A simplified subtractive hybridization protocol used to isolate DNA sequences specific to Xylella fastidiosa

Abstract

showing no hybridization to the driver DNA, was used as a probe specific to Xy. fastidiosa from citrus and oleander. This fragment was sequenced and the predicted protein showed 40% similarity to the central region of flagellin of Escherichia co/i serotypes H1 and H12. A pair of internal primers (f14-1 and f14-2) was designed for amplification of Xy. fastidiosa DNA. These primers detected Xy. fastidiosa strains isolated from citrus and oleander and yielded an amplification product of about 600 bp. They were also able to detect the bacteria in extracts from citrus plants with or without symptoms of disease. No amplification reaction was obtained using DNA extracted from other species and pathovars of Xanthomonas, Pseudomonas cichorii, Ennrinia carotovora, Agrobacterium tumefaciens and phytopathogens of citrus (Xanthomonas axonopodis pv. citri) and coffee (Burkholderia andropogonis, P. cichorii, Pseudomonas syringae pv. garcae). The isolation of a DNA fragment specific to Xy. fastidiosa from citrus showed that the simplified protocol of subtractive hybridization used in this work is potentially applicable to other microorganisms. I CTIODi

Comparison of Whole-Tissue and Xylem Fluid Collection Techniques to Detect Xylella fastidiosa in Grapevine and Oleander

“…Improving detection of the pathogen will enhance the study of the interaction between host plants and the pathogen. A better sample-collection technique could improve the sensitivity of current detection techniques (2,13,14,16,21,35). Vacuum extraction of xylem sap has been used to identify bacteria in grapevine (4).…”

“…Detection methods currently used include enzyme-linked immunosorbent assay (ELISA; 3,6,23,33,34), polymerase chain reaction (PCR; 2,7,8,14,[23][24][25]34), and culturing from infected tissue (10)(11)(12)29). Several problems still exist with these methods, mainly stemming from low titer of bacteria in samples, resulting in inconsistent detection (7,8,23,25,33,34).…”

Disrupção das ORFs XF-810, XF-818, XF-1940, XF-2359 e XF-2708 de >i<Xylella fastidiosa>/i<-CVC possivelmente relacionadas à patogenicidade da bactéria

“…O uso de métodos de diagnósticos para identificar a presença de X fastidiosa em plantas é um passo importante na implementação de medidas de restrição da disseminação da doença no campo e em áreas não infectadas (Ferreira et al, 1999).…”

Ash decline in Iowa

“…Acidovorax, Burkholderia, Erwinia, Herbaspirillum, Liberbactor, Pseudomonas, Ralstonia, Xanthomonas, and Xylella have been shown to be plant pathogens (Ferreira et al, 1999;). Some recent studies have shown that there are other prokaryotic pathogens found in the sieve tubes of plants (Ali et al, 1991;.…”

“…There are numerous other plant diseases that are caused by bacteria (Ferreira et al, 1999;Hocquellet et al, 1999, Hu and.…”