Isolamento e caracterização genômica de bacteriófago para biocontrole de Pseudomonas cichorii

Abstract

Pseudomonas cichorii is a Gram-negative soil phytobacterium, pathogenic to a wide variety of hosts and belonging to the Pseudomonas syringae complex. Due to its low specificity, its host range includes lettuce, eggplant, celery, mushrooms, tomatoes, coffee, wheat, soy, basil, as well as several ornamental plants. With the ideal environment for developing hot and humid areas, the bacteria spreads through rain, driven by the wind, or during sprinkler irrigation and suspended irrigation systems, infecting the surfaces of wet leaves. Its control includes a series of preventive humidity control interventions, in addition to the use of copper-based products or bactericides, which are not always successful in preventing the spread of the disease when conditions are wet. In the present study, a new virulent bacteriophage that infects phytobacteria P. cichorii was isolated in Brazil from leafy vegetables (lettuce, chicory and cabbage) that showed visible symptoms of bacterial diseases. The PCMW57 phage retained almost 100% of the infection activity after incubation for 60 minutes at temperatures ranging from 5 to 45 °C, and led a drastic reduction in titer above 60 °C. Its maximum stability was observed at pH 7.5, being stable in the pH range of 5.0 to 8.5. The one-step growth curve determined that the latent time period was approximately 60 minutes and the size of the phage burst was about 151 phage particles per infected cell. The phage showed the ability to lyse the bacteria in an antimicrobial activity test under different multiplicities of infection (MOI) (from 1 to 0.001). When tested on other phytobacteria, the phage was not able to induce the formation of lysis plaque, pointing out that none was susceptible to the phage. The purified phage proved to be viable when stored in glycerol in concentrations between 10% to 30% (v / v), at freezing temperature (-20 ºC). PCMW57 genome is a linear double-stranded DNA molecule of 40,117 base pairs in length and contains 49 Open reading frame (ORF). The phage is genetically similar to other Pseudomonas phages, sharing homology above 90% with WRT (94.6%), ΦPSA2 (92.7%), ΦPsa17 (92.3%) and KNP virus (91.7%) and 85.1% with the gh-1 phage, all belonging to the T7-virus genus. The results indicate that the isolated phage has high potential as an effective biocontrol agent for P. cichorii. As far as we know, this is the first report of a bacteriophage infecting P. cichorii. [resumo fornecido pelo autor]