Isolation And Characterization Of Root-Associated Endophytic Bacteria And Their Biocontrol Potential Against Fungal Phytopathogens Of Rice(oryza Sativa L.)

Rice(Oryza sativa L.)is one of the most important cereal crops worldwide,and its growth is affected by different phytopathogens.Several fungal phytopathogens including Magnaporthe,oryzae,Fusarium graminearum,Fusarium,moniliforme and Rhizoctonia solani,can cause serious rice diseases,outbreaks of these diseases result in great losses in rice yield.Currently chemical fungicides are still vital for the effective control of fungal plant pathogens.However,the long-term and excessive use of chemical pesticides increases the potential for the buildup of resistance in fungal phytopathogens,generating pathogen strains that are harder to control.Moreover,with the public concern over the hazardous effects of chemicals,it is very important to find and develop suitable alternatives for plant disease control.Biological control agents(BCAs),including microorganisms and microbial products,have been developed as promising and ecofriendly alternatives to chemical pesticides.Endophytic bacteria in plant roots displayed good inhibitory activity against many fungal phytopathogens,these root-associated bacterial endophytes improved plant resistance to fungal phytopathogens.Many endophytic bacteria were used as biological control agents for control of plant diseases.However,comprehensive assessment has not been performed for total bacterial endophytes against fungal phytopathogens in rice plants.In the present study,bacterial endophytes were isolated from rice cultivars roots,many endophytic bacteria were shown to possess good inhibitory activities against different pathogenic fungi.The results are as follow:1.122 bacterial endophytes were successfully isolated from three rice cultivars roots,16 S rRNA genes from the endophytic bacteria were PCR amplified,and the PCR products were sequenced using Sanger method.The phylogenetic tree constructed using 16 S rRNA revealed that these bacterial root endophytes belonged to six different bacterial genera,including Bacillus,Fictibacillus,Lysinibacillus,Paenibacillus,Cupriavidus,and Microbacterium.These six bacterial genera were further associated with the twenty-two different root-associated bacterial endophyte species.including Bacillus altitudinis,Bacillus aryabhattai,Bacillus cereus,Bacillus fusiformis,Fictibacillus phosphorivorans,Cupriavidus metallidurans,Bacillus marisflavi,Bacillus wiedmannii,Fictibacillus nanhaiensis,Bacillus subterraneous,Bacillus thioparans,Lysinibacillus mangiferihumi,Paenibacillus cucumis,Paenibacillus alvei,Paenibacillus uliginis,Bacillus pseudomycoides,Bacillus lehensis,Bacillus acidicele,Bacillus tequilensis sp.,Bacillus indicus,Bacillus toyonensis,and Microbacterium laevaniformans,respectively.Of these endophytic bacteria,15 isolates were clustered within Bacillus,which was the most dominant bacterial genera in the bacterial endophytes and account for 12% of total isolated bacteria.2.72 bacterial endophytes isolated from rice plant roots showed biocontrol potential against four different fungal phytopathogens by using the dual culture assay.Of the root-associated bacterial endophytes,10 isolates exhibited antagonistic activities against M.oryzae with rates ranging from 57.20% to 82.40%,and the isolate C-2B-2 reduced M.oryzae growth by approximately 82.40%,20 root-associated isolates showed inhibitory activities against F.graminearum with rates ranging from 58.00% to 72.60%,and the isolate C-3Y-2 reduced F.graminearum growth by approximately 72.60%,16 endophytic bacteria strains displayed antagonistic activities against Fusarium,moniliforme,with rates ranging from 47.50% to 68.90%,26 root-associated isolates showed antagonistic activity against R.solani with rates ranging from 37.80% to 64.70%.Furthermore,we PCR amplified fragments of gene clusters responsible for biosynthesis of secondary metabolites from the active bacterial endophytes,and therefore identified possible gene clusters in the bacterial endophytes,indicating that they have the potential for production of secondary metabolites.Together,122 bacterial endophytes were successfully isolated from three rice cultivars roots,and seventy-one root-associated bacterial endophytes exhibited inhibitory activities against four different fungal phytopathogens.C-2B-2 and C-3Y-2 showed good inhibitory activities against M.oryzae and F.graminearum,respectively,both endophytic isolates have the potential for application as biocontrol agents.