Evolutionary and Experimental Assessment of Novel Markers for Detection of Xanthomonas euvesicatoria in Plant Samples

• Tipo: Artigo em Revista Científica Internacional
• Data: 2012

Autores

Pedro Albuquerque (Autor) FCUP; Cristina M R Caridade (Autor) Outra; Arlete S Rodrigues (Autor) Outra; Andre R S Marcal (Autor) FCUP; Joana Cruz (Autor) Outra; Leonor Cruz (Autor) Outra; Catarina L Santos (Autor) Outra; Marta V Mendes (Autor) Outra; Fernando Tavares (Autor) FCUP

Revista

Título: PLoS ONE

Vol. 7 Nº 4

Página Final: e37836

ISSN: 1932-6203

Editora: Public Library of Science

Indexação

ISI Web of Knowledge - 16 Citações

Scopus - 18 Citações

Outras Informações

ID Authenticus: P-002-9YY

DOI: 10.1371/journal.pone.0037836

Abstract (EN): Background: Bacterial spot-causing xanthomonads (BSX) are quarantine phytopathogenic bacteria responsible for heavy losses in tomato and pepper production. Despite the research on improved plant spraying methods and resistant cultivars, the use of healthy plant material is still considered as the most effective bacterial spot control measure. Therefore, rapid and efficient detection methods are crucial for an early detection of these phytopathogens. Methodology: In this work, we selected and validated novel DNA markers for reliable detection of the BSX Xanthomonas euvesicatoria (Xeu). Xeu-specific DNA regions were selected using two online applications, CUPID and Insignia. Furthermore, to facilitate the selection of putative DNA markers, a customized C program was designed to retrieve the regions outputted by both databases. The in silico validation was further extended in order to provide an insight on the origin of these Xeu-specific regions by assessing chromosomal location, GC content, codon usage and synteny analyses. Primer-pairs were designed for amplification of those regions and the PCR validation assays showed that most primers allowed for positive amplification with different Xeu strains. The obtained amplicons were labeled and used as probes in dot blot assays, which allowed testing the probes against a collection of 12 non-BSX Xanthomonas and 23 other phytopathogenic bacteria. These assays confirmed the specificity of the selected DNA markers. Finally, we designed and tested a duplex PCR assay and an inverted dot blot platform for culture-independent detection of Xeu in infected plants. Significance: This study details a selection strategy able to provide a large number of Xeu-specific DNA markers. As demonstrated, the selected markers can detect Xeu in infected plants both by PCR and by hybridization-based assays coupled with automatic data analysis. Furthermore, this work is a contribution to implement more efficient DNA-based methods of bacterial diagnostics.

Idioma: Inglês

Tipo (Avaliação Docente): Científica

Nº de páginas: 15