Isolation And Identification Of Antagonistic Bacteria Against Phellinus Noxius And Analysis Of Its Antifungal Substances

Brown root rot disease is a broad-spread soil-borne disease and caused by Phellinus noxius.So far,there is still no efficient control agent and method to prevent tree brown root rot disease.Currently,the green agriculture advocates the agriculture methods from traditional transform into ecology,thus,using the low-toxic,high-efficiency and safe pesticide formulations for pest and disease control has enormous potential biological control because of its environmentally friendly and sustainability.Base on this,this study performed a plate confrontation method method to screen and identify a highly antagonistic strain HK25 from the rhizosphere soil.Further,we optimized the fermentation medium components and conditions,and detected the target lipopeptide gene.At the same time,we also used ESI-MS/CID to identify and analyze the antifungal substances to P noxius.The main results are as follows:1.Forty rhizosphere soil samples were collected from Guangdong,Guangxi,Fujian,Henan,Gansu,Tibet and Hong Kong,and total of 195 strains of bacteria were screened with the plate confrontation method.After primary and secondary screening,the results showed that the strain HK25 had the most significant stable antagonistic activity.It was identified as Bacillus velezensis based on morphological,physiological and biochemical characteristics,and gyrB gene sequence alignment analysis.2.In order to improve the antagonistic effect against P.noxius,the medium composition and fermentation conditions of B.velezensis strain HK25 were optimized.The single factor test was carried out by selecting five factors: carbon source,nitrogen source,inorganic salt,pH and fermentation time.We found that the optimal medium composition for the antagonistic effect of B.velezensis strain HK25 was: sucrose 10 g,Peptone 5 g,yeast extract 5g and NaCl 5 g.And the optimal fermentation time was 48 h and the optimal pH was 6.5-7.3.The crude extract of lipopeptide obtained from LB fermentation broth by hydrochloric acid precipitation and methanol extract was tested by blood plate and oil spreading test.The results showed that the crude lipopeptide was confirmed to produce biosurfactant.Through the detection of target lipopeptide genes fenB,ituD and lpa-14,combining with sequencing results and BLAST alignment analysis,the results showed that the genome of B.velezensis HK25 strain contain fenB,ituD and lpa-14 genes,and it had the ability to synthesize antimicrobial lipopeptide such as fengycins and iturins.In order to further clarify the types of antibiotics in antifugal substances in strain HK25,crude extracts of antifugal lipopeptides were prepared by hydrochloric acid precipitation method and purified by HPLC.The antifugalactivity of the three antibacterial grop obtained in 1-4 min,5-22 min and 26-34 min showed that the component 2 contained the major antifugal substances.The main inhibitory substances of component 2 in strain HK25 are fengycin B and bacillomycin D based on ESI-MS/CID,which have strong antifugal activity against P.noxius.