Abstracts - Hocelayne Paulino Fernandes

Metabolic profiling of leaves citrus species before and after the inoculation of P. citricarpa using 1H NMR and chemometric analysis

Hocelayne Paulino Fernandes*1, Thayana da Conceição Alves1, Rosana Gonçalves Pereira2, Geraldo José Silva Junior2, Antônio Gilberto Ferreira1, João Batista Fernandes1, Maria Fátima das Graças Fernandes da Silva1.

1 Department of Chemistry, Federal University of São Carlos, SP, 13565-905, Brazil. 2 Fundecitrus, Araraquara, SP, 14807-000, Brazil.
* hocelayne.fernandes@gmail.com

The citrus black spot (CBS) caused by the fungus Phyllosticta citricarpa is an important citrus disease, which affects most citric varieties, causing relevant economic damages. The challenge for citrus producers for control the disease has been increasing with the demand for products free of toxic chemical residues. Whereas of CBS symptoms may take up to one year, depending on the variety and environmental conditions1,2. This work presents the application of fingerprinting profiling by 1H NMR in studies of two Citrus species, C. lemon and C. latifolia infected for fungus P. citricarpa, susceptible and tolerant species, respectively. The obtaining form, preparation and analysis of leaves samples were planned carefully. The samples of susceptible and tolerant species were inoculated with P. citricarpa and the control samples were obtained in the same conditions. The samples were stored in freezer – 80 ºC until the analysis. The inoculated and control leaves were obtained in the times at 1, 2, 3, 4, 7, 15, 30 and 40 days. The methanolic extracts were obtained and the 1H NMR spectra were acquired at 298 K in a Bruker® AVANCE III 9.4 Tesla using a 5 mm BBI probe head with ATMA® and using SampleXpress™. AMIX® software was using for 1H NMR data. Some compounds (20) were identified in control and inoculated samples thought analysis 1D and 2D (COSY, HSQC, HMBC) NMR experiments, database and literature. The chemometric study showed a tendency of distinction between C. lemon (susceptible) and C. latifolia (tolerant) species. The susceptible specie showed strong distinction of inoculated and control samples from third day of inoculation. Already the tolerant specie not showed high changes in the chemical profile in the during inoculation time. The major changes in the chemical profile after infection with P. citricarpa on C. lemon leaves occurred in 2.7 - 3.5 ppm and aromatic region. These samples had high concentration of compounds as ethanol, choline, malonate and signal of organic acids. Already control samples showed high content of fatty acids, limonene, amino acids (threonine and proline) and some aromatics signals. So, the data obtained of fingerprinting profiling by 1H NMR associated chemometric tools provided a satisfactory result and confirmed that these methods were extremely useful to provide chemical information of leaves of Citrus species. For the C. latifolia specie we do not verify significant changes in the chemical profile of leaves, indicating their tolerance to fungus P. citricarpa. And for the C. lemon specie, the chemical profile of control and inoculated leaves showed difference from third day of inoculation. These results confirm that during this period the plant already showed a response to citrus black spot disease.
Key words: Citrus, Phyllosticta citricarpa, Metabolomics and 1H NMR.