Caracterização de células viáveis e não cultiváveis em Xanthomonas citri pv. anacardii

Abstract

The cashew tree is a plant originating from Brazil, concentrated mainly in the Northeast region, whose productivity is considerably affected by the incidence of diseases. Although most diseases are of fungal etiology, bacterial diseases such as angular leaf spot and Xanthomonas spot caused by Xanthomonas citri pv. anacardii, in cashew trees grown in the states of Ceará and Rio Grande do Norte. The main symptom of angular leaf spot is darkened necrotic spots, restricted by leaf limb veins, while in the case of Xanthomonas spot, the symptoms are necrosis in the fruits and the secondary veins of the leaves of the plant. The introduction of infected seedlings is the main form of dissemination among crops. X. citri's main reported means of survival are crop residues from previous host cycles, alternative hosts, epiphytic, endophytic and viable but nonculturable cells (VBNC) survival of the pathogen. In the latter case, VBNC are not observed in culture medium, but viable, allowing bacterial survival. Adverse environmental factors capable of inhibiting bacterial development may induce VBNC status, which tends to remain in low metabolic activity for long periods of time but may be reversed when under favorable conditions for bacterial growth. Therefore, this work aimed the characterization of VBNC in two pigmented isolates (CCRMTAQ13 and CCRMTAQ18) and one nonpigmented isolate (IBSBF2579) of X. citri pv. anacardii. To this end, the genomes of the three strains were obtained from the NCBI database, annotated and then analyzed for the identification of genes encoded under VBNC condition. To assess the state of VBNC, a bacterial growth curve was obtained, determining the exponential (log) and death phase of the isolates. These phases were evaluated by quantifying cell concentration by quantitative PCR (qPCR) with primers constructed based on relA gene, followed by the evaluation of pathogenicity of cells in VBNC state by artificial inoculation in cashew trees. Thirteen genes reported as expressed in the VBNC state were identified in the genomes of the three isolates, and the relA gene was selected for qPCR analysis. In vitro bacterial growth curves constructed in NYD culture media allowed the determination of the amount of total viable cells present in the exponential and death phases for CCRMTAQ13 (24/96h), CCRMTAQ18 (48/72h) and IBSBF2579 (96/168h) isolates). The characterization of the VBNC state in X. citri pv. anacardii occurred by comparing the number of colony forming units (CFU / mL) and the number of copies of the relA gene/mL at the death phase of the growth curve. In the serial dilution method, at the death phase, 0 CFU/mL were obtained in CCRMTAQ13, 0.2 x 108 CFU/Ml in CCRMTAQ18 and 0.07 x 108 CFU / mL in IBSBF2579. Through qPCR, 2.68 x 108 copies of the relA / mL gene in CCRMTAQ13, 2.38 x 108 copies of the relA/mL gene in CCRMTAQ18, and 7.34 x 108 copies of the relA/mL gene in IBSBF2579. Comparison of quantitative data showed up to 100-fold higher values in the qPCR assays for the death phase. For the incubation period there was no significant interaction between isolates and growth phases, with significant differences only between growth phases (exponential and VBNC), with the LOG phase taking an average of 1.91 days to cause the first ones. symptoms. For severity, evaluated at twenty days after inoculation, interaction between isolates and growth phases was observed, being the CCRMTAQ18 isolate in the LOG phase, the lowest average (3.95 mm²), and the IBSBF2579 isolate, the highest average. (7.17 mm2). The characterization of VBNC allows a new perspective for the diagnosis of phytobacteria, and in this study the first report of the occurrence of VBNC in X. citri pv. anacardii, an important phytopathogenic bacterium for northeastern Brazil.