Screening And Study On The Biological Control Factors Against Isatis Indigotica Root Rot Disease

Isatis indigotica root rot disease is found to be more and more serious in production areas of China recent years.In the present study,we tried to screen plant extract and effective antagonistic microorganisms from soil samples collected from different crop rhizosphere.The aim is to provide a new clue for controllingâ… .indigotica root rot disease effectively.Fusarium solani was confirmed as the main causal agent ofâ… .indigotica root rot disease in previous study.One hundred and seventy two out of the tested 201 isolates are bacteria isolates,the other 29 are fungal isolates.All the selected isolates were tested against F.solani for their antagonistic effect.Bacteria isolates named as Bs-28,Bs-84 and B1-105 showed strong antagonistic effect against F.solani.Antagonistic isolates Bs-28,Bs-84 and Bl-105 were identified by traditional and molecular biology methods.Bs-28,Bs-84 and Bl-105 strains were identified as Bacillus spp.by 16S rDNA sequence analysis.Furthermore,Bs-28 and Bs-84 were finally identified as Bacillus subtilis,Bl-105 was identified as Bacillus licheniformis based on the morphological characterization,cultural characters,physiology and biochemical characteristics according to Bergey's Manual of Determinative Bacteriology(Eighth Edition).The selected three isolates were fermentated.The optimized fermentation condition for Bs-28 was 2%corn meal,2%yeast extract,0.2%sodium dihydrogen phosphate (NaH₂PO₄) and disodium hydrogen phosphate(Na₂HPO₄),broth content 10 ml/300 ml flask,initial inoculum 1 ml and initial pH 7.5;The optimized fermentation condition for Bs-84 was 2%corn meal,2%yeast extract,broth content 10 ml/300 ml flask,initial inoculum 1.5 ml and initial pH 8.0.The optimized fermentation condition for Bl-105 was 0.5%corn meal,1%yeast extract,0.4%magnesium sulfate(MgSO₄),broth content 10 ml/300 ml flask,initial inoculum 1.5 ml and initial pH 6.0.The antifungal activity of ethanol extract of 123 species of Chinese herbs against F. solani was tested on in vitro.Results indicated that ninety nine species of Chinese herbs had significant inhibitory effect against colony growth at the concentration 0.01 g/mL.The inhibition ratio of Glehnia littoralis extract reached 100%,and that of Cortex meliae and Clematis chinensis were over 50%.One hundred and twelve species of Chinese herbs had significant inhibitory effect against conidial germination,inhibition rates of eleven extracts were over 50%,the inhibition rates of Glehnia littoralis and Radix Achyranthis bidentatae were 100%.Those extracts with inhibition rates over 50%were chosen for the secondary screening tests at the concentration of 0.005 g/mL.G.littoralis had 100%inhibitory effect in the second screening tests against colony development,while the the inhibition rate of G. littoralis against conidial germination was 74.50%.The corresponding inhibition rates of Cortex meliae were 59.59%and 53.46%.EC₅₀ of G.littoralis and Cortex meliae were 3.621 mg/mL and 6.631 mg/mL seperately.Three tested antagonistic microorganism isolates were fermentated together with G. littoralis and/or Cortex meliae extracts.The inhibition of the mixtures and single fermentation were tested against the mycelium growth of F.solani.The optimized fermentation was the combination Bs-28+Bs-84+Bl-105+G.littoralis.In field trial of 2008,the selected three isolates and two plant extracts were tested to controlâ… .indigotica root rot disease in Tangshan of Hebei province.All the tested isolates and plant extracts showed control efficacy againstâ… .indigotica root rot disease, antagonistic isolates Bs-28 and Bs-84 improvedâ… .indigotica yield significantly. Antagonistic isolate Bs-28 gave the best control effect 49.67%and 47.03%yield increasing.