Research On Control Of Dickeya Dadantii Causing Stem And Root Rot Of Sweet Potato Through Bacillus And Biosynthesized Nanoparticles

Stem and root rot disease of sweet potato caused by the soft rot bacterial pathogen Dickeya dadantii recently broke out in the major sweet potato growing regions in China and calls for effective approaches to control the disease.There is a lack of crop varieties resistant to the pathogen.Some antibiotics are effective in controlling plant diseases caused by bacterial pathogens but are no longer permitted to use at large-scale in fields due to the risk of the selection of multi-drug resistant human or animal pathogens.There is an increasing demand to find alternative methods to combat the plant disease.Biocontrol using antagonistic microbes against plant pathogens has been accelerated.Moreover,novel nanopesticides made by the cutting-edge nanotechnology have emerged as promising antimicrobial agents.Green synthesized metal nanoparticles can be used as substitutes to the conventional chemical pesticides and may develop to highly efficient,sustainable,cost-effective and environmentfriendly approaches to control the plant diseases.This study developed biogenic methods(screening of antibacterial Bacillus,and green synthesis of metal nanoparticles using bacterial culture supernatants or plant extracts)and the products(Bacillus and nanoparticles)to control the pathogen D.dadantii,aiming to control the stem and root rot disease of sweet potato.Plant-associated Bacillus strains against D.dadantii were screened using an in vitro overlay culture assay and an in vivo tuber slice assay.All Bacillus strains inhibited D.dadantii growth and maceration of sweet potato tubers.Two most effective strains B.amyloliquefaciens A3(in vitro inhibition rate 55% and in vivo inhibition rate 82%)and B.velezensis A2(in vitro inhibition rate 50% and in vivo inhibition rate 77%)were selected for further studies.They showed broadspectrum antibacterial activities against other type strains and field isolates of Dickeya and Pectobacterium species.They produced lipopeptides determined by matrix–assisted laser desorption/ionization coupled with time of flight mass spectrometry.Their culture supernatants inhibited D.dadantii growth,swimming motility and biofilms and damaged D.dadantii cells.B.amyloliquefaciens A3 is more effective than B.velezensis A2 in producing surfactins(wellknown lipopeptide antibiotics and biosurfactants)and inhibiting D.dadantii.Consequently,an effective method was developed to screen effective Bacillus strains to control the soft rot pathogen D.dadantii and screened out a strong surfactin producer B.amyloliquefaciens A3,which can be used as a reference strain for further screening of more effective Bacillus strains and to develop an agent to control the stem and root rot disease of sweet potato.A simple method for green synthesis of silver nanoparticles(Ag NPs)was developed using the bacterial cell-free culture supernatant(CFCS).Silver nanoparticles were first synthesized with the CFCS of a Pseudomonas rhodesiae strain and displayed the characteristic surface plasmon resonance peak at 420–430 nm and as nanocrystallites in diameters of 20–100 nm determined by transmission electron microscopy,scanning electron microscopy,and X-ray diffraction spectroscopy.Functional groups associated with proteins in the CFCS may reduce silver ions and stabilize Ag NPs.The silver nanoparticles showed antibacterial activities against D.dadantii growth,swimming motility,biofilm formation,and maceration of sweet potato tubers whereas the CFCS of P.rhodesiae did not.The silver nanoparticles at 12 μg?ml-1 and Ag NO3 at 50 μg?ml-1 showed similar antibacterial activities.The antibacterial activities increased with the increase of the Ag NP concentrations;Ag NPs at 50μg.m L-1 inhibited 70% of D.dadantii activities.Extensive use of Ag NPs may cause excessive release of silver ion in the environment and detrimental effects on the environment.Here,the CFCS of B.amyloliquefaciens A3 and Ag NPs synthesized with the CFCS of B.amyloliquefaciens were combined to enhance the antibacterial activities and reduce the working concentration of Ag NPs.The Ag NPs,CFCS,or Ag NPs and CFCS in combination inhibited D.dadantii growth,swimming motility,biofilm formation and maceration of sweet potato tuber.The antibacterial activities increased with the increase of the concentration of Ag NPs and CFCS.The Ag NPs(50μg.m L-1),CFCS(50%),or the combination of Ag NPs(12μg.m L-1)and CFCS(12%)inhibited 70% of D.dadantii activities.The combination of Ag NPs at a low concentration and CFCS of B.amyloliquefaciens can be a costeffective and ecofriendly approach to control the soft rot disease caused by D.dadantii.Edible plant fruits are safe raw materials free of toxicants and rich in biomolecules for reduction of metal ions and stabilizing nanoparticles.Zinc oxide nanoparticles(Zn ONPs)and titanium dioxide nanoparticles(Ti O2NPs)are the most produced consumer nanomaterials.Here,Zn ONPs and Ti O2 NPs were synthesized by mixing Zn O or Ti O2 solution with lemon fruit extract at room temperature around 25℃.The physiochemical characterizations of synthesized Zn ONPs and Ti O2 NPs were confirmed by ultraviolet-visible spectrometry and energy dispersive spectroscopy.Polymorphic crystalline structures of the Zn ONPs and Ti O2 NPs were determined by transmission electron microscopy,scanning electron microscopy and X-ray diffraction spectroscopy.The surface stabilization groups of nanoparticles from the lemon fruit extract were discovered by fourier-transform infrared spectroscopy.In contrast to Zn O,Ti O2 and the lemon fruit extract showing no antibacterial activities,Zn ONPs and Ti O2 NPs showed similar extent of antibacterial activities against D.dadantii growth,swimming motility,biofilm formation and maceration of sweet potato tuber slices and the antibacterial activities increased with the increase of nanoparticle concentrations.Both Zn ONPs and Ti O2 NPs at 50μg.m L-1 inhibited 60% of D.dadantii activities.Together,the methods of screening effective Bacillus and green synthesis of antibacterial metal nanoparticles developed here,and the resulted effective Bacillus,metal nanoparticles and in combination of nanoparticles with Bacillus culture supernatants are promising in developing biogenic agents to reducethe pathogen contamination of the seed tubers,promoting the control of the stem and root rot disease of sweet potato.