Isolation, Diversity Of The Endophytic Bacteria In Poplar And The Antibiotic Activity Of Bacillus Amyloliquefaciens PEBA20 On Phytopathogens

Poplar canker caused by Botryosphaeria dothidea, was a key stem disease which occurred widespreadly and was difficult to control.Biological control was an envirorment friendly method for the disease management with great potential development. There was little reports about biological control of Botryosphaeria dothidea. As a new type of biocontrol factors, plants endophytic bacteria were a class of microorganisms which colonied in a variety of tissues of healthy plants with mutually beneficial co-existence between plants. There was significance of controlling the diseases of poplars with endophytic bacteria isolated from the poplar body.In this study, we isolated endophytic bacteria from the poplar trees and analysed the bacteria diversity, selected the endophytic bacteria with high antagonistic activity, identificated the endophytic bacteria strain of PEBA20 with systematic taxonomy identification, determined its optimization of fermentation conditions and antibacterial activity, prelimary purified the proteins. The main findings showed as follows:1. 126 endophytic bacteria were isolated from branches of triploid Populus from Tai-an city in march, june, september and december, 2007 to 2008. We selected 12 poplar endophytic bacteria showed good antagonist activity to Botryosphaeria dothidea with flat antagonist screening method. We found a lot of poplar endophytic bacteria in poplars from these separated bacteria. Using Shannon-Wiener analysis with diversity index (H), abundance (D), evenness (J), the endophytic bacteria from poplar in different time showed significant difference.2. We identificated PEBA20 as Bacillus amyloliquefaciens through cultivation, staining reaction, physiological or biochemical characteristic analysis and 16SrDNA sequence analysis. PEBA20 showed good antimicrobial effect on Botryosphaeria dothidea. The results showed that the antibacterial spectrum of PEBA20 was broad, the antibacterial activity were high against Valsa sordida,Phytophthora capsiciLeon,Glomerella cingulata, Fusarium oxysporum and other 12 kinds of pathogenic fungi, the inhibitory rate of the bacteria and fermentation filtrate against Valsa sordidaI was 79.53% and 67.32% respectively, while the inhibitory rate against Botryosphaeria dothidea was 53.27% and 40.48% respectively. This indicated that PEBA20 has good effect to control the disease of poplar trees. PEBA20 also showed good antibateria activity against Xanthomonas jugandis,Ralstonia solanacearum,Clavibacter sepedonicum,Bacillus megaterium , the inhibition zones to bacteria were more than 12mm.3. Antifungal activitives of different fermentation conditions of PEBA20 were determined. The results showed that culturing for 120h and cultured in NB medium were the favorable conditions for the antibiotic activity. We calculated the most suitable fermentation conditions for PEBA20 were T = 28.5℃, rpm = 140rpm, pH = 5, ventilation volume 100mL, t = 120h.4. Crude protein of PEBA20 under different culture conditions had better effects to Botryosphaeria dothidea,indicating the existence of th antibiotic proteins. The column chromatography with CM-Sepharose Fast Flow and DEAE-Sepharose Fast Flow demonstreated that the protein with antibiotic activity could be purified.