Effect Of Biocontrol Agent Bacillus Amyloliquefaciens SN16-1 On Tomato Rhizosphere Bacterial Community And Its Biocontrol Mechanisms

Fusarium oxysporum is a soil borne fungus,which can infect more than 100 varieties of plants and cause serious economic losses.It makes plants wither,and even death through the invasive plant vascular bundle.Until now,strategies for controlling Fusarium-associated wilt disease include breeding resistant crops,crop rotation,chemical fungicides application,soil fumigation,and biological control.Among these,biological control is increasingly favored as an effective and environmental safe approach to manage wilt.Research on the prevention mechanism of biological control agent(BCA)mainly focused on bacterial itself,such as secondary metabolites production,spore and biofilm formation,and induction plant systemic resistance,but synergistic interaction of BCA and rhizosphere bacterial for surpression plant disease is still not common.This project took Bacillus amyloliquefaciens SN16-1 as the maily study object,and Pseudomonas fluorescens SN15-2 as control,first to explore biological activity of SN16-1,and explored effect of SN16-1 and Fusarium oxysporum f.sp.lycopersici(FOL)on the growth of tomato plants through greenhouse experiment.Terminal restriction fragment length polymorphism(T-RFLP)and Illumina MiSeq sequencing technology were used to in-depth analysis of the effect of SN16-1 and FOL on tomato rhizosphere bacterial community.Firstly,the isolated biocontrol strain SN16-1 from soil was identified by the 16S rRNA and gyrB gene amplification,and results showed SN16-1 belonging to Bacillus amyloliquefaciens.HPLC technology and PCR amplification were taken to obtain the types of antibiotics produced by SN16-1,we found SN16-1 could produce surfactin,iturin And fengycin.In addition,the antagonistic activity of SN16-1 was also studied,which showed SN16-1 had a broad-spectrum antagonistic activity to various pathogen,and inhibition rate against FOL was 45.73%.The greenhouse experiment showed that SN16-1 and SN15-2 promoted the growth of plants,while FOL significantly inhibited.The biological control effect of SN16-1 and SN15-2 were 55.29%and 41.69%,respectively.Secondly,through collecting the different time of tomato rhizosphere soil,analysed effect of SN16-1 and FOL on tomato rhizosphere bacterial community using T-RFLP technology.Principal component analysis(PCA)and diversity indices analysis showed the treatment with SN16-1 and SN15-2 had a transient influence on the indigenous community,while FOL influenced durable.As for the significantly changed T-RFs in 10 days,the corresponding genus obtained using RDP database comparison.The genera of antibiotic-producing and plant-promoting were activated in SN16-1 and FOL treatments,which indicated that the SN16-1 had a quick response to pathogen FOL invaded.Moreover,in FOL group,the genera owned activity of bioremediation,the relative enzymes of degrading the pathogen cell wall,sensitive to pathogen were decreased,which could convenient for the pathogen colonization to plant,and simultaneously,the bacterial of nutrient cycling and plant growth promoting were enhanced to protect plant.Thirdly,according to the results of T-RFLP,the two periods of drastic change and final recovery stages were selected and analyzed by Illumina MiSeq technology of the 16S rRNA V4 region.The sequences were used to establish operational taxonomic units and were classified into 22 phyla and 323 genera,respectively.Proteobacteria,Acidobacteria,Actinobacteria,Gemmatimonadetes,Bacteroidetes,and Proteobacteria were the most highly represented phyla in each sample.Pseudomonas and Massilia were found in significantly higher abundance in the SN16-1 group,while sequences of Chthoniobacter and Haloferula were higher in FOL group.The results of diversity indices and PCA analysis were in accordance with T-RFLP.In summary,the research revealed the effect of SN16-1,SN15-2 and FOL on tomato plant growth and rhizosphere bacterial,and through the function analysis of the change genera to disccuss the ecological biocontrol mechanism of SN16-1.These results provide important insight into risks associated with using SN16-1 for the control of tomato wilt.