Studies On Aspergillus Niger Y-61 With Biological Control Action Against Root-knot Nematode

Aspergillus niger Y-61,as an excellent biocontrol strains to root-knot nematode, was screened by Beijing Academy of Agriculture and Forestry.The secondary metabolites had a strong cytotoxicity to plant nematodes. Its physical and chemical properties, purification process, and structure identification were determined. Effect on root-knot nematode of fermented filtrates and the combination toxicity of the active compounds were conducted too. Results were as follows:1. The hatching rate of individual eggs and egg mass were suppressed by 67.38% and 95.96% after treated by original fermentation for 7 days. The hatching rate of individual eggs and egg mass were suppressed by 82.11% and 98.47% treated by fermentation concentrated to 5 times of that for 7 days.Knock down rate was 74.08% when juveniles of Meloidogyne spp were treated by the fermentation filtrate for 10 minutes, and corrected mortality rate was 97.25% after treated for 12 h.2. The active components possessed thermal and acid-base stability. The activity of cytotoxicity had no influence after the fermentation filtrate was treated with 60℃for 2h, the cytotoxic activity to nematode of active components was little change under pH2.0～9.0, the cytotoxic activity was significantly decreased when fermentation filtrate at pH1 or pH10. The nematode's death rate remained above 95% after fermentation filtrate treated with UV for 10 hours,it suggested that the active components had a tolerance to UV.3. Separation and purification process of the active components were conducted. Results were as follows, crude extracts were obtained from the elvant of silica gel column chromatography using ethyl acetate, ethyl acetate and methanol, methanol as elvent after fermentation filtrate treated with resins. Two active components were obtained by means of Sephadex LH-20 column chromatography with ethanol as eluent.4. Molecular weight of active components was 90 Da and 192 Da by the MS of determination,respectively. A single absorption peak (-COOH) from active component (Ⅰ) could be observed in the spectrum of IR (1000 ~ 4000nm),a serious of peaks (-COOH, -OH, -CH2, -C = O ) from the active component (Ⅱ) could observed too. The formula of active component (Ⅰ) and (Ⅱ) is C2H2O4 and C6H8NO7, respectively, based on MS,IR,1H and 13C NMR analysis results. C2H2O4 is oxalic acid and C6H8NO7 is citric acid.5. Combination toxicity of the active compounds was assayed using two-factor repeated analysis of variance and contour diagram method. Results showed that oxalic acid and oxalic acid showed synergy action because there is significantly interaction between in the process of acting on the root-knot nematodes. Experimental LC50,mix is 0.09738(g/L), and theoretic LC50,add of mixture both citric acid and oxalic acid by 1:1(V/V) is 0.1468(g/L) according to calculating LC50 of oxalic acid and citric acid. Suggested that citric acid and oxalic acid played synergistic effect on cytotoxic action to nematode because of LC50,add > LC50,mix. Student t test statistical analysis of experimental LC50,mix and theoretical LC50,add from agent mixed with different proportions of two active components was conducted. Lethal rates to root-knot nematodes were significantly different between citric acid and oxalic acid mixed in different proportions. Therefore, oxalic acid and citric acid displayed synergy action to root-knot nematodes.