Isolamento e caracterização de fagos bacteriolíticos contra ralstonia solanacearum e xanthomonas perforans causadores da murcha e mancha bacteriana em tomateiros

Abstract

Tomato plants (Solanum lycopersicum L.) and their fruits are one of the crops that most suffer from attacks by pests and pathogens in Brazilian crops. Climatic factors such as high temperatures and high humidity favor the establishment of diseases, and the use of pesticides that do not combat phytopathogens is becoming limited due to the resistance that these microorganisms have been showing to these products. In this sense, the use of phagotherapy, a technology that uses formulations of lytic phages that are viruses that infect and kill bacteria, is now considered a promising alternative for the control of bacterial pathogens. Therefore, in the present work, phages from samples of soil and leaves of tomato plants with symptoms of wilting and bacterial spot were isolated, aiming at applications for the biocontrol of phytopathogens that cause the diseases in question. For isolation, Ralstonia solanacearum RS705 and Xanthomonas perforans IS3FMA strains were used as host lines, and phage B3 (RS705) and phage X1, X2, X3 and X4 (IS3FMA) were isolated respectively. Subsequently, the 5 phages obtained were characterized at the morphological level by transmission electron microscopy (TEM). The 4 X. perforans phages showed the same morphology and size and the other characterizations followed for phage X1. This together with the B3 phage of R. solanacearum was classified as belonging to the class Caudoviricetes and a genomic analysis for better definition of the current families is in progress. In addition, phages were evaluated for multiplicity of infection (MOI). Phage B3 showed greater evolution against RS705 at the lowest MOI demonstrated (0.01), while X1 was more efficient in the evolution of IS3FMA at MOI 10. At larger scales, the bacteriolytic activity of phages was also transmitted in liquid medium using the best MOI's occurring with a decrease of 49.23% in the OD 600 nm of RS705 and of 76.2% in the OD 600 nm of IS3FMA when both cultures were grown with their respective phages. In addition, evaluate the stability of the phages against variations in pH, temperature, UV light and storage time. Both phages demonstrated good stability between pH's 3.0 and 8.0 (B3) and between pH's 3.0 and 9.0 (X1) for a period of 24 hours. This same stability was maintained between temperatures of 20 and 40°C for 72 hours for both phages, as well as there were no experimental variations in their titles when exposed to UV-A light for 2 (B3) and 1 hour (X1). In addition, phage B3 showed excellent stability, not showing any negative and significant effect on the titer until the 4th month of storage when kept in SM at 4°C. The last parameter evaluated in vitro was the phage specificity across the host spectrum. Phage B3 showed lytic activity only against species of Ralstonia sp., while X1 did not show activity against any of the species, so far being specific to its host lineage (IS3FMA). At the end, an evaluation of the bacteriolytic potential of the B3 phage in vivo was conducted using groups of plants infected with RS705 over 14 days. It was observed that B3 inhibited disease symptoms by 100% in treatments in which the phage was inoculated 2 and 3 times after infection with the pathogen, in addition to lower bacterial densities (CFU/mL) in the soil and stem of all the groups treated with the phage at least once when compared to the control group that received only the pathogen. In addition, the highest titers (PFU/mL) of B3 in the soil were found in groups that presented lower rates of disease progression at the end of the experiment. In short, the B3 and X1 phages enjoyed a great potential for the biocontrol of phytopathogens that cause bacterial wilt and spot in tomato plants.