Detection and Diversity Assessment of Xylella fastidiosa in Field-Collected Plant and Insect Samples by Using 16S rRNA and gyrB Sequences

Abstract

The causal agent of diseases in many economically important plants is attributed to the xylem-limited bacterium Xylella fastidiosa. The detection of this plant pathogen has been hampered due to its difficult isolation and slow growth on plates. Nearly complete nucleotide sequences of the 16S rRNA gene and partial sequences of the gyrB gene were determined for 18 strains of X. fastidiosa isolated from different plant hosts. A phylogenetic analysis, based on gyrB, grouped strains in three clusters; grape-isolated strains formed one cluster, citrus-coffee strains formed another cluster, and a third cluster resulted from all other strains. Primer pairs designed for the 16S rRNA and gyrB genes were extensively searched in databases to verify their in silico specificity. Primer pairs were certified with 30 target and 36 nontarget pure cultures of microorganisms, confirming 100% specificity. A multiplex PCR protocol was developed and its sensitivity tested. Sequencing of PCR products confirmed the validity of the multiplex PCR. Xylella fastidiosa was detected in field-collected plants, disease vector insects, and nonsymptomatic but infected plants. Specific detection of X. fastidiosa may facilitate the understanding of its ecological significance and prevention of spread of the disease.The insect-transmitted bacterium Xylella fastidiosa (38) causes diseases in many economically important plants, including citrus, coffee, grapevine, peach, plum, almond, alfalfa, elm, oak, mulberry, maple, and oleander, by clogging the xylem and affecting water and nutrient transport (26). Control of these diseases, particularly citrus variegated chlorosis and Pierce's disease in grapevines, often relies on management of insect vectors and on inoculum reduction by elimination of diseased and surrounding plants or pruning of infected branches (26). Therefore, early detection and identification of the pathogen is a cornerstone in preventing the spread of disease.Current methods of detecting the slow-growing microorganism X. fastidiosa rely mainly on isolation and cultivation to pure culture, followed by biochemical and serological analysis. These methods are labor-and time-consuming and are complicated by the presence of many contaminant bacteria associated with the plant host (19). Other methods include scanning electron (7, 11), phase-contrast (18), and fluorescence (14) microscopy. Primer sets based on an EcoRI restriction (23) and RAPD [random(ly) amplified polymorphic DNA] fragments (25) were reported previously. However, these primers do not target any coding sequence, and to date no coherent phylogenetic analysis of their amplicons has been performed.Advances in molecular microbial ecology opened avenues for designing taxonomically meaningful highly specific PCR primers (3). The 16S rRNA gene has been widely used for this purpose, providing enough sequence information to allow the analysis of both close and distant phylogenetic relationships among microorganisms (30). However, when the resolution of the 16S rRNA gene-based analysis ...

Short communication. A survey of potential insect vectors of the plant pathogenic bacterium Xylella fastidiosa in three regions of Spain

“…Uneven amplifications of 16S or gyrB genes were found in some cases for the positive control samples. A direct explanation for this result is the presence of only one gyrB gene in the X. fastidiosa genome, whereas two copies of the 16S rRNA are found (Rodrigues et al, 2003).…”

“…The presence of X. fastidiosa in specimens of the most frequent leafhopper and spittlebug species collected by sweep net and stem tap in this survey was tested either using nested-PCR (Ciapina et al, 2004) or multiplex-PCR (Rodrigues et al, 2003) assays. For extraction of total DNA, insect tissues combined in samples from the same plot (Table 1) were placed directly in a 1.5-mL Fast DNA tube containing lysing matrix A, 500 µL of CLS-VI solution (Qbiogene), and 200 µL of Protein Precipitation Solution (PPS) (for insect material).…”

“…For the multiplex-PCR assay two sets of primers were used in same reaction tube to amplify the gyrB gene (FXYgyr499/RXYgyr907 primers) and the 16S rRNA (S-S-X.fas-0067-a-S-19/S-S-X.fas-0838-a-A-21 primers) using same amplification conditions described in Rodrigues et al (2003). The PCR products were separated by 1.5% ethidium bromide agarose gel electrophoresis at 100 V, in 1X TBE buffer for 1.5 h.…”

Nutritional deficiency in citrus with symptoms of citrus variegated chlorosis disease

“…Pera) trees. The procedure to collect samples was the same used by Rodrigues et al (2003). The leaves were randomly collected from plants with and without symptom from 4 orchards.…”

A Multigene Phylogenetic Study of Clonal Diversity and Divergence in North American Strains of the Plant Pathogen Xylella fastidiosa

“…The amount of diversity within these plant-host strains, however, was contradictory, with one study indicating limited diversity (12) and another indicating moderate amounts (20). Several single-gene trees and noncoding regions have also been analyzed, including 16S rRNA (25, 32), 16S-23S intergenic spacer sequences (20,25,35), and gyrB (32). The resulting trees had conflicting topologies, due to a lack of informative sites and phylogenetic signal.…”