Isolation Of Soybean Endophytic Bacteria As Candidate Biological Control Agents For Soybean Root Rot Disease Control

Soybean root rot is one of the main factors that cause serious losses in soybean production of China.The control of this disease mainly depends on resistant varieties and chemical pesticides.However,pathogen resistance and environmental pollutiori usually restrict the continuous use of chemical pesticides;while biological control is an alternative green way for disease control.Plant endophytic bacteria can colonize in host plants and are not easily affected by the external environment.Some endophytic bacteria can further benefit the plants,such as producing anti-pathogen substances,competing niche and nutrition with pathogens,and inducing plant resistance.Therefore,endophytic bacteria have been widely applied in plant disease control.The purpose of this study is to isolate endophytic bacteria from soybean tissues and identify candidate biological control agents for soybean root rot disease control.As the representative major pathogens of soybean root rot,Phytophthora sojae,Fusarium equiseti,Fusarium solani,and Fusarium oxysporum were inoculated onto soybean seedlings,respectively.Those soybean seedlings with no disease symptom were selected to isolate endophytic bacteria.We obtained 126 endophytic bacteria strains and found 11 of them had obvious antagonistic effect against all the four pathogens.Species of the 11 strains were further identified by 16S rDNA and gyrB sequencing.The results showed that strains 3,6,9,10,15,17,18,and 19 were Paenibacillus polymyxa,strain 41 was Bacillus safensis,strain 1 was Bacillus amylolibacillus,and strain 4 was Klebsiella sp.By using more pathogen species or strains for antagoaism test,we found strains 10 and 19 had broad-spectrum effects on various pathogens.The inhibition rate of strain 10 on Phytophthora sojae,Fusarium equiseti,and Phytopythium helicoides was 84%,71%,and 75%,respectively,and that of strain 19 were 83%,72%,and 70%,respectively.To investigate the colonization ability and dynamics of the bacteria strains in soybean tissues,we firstly induced and obtained Rifampin-resistant strains 10R,19R.and 41R based on the endophytic bacteria 10,19,and 41.We found the strain 4lR could colonize soybean roots.Its colonization amount were increasing at the early stage of colonization,with a peak at 4 days(9.6×103 cfu/g),and then reduced to 3.4×103 cfu/g at 32 days.Strain 41R can also transfer upward from roots to the stems and leaves of soybean.All three strains had control effect on some soybean root rot pathogens.The control effect against F.equiseti was 70%-95%,Phytopythium helicoides was 2.5%-33%,and P.sojae P6497 was 23%-58%.By detecting the relative biomass of endophytic bacteria in infected tissues,we further proved that 10R,19R,and 41R could reduce the infection of F.equiseti,P.sojae strain 6497,and Phytopythium helicoides on soybean.In summary,endophytic bacteria with antagonistic effect against soybean root rot pathogens were isolated from soybean tissues,and their inhibitory spectrum was tested.The colonization ability and dynamics in soybean tissues were analyzed,and the control effect of endophytic bacteria on soybean root rot in greenhouse was evaluated.The results provided research materials and experience to control soybean root rot by using pathogen-antagonistic soybean endophytic bacteria.