Screening Of Biocontrol Agents And Antibacterial And Nematicidal Genes From Isatis Indigotica

Biological control can be defined generally as the usage of natural or modified organisms,genes,or gene products to reduce or diminish the population of pests or effects of diseases.To develop a sustainable agriculture system obliges the sensible use of numerous practices that are integrated and compatible with pest control.Biological control can perform a key role in minimizing the occurrence of prevailing pest species within an integrated pest management.In the future,bio-control agents might replace the use of inorganic chemicals in disease and pest control management in agriculture because of their versatile and effective natures.Most biocontrol agents used in agriculture are of bacterial origin.There are many genera with capabilities to control phytopathogens,but this paper concentrates on the Bacillus genus –Bacillus amyloliquefaciens.Bacillus amyloliquefaciens shares numerous features in common with Bacillus subtilis with regard to their biocontrol capabilities.However,the chapter’s mainstay includes four major themes.One of the themes is the isolation,screening as well as identification of Bacillus amyloliquefaciens as a biological control agent,while another theme is covered in the chapter is the isolation as well as the evaluation of the proteins produced by the genes of plant Isitis indigotica against the potential phytopathogens while the fourth theme is the evaluation of the nematocidal activity of the extracted proteins.The respective strains of bacteria were isolated from wheat leaf,root and stem samples.The samples were collected from different locations in Wuhan,China.The strains were evaluated against Rhizoctonia solani by dilution culturing method and 37 strains were found to be resistant against R.solani.25 strains which had better resistance were selected for identification by16 s DNA gene,22 of them belong to Bacillus genus.Trichoderma genus,which is a worldwide soil-born fungi,have been studied as potential biological control agent for more than eighty years since Trichoderma lignorum was found can kill plant pathogen Rhizoctonia solani and control plant diseases.Trichoderma harzianum was transformed with IAP20 gene(anti-apoptosis gene).After transformation the fungus was evaluated against different temperature and UV treatments.Fungi was treated at different temperature i.e.,35℃,45℃,55℃ for the time interval 120 min,60 min,30 min,respectively.The plates were incubated at 28℃ after treatment.Wild strain was used as control.Data was recorded after 8,16 and 24 hours of incubation at 28℃,but no significance difference was found in growth after the treatments.The fungus was exposed to UV for 30,60,and 120 min,respectively.Then was incubated at 28℃.The growth was recorded after 8,16,24 and 32 hours of incubation.Wild strain was used as control but no significance difference was found in the growth of wild and transformed after treatment.A c DNA library was constructed by using recombinant technology.The genes of source plant ‘Isatis indigotica’ were incorporated in the vector and transfected into the target host(Bacillus subtilis WB800).The clones were selected on the basis of their phenotype for further evaluation.The selected clones were cultured and their extra and intra cellular proteins were applied against test pathogens after purification.The extracted proteins were evaluated against bacterial plant pathogens Xanthomonas oryzae.pv.oryzae,Xanthomonas oryzae.pv.oryzaecola,R21-5,pslbtw,168,DH5α,YCKYB1.The proteins extracted from the clones named as 205 and505 found to be inhibiting the growth of Xanthomonas oryzae.pv.oryzae,Xanthomonas oryzae.pv.oryzaecola,R21-5,and YCKYB1.The intracellular proteins of 205 were found to inhibit the growth of test bacteria i.e,WB800,Empty vector,R21-5,Xanthomonas oryzae.pv.oryzae,Xanthomonas oryzae pv.oryzaecola,R21-5,and YCKYB1.While in case of extracellular proteins extracted from 205 were showed inhibition against R21-5 and YCKYB1.The intracellular proteins extracted from clone named as 505 inhibted the growth of only R21-5 but the extracellular proteins inhibited the growth of WB800,Empty vector,R21-5,Xanthomonas oryzae pv.oryzae,Xanthomonas oryzae pv.oryzaecola,R21-5,and YCKYB1.The clones from c DNA library were evaluated for their nematicidal activity against Caenorhabditis elegans.Different bioassays like “food choice” and “killing” assay were performed to check the efficiency of clones.The clones named as 177 and 205 were found to be effective against nematodes.