Characterization And Biocontrol Function Of Phages From Pathogenic Pseudomonas Syringae

Pseudomonas syringae is one of the main plant pathogenic bacteria which belongs to gram-negative bacterias.Due to its high pathogenicity,extensive,rapid spreading and difficult to eliminate.Phage is a general term for the virus which infects bacteria,and anti-infection therapy base on phage has been thought to be a potential biological therapy to substitute traditional antibiotics,due to its high safety and low cost in agricultural applications.In this paper,the Pseudomonas syringae pv.syringae 1.3070 was used as materials.Several phages have been screened and isolated from environmental samples using Pss as a host.The isolated phages were characterized and the therapeutic effects was evaluated in vitro and in vivo.First of all,two virulent bacteriophages were isolated by using Pss as the host,named PSV6 and PSV3.Both phages showed high lytic activity,and their highest titers were determined as 10¹² and 10¹¹ PFU/ml,respectively.The PSV6 and PSV3 were classified to the Podoviridae and Siphoviridae according to their morphological aspects under transmission electron microscopy,respectively.Their multiplicity of infection（MOI）value were determined as 1,and the latent period is 40 min and 45 min,the lysis period is 40 min and 75 min,respectively.Both of them remain stable at p H 6-9and p H 5-10,as well as at temperature of 4～30℃and 4～60℃;the sensitivity of the two to organic solvents is different,PSV6 is not sensitive to acetone,and its sensitivity of other organic solvents ranges from strong to weak:ether>ethanol>chloroform,PSV3 is not sensitive to ether and chloroform,and its certain sensitivity to acetone and ethanol.The whole genome of the isolated phages were sequenced and analyzed,and results showed that PSV6 had a double-stranded circular DNA with full length of 40,070 bp,GC content of 57.96%,encoding 48 ORFs,and high homology to PCS5、PCS4、UNO-SLW1～UNO-SLW4.The PSV3 had also a double-stranded DNA,with full length of229,871 bp,49.08%GC content,encoding 376 ORFs,and it has high homology to PSN9,Kakheyi25,Pae S C1,Pae S SCH Ab26 and PA73.Neither toxin nor antibiotic resistance genes was identified in both genomes of phage PSV6 and PSV3.At the end of the paper,the efficacy of phages PSV6 and PSV3 against host bacteria Pss was evaluated in vitro and vivo.The results showed that both phages can effectively inhibit the proliferation of host bacteria in vitro,but there were differences in the dynamics of lysis of the host bacteria.The higher titer of phage had stronger ability to inhibit the proliferation of host bacteria in short time.By in vivo study,the results showed that the phage’s inhibition ability was increased with the phage titer,when samples were treated with different PFU phages,thus the prevention and control effect were improved.Compared to using PSV6 alone,mixed phages showed a better effect,and PSV3has the best control effect on Pss when treated with phage at MOI of 100 for 15 minutes.This thesis provides a useful exploration of the phage method for the control of bacterial diseases caused by Pss in agricultural applications.