Differentiation of Strains of Xylella fastidiosa by a Variable Number of Tandem Repeat Analysis

Abstract

Short sequence repeats (SSRs) with a potential variable number of tandem repeat (VNTR) loci were identified in the genome of the citrus pathogen Xylella fastidiosa and used for typing studies. Although monoand dinucleotide repeats were absent, we found several intermediate-length 7-, 8-, and 9-nucleotide repeats, which we examined for allelic polymorphisms using PCR. Five genuine VNTR loci were highly polymorphic within a set of 27 X. fastidiosa strains from different hosts. The highest average Nei's measure of genetic diversity (H) estimated for VNTR loci was 0.51, compared to 0.17 derived from randomly amplified polymorphic DNA (RAPD) analysis. For citrus X. fastidiosa strains, some specific VNTR loci had a H value of 0.83, while the maximum value given by specific RAPD loci was 0.12. Our approach using VNTR markers provides a high-resolution tool for epidemiological, genetic, and ecological analysis of citrus-specific X. fastidiosa strains.Xylella fastidiosa has been associated with diseases in economically important crops such as grapevine, plum, almond, peach, citrus, and more recently, coffee (13, 17) as well as some diseases in ornamental plants (3). Reciprocal transmission tests including X. fastidiosa from several hosts evidenced the occurrence of host infection groups (for details, see reference 10). In Brazil, X. fastidiosa has been responsible for causing diseases in important crops such as citrus and coffee (5, 13), although it has also been observed in plum (7) and Hibiscus schizopetalus (E. W. Kitajima, H. D. Coletta-Filho, M. A. Machado, and Q. S. Novaes, 33th Congr. Brazil Phytopathol. Soc., abstr. 323, 2000). The major economic damage is in the sweet orange crop (Citrus sinensis Osb.) which has suffered an annual loss of about $100 million. However, the total cost to Brazilian agriculture is probably higher because the economic damage caused to the coffee crop by X. fastidiosa has not been estimated.Methods for distinguishing between bacterial strains are important for detecting disease outbreaks for epidemiological analysis, and for understanding the genetic structure of microbial populations. Molecular techniques of DNA profiling based on PCR of randomly amplified polymorphic DNA (RAPD) and repetitive element polymorphism PCR (rep-PCR) have been used with great success for studies of genetic variation and the relationships between X. fastidiosa strains (2,6,8,16). However, the data produced by these methods are biallelic, which limits the amount of genetic information per locus and thus the use of these methods in estimating genetic diversity. Interlaboratory reproducibility is also a weakness of RAPD and rep-PCR analysis (20). Short sequence repeats (SSRs) with a potential variable number of repeats (VNTR) within prokaryotic DNA have been used as markers for discrimination between strains (1, 9, 12, 23). Some of these repetitive regions are located within or near DNA coding regions and so could have the potential to affect gene expression (22).The complete sequencing of the genome of the pat...

Temporal and Spatial Scaling of the Genetic Structure of a Vector-Borne Plant Pathogen

“…Bacterial repeats in protein coding or upstream of regulatory regions are responsible for phenotypic switches as well as cell adaptations to specific environmental challenges (3,32). Here, most SSRs were located in housekeeping genes, noncoding regions, and hypothetical proteins (14,31). An understanding of the functional role of SSR variation in X. fastidiosa may yield interesting insights.…”

Genetic Structure and Biology of Xylella fastidiosa Strains Causing Disease in Citrus and Coffee in Brazil

“…We used the methods described by Coletta-Filho et al (8) to conduct a study on the population genetic structure of citrus and coffee X. fastidiosa isolates. This method is based on fast-evolving microsatellites (also known as short sequence repeats [SSR] and variable number tandem repeats) that permit genetic studies at the pathogen population level.…”

DNA Fingerprinting ofSalmonella entericasubsp.entericaSerovar Typhimurium with Emphasis on Phage Type DT104 Based on Variable Number of Tandem Repeat Loci

“…Characterization of 426 B. anthracis isolates with this method gave 89 distinct genotypes (22). Polymorphic VNTR regions which can be used for typing purposes have been identified and tested in Yersinia pestis (1,23), Francisella tularensis (21), Mycobacterium tuberculosis (10-12, 37, 40, 41), Xylella fastidiosa (6), Haemophilus influenzae (43,44,49), and Bacillus anthracis (2,8,18,22,38). The VNTR-based typing approach is promising in all of these strains.…”