Isolation And Biological Control Of An Antagonistic Bacterial Strain Against Medicinal Chrysanthemum Fusarium Wilt

Chrysanthenum Fusarium wilt, caused by soil-borne pathogen of Fusarium oxysporum, is one of the most prevalent diseases, which typically occurs in Chrysanthenum morifolium fields with continuously cropping obstacles. Nowdays, biological control has become the most important way in control of soil-borne plant diseases.In this study, one pathogen strain (F1) was isolated from the diseased parts of chrysanthenum plants collected from the Chrysanthenum morifolium field. F1 was identified as Fusarium oxysporum f. sp. Chrysanthemi by methods of traditional culture, microscopical observation, reinoculated test and molecular procedures.One antagonistic strain (a4) was isolated from the rhizosphere soil of health chrysanthenum plants in the field severely diseased by Chrysanthenum morifolium fusarium wilt. Strain a4 was identified as Bacillus amyloloquefaciens stain by methods of traditional culture, microscopical observation, characteristication of physicobiochemical properties and molecular procedures.The in vitro inhibition rates of strain a4 to the pathogen in petri dishes reached to 71.0%±2.7%. While the inhibition by fermentation liquids of the strain were pronounced and the inhibition rates were more than 90% when 50 fold fermentation dilutions were used in in vitro tests.A plasmid, pHApII, carrying green fluorescent protein (gfp) was transformed into Bacillus amyloliquefaciens a4 by electroporation method. The transformants grew normally(a4-gfp) and gfp flouresence was detected. Strain a4-gfp was inoculated to rhizosphere of medicinal chrysanthemum to study its colonization. The total number of a4-gfp reduced apparently as time goes by. The number of a4-gfp in rhizosphere soil was 1.3x103 CFU·g-1 soil after 30 days root dipping treatment. At the same time, the number of a4-gfp was 1.8×103 CFU·g-1 soil in rhizosphere soil in the treatment of root watering.Results from seedlings nursery experiment showed that application of BOF promoted the root growth of chrysanthenum morifolium seedling. Fresh weight of chrysanthenum morifolium seedlings was increased by 205%, and height of the plants was increased by 49.6%, respectively, as compared with the control. The treatment of BOF increased the number of bactrias, which was increased by 65 times more than the common level, and it also reduced the number of F oxysporum.The field experiment validated that application of BOF both in nursery and transplanting could effectively control the incidence of Fusarium wilt of chrysanthemum and regulate the composition of microbial flora in rhizosphere soil. Results from field experiment showed that control efficiency against the wilt was 89.1%, when the nursery soil and the growing pot soil were both amended with BOF (n+F), and the highest control efficiency (93.4%) was obtained in the treatment of adding a4 strain(n+F+a4). Application of BOF increased the activities of peroxidase (POD), polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL) by 200%,200%, and 300%, respectively, when compared with the control. Thus, BOF enhanced the plant resistence to phytopathogens. Counting of microbial flora of treatment n+F+a4 in rhizosphere soils of chrysanthemum showed that bacteria was increased by more than 10-fold and F. oxysporum were reduced by more than 100-fold in comparison with the control.