Evaluation of antimicrobial activity of peptides on citrus bacterial pathogen growth

Author(s)

Rosangela Naomi Inui Kishi

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2014-08-19

Examining board members

Marcos Antonio Machado; Camila Caldana; Helvecio Della Coletta Filho; Jose Camillo Novello; Eneida de Paula

• Advisor: Marcos Antonio Machado

Abstract

The citrus culture has been affected by various pathogens such as, Xylella fastidiosa (Xf), Xanthomonas citri subsp. citri (Xcc) and Candidatus Liberibacter asiaticus (CaLas).To control these pathogens, large quantities of pesticides are needed. In this context, are important new strategies for sustainable management, such as the use of antimicrobial peptides (PAM), which can control the growth of these pathogens. Here we have prospected at coding peptide sequences in the citrus database of expressed sequence (CitEST) and used other peptides previously tested. To verify the AMPs action on the microorganisms, in vitro tests were performed. Among the peptides evaluated in vitro, CJ0714, EC0604, SC0902 and PE1105 - citrus peptide showing bactericidal activity against Xcc and S. meliloti (phylogenetically close to CaLas) and bacteriostatic effect on Rhizobium radiobacter. All peptides evaluated in vitro for Xcc, had their activities evaluated in a detached leafs, under controlled conditions, and plants placed in the greenhouse. The results in detached leaf results corroborated with the in vitro data. In the greenhouse, results for CJ0714, AR0610 and PE1105 peptides were not similar with those obtained in vitro and in detached leaf. Nevertheless, the presence of symptoms was reduced as compared with the positive control (bacteria only). This may be due to factors and compounds present in the plant and to the instability of the peptide at conditions close to the natural ones. Peptides that showed promising results in vitro and in detached leaf or in previous works were cloned into the CTV (Citrus Tristeza Virus) vector to evaluate the transient expression and its action on CaLas. Despite the satisfactory results in previous experiments, it was not possible to observe the PAM effect on CaLas population when expressed through CTV vector. Nevertheless, reduction of the frequency of the bacteria in the insect vector was observed when Diaphorina citri fed on plants with AMP PE1106, since the pathogen can be transmitted from one generation to another by transovarial mode. None peptides evaluated was efficiently on the growth of Xf. To evaluate the effects of the AMPs on endophytic microorganisms, Methylobacterium sp. was used and it was observed that peptides exhibit activity against this bacterium. Tests to check the cytotoxicity of these AMPs for humans were performed. The hemolysis test allows checking the activity of the peptide on human erythrocyte. The AMPs that showed the lowest hemolytic activities were SC0902, PE1105 and PE1106. Furthermore, we noticed the proliferation of lymphocytes and mast cell desgranulation. Thus, this work presents PAM, PE1105 as potential for the control of these bacterial pathogens of citrus because it showed antimicrobial activity and no cytotoxic effect on human cells (AU)

• FAPESP's process: 10/11072-4 - EXPLORATION AND ANALYSIS OF ANTIMICROBIAL PEPTIDES ACTIVITY TO CONTROL PATHOGENS OF CITRUS
• Grantee: Rosangela Naomi Inui Kishi
• Support Opportunities: Scholarships in Brazil - Doctorate