Screening And Identification Of Peach Endophytic Bacteria With Antagonism Against Agrobacterium Tumefaciens

Peach crown gall disease is caused by Agrobacterium tumefaciens,a bacterial disease that causes serious damage to peach production.Biological control is the most effective way to prevent crown gall disease.Endophytic bacteria colonizes in plant tissue,has been widely used in biological control of plant disease due to a variety of biological functions,such as disease resistance,growth promotion,nitrogen fixation and so on.It also has advantages in occupying favorable niche,induce plants to produce systemic resistance,and directly antagonism against pathogens.The objectives of this study were to isolate and analyse the population composition of culturable endophytes in twigs and roots of 4 different peach cultivars,and to screen the new antagonists for biological control of crown gall disease.Twigs of 4 different peach cultivars with different treatments were collected at different time and the roots were collected without treatment.Endophytes were isolated after surface sterilization,and their populations were counted.Then the quantity and diversity of endophytic bacteria of all the samples were analyzed.A total of 635 endophytic bacteria were isolated from peach of ‘Honggengansutao',‘Xibei 13-1',‘Zhongtaokangzhen 1',and ‘Okinawa' cultivar,which proved that endophytic bacteria were abundant in peach trees.The abundance and quantity of endophytic bacteria in twigs and roots of four peach cultivars under different treatments were different,and the number of endophytic bacteria in twigs significantly increased after inoculation with A.tumefaciens.This suggests that the resistance of peach trees against crown gall disease may be related to its endo-bacteria.The antagonisitic avtivity of all the isolates against A.tumefaciens were tested by pair co-culturing method,and the greenhouse test using sunflowers as a susceptible plant.And the antagonists with strong inhibitory ability were identified by physiological and biochemical and molecular methods.In vivo,strains 10DM4-1 and 10DI2-2 showed good performance in disease control with the efficacy of 86.08% and 89.89%,respectively.Strains 10DM4-1 and 10DI2-2 were then identified to be Pantoea deleyi and Enterobacter cowanii by their 16 S rDNA sequences in combination with their biochemical and physiological characteristics,respectively.These two bacterial strains can produce clear zone around the bacterial colony on the YEB plates inoculated with A.tumefaciens,indicating that the production of antagonistic substances is an important mechanism for its role in biocontrol.The antagonistic strains were tagged with GFP by electroporation.Fluorescence microscope and the method of plate dilution were used to detect the colonization of tagged strains in tomato roots.The transformants 10DM4-1-gfp and 10DI2-2-gfp could stably colonize and survive in the intercellular of tomato roots.This suggests that Pantoea deleyi 10DM4-1 and Enterobacter cowanii 10DI2-2 could colonize the favorable ecological niche and has high potential application value for biological control of crown gall disease.In conclusion,antagonistic strains 10DM4-1 and 10DI2-2 may be developed into new biocontrol agents for the prevention of crown gall disease.Therefore,it can be said that the above results provide a new experimental material for the further biological control of crown gall disease by using endophytic bacteria resources.In addition,this study provides two potential bacteria species for biological control of crown gall disease.