The Role Of Plant Growth Promoting Bacillus Subtilis Strain And Its Antagonistic Genes Identified By Removing PCR

The development of new and better methods to enhance crop health and yield has become more and more important,as the world's population is increasing,and the demand of more food production increases.The use of traditional chemicals is damaging to the human health,plants and environment.Focus of the modern world is escalated on eco-friendly methods in the last decades.One of the best method is to use plant growth promoting bacteria(PGPB).PGPB may play a crucial role to trigger the tolerance against biotic/abiotic stresses alongwith growth and health enhancement in plants.This study was conducted to isolate plant growth promoting Bacillus strains that have broad-spectrum pathogen suppression ability and several physiological activities to promote plant growth promotion.In all,277 strains were isolated and tested against Rhizoctonia solani AG1-IA.Seventy elite strains suppressed the growth of R.solani,while 5 strains highly inhibited the growth of R.solani.Our prime aim was to isolate and select the best Bacillus sbtilis strain.An endophytic Bacillus subtilis 330-2 isolated from rapeseed was selected for further studies in accessing its role in enhancing plant growth and tolerance to abiotic stresses and banded leaf and sheath blight disease in maize.The strain 330-2 produced the IAA,siderophore,hydrolytic enzymes and solubilized different sources of organic/inorganic phosphates and zinc.Furthermore,the strain strongly suppressed the in vitro growth of R.solani AG1-IA,Botrytis cinerea,Fusarium oxysporium,Alternaria alternata,Cochliobolus heterostrophus,and Nigrospora oryzae.The strain also significantly increased seedling growth(ranging 14-37%)of rice and maize.To our knowledge,the removing PCR(R-PCR),established in our lab,is one of the simple cost-effective method for generating deferentially expressed genomic DNA fragments distinguishing the samples under comparison.In this study,R-PCR was used to isolate and identify 147 differentially expressed genes from B.subtilis 330-2.Majority of these differentially expressed genes are involved in biotic and abiotic stresses.The results showed that the R-PCR efficiency was 92.52%.Among which,10%,32%and 11%genes were involved in antibiotic productions(srfAA,bae,fen,,mln,dfnl,etc.),metabolism(gltA,pabA,ggt,etc.)and transportation of nutrient(fhu,glpT,gltT,etc.),respectively.In summary,these results clearly indicated the effectiveness and mechanisms of B.subtilis strain 330-2 in enhancing plant growth as well as tolerance to biotic/abiotic stresses,which suggested that it has great potential for commercialization as a vital biological control agent.Caenorhabditis elegans is free-living soil nematode and commonly used as an infectious model organism for toxicological studies due to its well-known biology,large brood size,readily scorable life traits,and short generation time.C.elegans encounter various kind of bacterial species present in the natural environment,which may vary from being palatable to pathogenic.These bacteria serve as food source for nematodes and influence behavior and physiology.Spore-forming bacillus species can be prolific producers of economically important secondary metabolites,other biologically active compounds and affect the lifespan of C.elegans.The nematodes are able to differentiate the food quality of bacteria and try to minimize the general exposure to the pathogens via avoidance behavior.The recent study has shown that the interactions between C.elegans and its environment may influence aging and longevity of the host.In this regards B.subtilis strain 330-2 genomic library was constructed with the expression vector pET22-b,transformed to E.coli DE3 and genes were induced by IPTG.These induced genes were screened for feeding behavior against C.elegans.After feeding to nematodes on initial basis 80 induced genes were selected as food sources.Among them 8 induced genes were selected for further study.The results showed that growth rate and progeny was significantly reduced when C.elegans were fed with induced genes,and notably marked attenuation and extension in the lifespan of the worms were observed.The spoIIIE gene significantly decreased the brood size up to 27%.B.subtilis strain 330-2 induced genes,yqeK and spoIIIE significantly enhanced the longevity of C.elegans by 33%and 30%,respectively,while ltaS1 and bmr decreased the lifespan of C.elegans by 11 and 9%,respectively,as compared to control E.coli strain DE3.The experimental evidence supports a role for the B.subtilis strain 330-2 induced genes in affecting the aging and lifespan through distinct mechanism,such as food source.This study provides the first evaluation of new potential genes from B.subtilis 330-2 that was not identified in previous studies against C.elegans.In this research,the R-PCR protocol was also optimized and improved with efficiency increased from 73.6 percent to 92 percent.The finalized step wise protocol has been put in the appendices.