatography with Shimadzu SPD-M20A Infinity II Diode Array Detector (DAD); A 150 mm×4.6 mm C18 column with a particle size of 5 μm from Phenomenex was used; The mobile phase comprised methanol and water (18:82, v/v) and was set at a flow rate of 0.8 mL/min; The column temperature was 35°C, the injection volume was 20 μL, and the total run time was 15 min for mycotoxin analyses. The infection lengths of wheat seedlings were used to test the infectivity of F. graminearum, and the lesion length was measured with a digital calliper. Significant differences were not found for the growth rate of F. graminearum treated by different nitrogen levels deficiency after nitrogen recovery for 24 h (P<0.05). The biomass of total nitrogen deficiency treatment was significantly higher than the other treatments (P<0.05), reaching 0.354 g/mL liquid media. The biomass of each treatment was not significantly different after 48 h culture. However, after 72 h of nitrogen recovery, the biomass of nitrogen treatment (25%) was significantly higher than that of the other treatments (P<0.05). The treatment of total nitrogen deficiency only produced 8.2×105 spores/mL, but it had the highest spore germination rate, reaching 46.00%. The DON production of F. graminearum of total nitrogen deficiency treatment was always low after the nitrogen recovery. The nitrogen treatment (25%) for 24 h and the nitrogen proportion 50%, 75% and 100% treatments for 72 h had the higher toxin production compared to the other treatments at the same time (P<0.05), the toxin content were 1.361, 0.515,1.409 and 0.540 μg/g. After the nitrogen recovery, the pathogenicity of F. graminearum showed a trend of decreasing firstly and then increasing. At the initial stage of nitrogen recovery, the infection length of nitrogen proportion 100% treatment reached 13.28 mm. Then, the infection length was decreased significantly from 2 h to 48 h, and increased to 11.96 mm at 72 h. The infection lengths of the three treatments (nitrogen proportion 25%, 50% and 75%), reached the lowest at 24 h, and then increased to the maximum at 72 h after nitrogen recovery. Cultured for 72 h, the infection length of the treatment with total nitrogen starvation was 7.73 mm, significantly lower than the treatments of nitrogen proportion of 25%, 50% and 100% (P<0.05). The results would provide valuable information for establishing rational strategy of fertilizer application after straw incorporation into soils. [ABSTRACT FROM AUTHOR]