An Optimized Microsatellite Scheme for Assessing Populations of Xanthomonas phaseoli pv. manihotis.

Diverse molecular markers have been used to analyze the genetic diversity of plant pathogens. Compared with traditional fingerprinting methods, multiple loci variable number of tandem repeat analyses (MLVAs) have gained importance recently due to their reproducibility, high discriminatory power, ease of performance, low cost, and throughput potential. These characteristics are desirable for continuous pathogen monitoring, especially for pathogens with relatively low genetic diversity, and for disease epidemiology studies. Genetic diversity studies of Xanthomonas phaseoli pv. manihotis, which is the causal agent of cassava bacterial blight, have shown variability and changes in the bacterial population over time. Thus, an easy and fast method needs to be developed to type populations of this pathogen in different countries of the world, especially on small scales. In this study, we developed an MLVA scheme to analyze X. phaseoli pv. manihotis variability on a local scale. The MLVA-15 scheme comprises 15 variable number of tandem repeat loci grouped into four multiplex polymerase chain reaction pools. We showed that the MLVA-15 scheme had slightly higher discriminatory ability at the locality level when compared with amplified fragment length polymorphisms. The MLVA-15 scheme allowed for an accurate determination of the number of genotypes in the sample and showed reproducibility and portability. Additionally, this scheme could be used to analyze numerous strains in a reasonable timeframe. The MLVA-15 scheme was highly specific to X. phaseoli but up to eight tandem repeat loci could be amplified from other Xanthomonas spp. Finally, we assessed the utility of the scheme for analyses of X. phaseoli pv. manihotis genetic variability in the Colombian Caribbean region. MLVA-15 distinguished 88.9% of the haplotypes in our sample. Strains originating from the same field and isolated at the same time could be discriminated. In this study, the advantages of the MLVA-15 scheme targeting 6- or 7-bp repeats were demonstrated. Moreover, this scheme was a fast method that was appropriate for routine monitoring of X. phaseoli pv. manihotis populations on a local scale and, thus, was useful for addressing epidemiological questions.

A minisatellite-based MLVA for deciphering the global epidemiology of the bacterial cassava pathogen Xanthomonas phaseoli pv. manihotis.

Cassava Bacterial Blight (CBB) is a destructive disease widely distributed in the different areas where this crop is grown. Populations studies have been performed at local and national scales revealing a geographical genetic structure with temporal variations. A global epidemiology analysis of its causal agent Xanthomonas phaseoli pv. manihotis (Xpm) is needed to better understand the expansion of the disease for improving the monitoring of CBB. We targeted new tandem repeat (TR) loci with large repeat units, i.e. minisatellites, that we multiplexed in a scheme of Multi-Locus Variable number of TR Analysis (MLVA-8). This genotyping scheme separated 31 multilocus haplotypes in three clusters of single-locus variants and a singleton within a worldwide collection of 93 Xpm strains isolated over a period of fifty years. The major MLVA-8 cluster 1 grouped strains originating from all countries, except the unique Chinese strain. On the contrary, all the Xpm strains genotyped using the previously developed MLVA-14 microsatellite scheme were separated as unique haplotypes. We further propose an MLVA-12 scheme which takes advantage of combining TR loci with different mutation rates: the eight minisatellites and four faster evolving microsatellite markers, for global epidemiological surveillance. This MLVA-12 scheme identified 78 haplotypes and separated most of the strains in groups of double-locus variants (DLV) supporting some phylogenetic relationships. DLV groups were subdivided into closely related clusters of strains most often sharing the same geographical origin and isolated over a short period, supporting epidemiological relationships. The main MLVA-12 DLV group#1 was composed by strains from South America and all the African strains. The MLVA-12 scheme combining both minisatellite and microsatellite loci with different discriminatory power is expected to increase the accuracy of the phylogenetic signal and to minimize the homoplasy effects. Further investigation of the global epidemiology of Xpm will be helpful for a better control of CBB worldwide.