Development of a scar marker for Pierce's Disease strains of Xylella fastidiosa

Abstract

The objective of this research was to develop a primer for a polymerase chain reaction specific for Xylella fastidiosa strains that cause Pierce's Disease (PD) in grapes (Vitis vinifera). The DNA amplification of 23 different strains of X. fastidiosa, using a set of primers REP1-R (5'-IIIICGICGIATCCIGGC-3') and REP 2 (5'-ICGICTTATCIGGCCTAC-3') using the following program: 94 ºC/2 min; 35 X (94 ºC/1 min, 45 ºC/1 min and 72 ºC/1 min and 30 s) 72 ºC/5 min, produced a fragment of 630 bp that differentiated the strains that cause disease in grapes from the other strains. However, REP banding patterns could not be considered reliable for detection because the REP1-R and REP 2 primers correspond to repetitive sequences, which are found throughout the bacterial genome. The amplified product of 630 bp was eluted from the agarose gel, purified and sequenced. The nucleotide sequence information was used to identify and synthesize an specific oligonucleotide for X. fastidiosa strains that cause Pierce's Disease denominated Xf-1 (5'-CGGGGGTGTAGGAGGGGTTGT-3') which was used jointly with the REP-2 primer at the following conditions: 94 ºC/2 min; 35 X (94 ºC/1 min, 62 ºC/1 min; 72 ºC/1 min and 30 s) 72 ºC/10 min. The DNAs isolated from strains of X. fastidiosa from other hosts [almond (Prumus amygdalus), citrus (Citrus spp.), coffee (Coffea arabica), elm (Ulmus americana), mulberry (Morus rubra), oak (Quercus rubra), periwinkle wilt (Catharantus roseus), plums (Prunus salicina) and ragweed (Ambrosia artemisiifolia)] and also from other Gram negative and positive bacteria were submitted to amplification with a pair of primers Xf-1/REP 2 to verify its specificity. A fragment, about 350 bp, was amplified only when the DNA from strains of X. fastidiosa isolated from grapes was employed.

Xylella fastidiosa: Host Range and Advance in Molecular Identification Techniques

“…In Pooler and Hartung ( 1995a ) developed a series of specific primers capable to detect X. fastidiosa in general and X. fastidiosa strains that cause CVC by cloning and sequencing randomly amplified polymorphic DNA (RAPD) products (Pooler and Hartung, 1995a ). Since then a number of reports have been published describing the development of specific PCR primers for the identification of X. fastidiosa (Table 3 ) and if the 16S rDNA and the 16S-23S intergenic spacer region (ITS) are the most common targets (Rodrigues et al, 2003 ; Chen et al, 2005 ; Martinati et al, 2007 ), also several other genomic sequences, with or without a known function, have been used (Travensolo et al, 2005 ; Huang, 2009 ). The use of different genes, alone or in combinations, can be exploited in order to increase the level of sensitivity and specificity of the test.…”

Non-specificity of sequence characterised amplified region as an alternative molecular epidemiology marker for the identification of Salmonella enterica subspecies enterica serovar Typhi

“…However, successful use of SCAR markers has been reported for Trypanosoma cruzi [ 42 ] most probably due to the conservative nature of its genome and for the analysis of genomic instability in breast cancer tissues [ 43 ]. Other successful applications of SCAR markers have been for the detection of Agrobacterium vitis in rice [ 44 ], Pseudomonas brassicacearum as a biological control agent of snow mould in winter wheat [ 20 ] and Xylella fastidiosa in grape vine disease [ 45 ]. Similarly, it has been utilised in the identification of strawberry genotypes carrying red stele resistance gene for mass breeding [ 46 , 47 ], preservation of an endangered ornamental tree species [ 48 ] and for adulteration detection [ 49 , 50 ].…”