Preparation and evaluation of alkyl dihydroxybenzoates against xanthomonas citri subsp. citri and control of citrus canker

Author(s)

Ana Carolina Nazaré

• Document type: Doctoral Thesis
• Press: São José do Rio Preto. 2019-08-14.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Biociências Letras e Ciências Exatas. São José do Rio Preto
• Defense date: 2019-07-19
• Advisor: Luis Octávio Regasini; Henrique Ferreira; José Belasque Junior

Abstract

Citrus cancer affects citrus varieties of worldwide economic importance, being caused by Xanthomonas citri subsp. citri. (Xcc), a Gram-negative bacterium. The control of citrus canker is accomplished through integrated management with applications of cupric substances, planting of less susceptible cultivars, use of windbreaks and control of citrus-mining larva. However, excessive use of copper can cause soil accumulation and negatively affect root growth of citrus plants, and consequently nutrient absorption. For this reason, the search for environmentally safe and efficient anti-Xcc substances is of crucial importance. In this paper we use the planning of anti-Xcc synthetic substances. Thus, 36 alkyl dihydroxybenzoates (DHB) were synthesized, which showed anti-Xcc activity. Among them, three esters with the lowest MIC values and seven carbons in the side chain (heptyl) were selected, 4 (52 μM), 16 (80 μM) and 28 (88 μM). The Xcc growth curve assay showed that the selected DHB promoted exponential phase delay, extending the time for Xcc to reach the stationary phase. The effect of DHB 4, 16 and 28 on Xcc membrane permeability was compared to nisin control, with DHB 28 being the most effective derivative (93.8%) than 16 (41.3%) and 4 (13, 9%). Data from the GTPase inhibition assay suggest that esters 4, 16 and 28 may promote changes in protofilaments, leading to depolymerization of the Filamentous temperature sensitive protein Z (FtsZ) and thus preventing bacterial cell division. The viability of application of DHB 4, 16 and 28 was confirmed by cytotoxicity assays, with IC50 values greater than 100 μM against eukaryotic cells tested. Xcc cells lost the ability to colonize the host after treatment with coumpounds 4, 16 and 28 at ½ MIC and MIC, inhibiting the appearance of symptoms of citrus canker on sweet orange leaves (Citrus sinensis). DHB 4, 16 and 28 were evaluated for the protective capacity of sweet orange young plants and the incidence of symptoms decreased by 33.3% (4), 15.3% (16) and 28.6% (28) to copper oxychloride (26.7%). The esters maintained copper oxychloride-like behavior with respect to the severity of citrus canker symptoms. The phytotoxicity of the three DHB was evaluated in a Lactuca sativa assay, causing less interference in seedling growth when compared to copper oxychloride present in the copper base formulations used in citrus crops. The esters did not cause changes in photosynthetic parameters, such as transpiration rate, stomatal conductance, maximum assimilation, PSII photochemical conversion effective quantum yield, PSII photochemical extinction coefficient and PSII non-photochemical extinction coefficient in young C. sinensis. In the evaluation of toxicity against stingless bees (Scaptotrigona postica), esters 4, 16 and 28 caused similar mortality to copper oxychloride. (AU)

• FAPESP's process: 15/08089-6 - Preparation and biological evaluation of alkyl dihydroxybenzoates (AD) as useful agents to combat citrus canker
• Grantee: Ana Carolina Nazaré
• Support Opportunities: Scholarships in Brazil - Doctorate