Diversity of AVR and leucin rich protein genes of Xanthomonas axonopodis pv. citri, causal agent of citrus canker

Abstract

The technological progress that has been observed during the last decades has contributed to the generation of relevant information regarding the genetic material (DNA/RNA) of several microorganisms. The study of the genetic diversity of a vegetal pathogen can provide subsidies to the knowledge related to its interaction with the host, to establish hypothesis regarding its evolution history and to define strategies of control, among others. Citrus canker is one of the main diseases of citrus crop. As a result of its presence in Brazil since 1957, a campaign to eradicate the pathogen and, among other actions, exclusion and eradication strategies have been applied at different scales. The causing agent, Xanthomonas axonopodis pv. citri (Xac), is a gram-negative, aerobic, rod-shaped bacterium showing a polar flagellum that can induce symptoms in virtually all citrus species and also other genus of the Rutaceae family. The bacterial pathogens that are genetically similar (clone populations), aiming the characterization of the genetic structure of the population, has been studied for human and vegetal pathogens by Southern blot using insertion sequences or avr genes as probes. The best examples of this technique are the studies conducted with X. oryzae pv. oryzae, X. pv. mangiferaeindicae and X. axonopodis pvs. citri and manihotis. The molecular characterization of pathogenic isolates of Xanthomonas in citrus has been made aiming the differentiation of the existing pathovar preferably. Recently, the genotypic determination of a worldwide collection of Xac isolates by BOX (BOX elements) and ERIC-PCR (enterobacterial repetitive intergenic consensus) allowed for a relative characterization of genetic groups and partially separated geographical distant isolates, such as those originated from Asia and America, and also those with differentiated pathogenicity. Another adequate characterization of Xac genetic structure was conducted by Southern blot, using insertion sequences as probes, and also fluorescent amplified fragment length polymorphism (fAFLP). The results obtained by Southern blot allowed for a relative division of the isolates according to the geographical origin. On the other hand, the genotype determination by fAFLP showed a limited polymorphism. In spite of these recent results, information that make a correlation between the genetic diversity and pathogenicity and/or the dynamic interaction between Xantomonas citri-citrus, such as those provided by studies based on avr/pth genes, continue being rare in the literature. The recent information generated by the Genoma/FAPESP programs facilitates this type of study, and new hypothesis about the dynamics of this interaction can be established, which will reflect in the future on citrus canker control. In this sense, the present research proposal aims to develop DNA probes and primers based on pathogenicity genes (avr and leucin rich protein) to be used in the analysis by Southern blot and restriction fragment length polymorphism (RFLP) The information will be generated at the following levels: a) populational (organism), considering a collection of Xac isolates to be evaluated as to the frequency and relative gene position by Southern blot; and b) genetic, considering the polymorphism existing in the nucleotide sequence of genes by RFLP. With these considerations, it will be possible to formulate hypothesis referring to the dynamics of Xac-citrus interaction, of pathogenic genes of Xanthomonas, and of isolates of Xac originated from regions that differ among themselves as to citrus canker control strategies. (AU)