Comparison of Randomly Amplified Polymorphic DNA with Amplified Fragment Length Polymorphism To Assess Genetic Diversity and Genetic Relatedness within Genospecies III of Pseudomonas syringae

Abstract

Recently, DNA pairing analyses showed that Pseudomonas syringae pv. tomato and related pathovars, including P. syringae pv. maculicola, form a genomic species (Pseudomonas tomato) (L. Gardan, H. L. Shafik, and P. A. D. Grimont, p. 445-448, in K. Rudolph, T. J. Burr, J. W. Mansfield, D. Stead, A. Vivian, and J. von Kietzell, ed., Pseudomonas syringae Pathovars and Related Pathogens, 1997). The genetic diversity of 23 strains belonging to this genomic species and 4 outgroup strains was analyzed with randomly amplified polymorphic DNA (RAPD) and amplified fragment length polymorphic (AFLP) techniques. Simple boiling of P. syringae cells was suitable for subsequent DNA amplification to obtain reliable patterns in RAPD and AFLP analyses. In general, the grouping of P. syringae strains by both analysis techniques corresponded well with the classification obtained from an RFLP analysis of ribosomal DNA operons, DNA pairing studies, and an analysis of pathogenicity data. However, two strains of P. syringae pv. maculicola produced distinct DNA patterns compared to the DNA patterns of other P. syringae pv. maculicola strains; these patterns led us to assume that horizontal transfer of DNA could occur between bacterial populations. Both techniques used in this study have high discriminating power because strains of P. syringae pv. tomato and P. syringae pv. maculicola which were indistinguishable by other techniques, including pathogenicity tests on tomato, were separated into two groups by both RAPD and AFLP analyses. In addition, data analysis showed that the AFLP method was more efficient for assessing intrapathovar diversity than RAPD analysis and allowed clear delineation between intraspecific and interspecific genetic distances, suggesting that it could be an alternative to DNA pairing studies. However, it was not possible to distinguish the two races of P. syringae pv. tomato on the basis of an analysis of the data provided by either the AFLP or RAPD technique.

FIG. 1

Synthesis of 11-bp adaptors by digestion of annealed, symmetrical, 22-bp oligonucleotides with TaqI.

FIG. 2

Dendrograms showing the genetic relatedness of P. syringae and P. viridiflava strains based on RAPD and AFLP analyses. The trees were constructed by the neighbor-joining (NJ) method. A, D, M, N, and Q indicate genomic groups determined by PCR-RFLP analysis of the rrn operon (12), and sp. I, sp. II, sp. III, and sp. VI indicate the genomic species determined by DNA-DNA hybridization (18). The scale indicates the amount of difference. The numbers on the branches indicate the bootstrap percentage at each node. +, strain pathogenic on tomato; −, strain not pathogenic on tomato.

FIG. 3

Correlation between the genetic distances calculated with AFLP data and the genetic distances calculated with RAPD data. Data points indicate combinations of genetic distances calculated for pairs of isolates. A total of 27 strains were tested in the study (linear correlation, P < 0.0001, r² = 0.76).

FIG. 4

Histograms of intraspecific and interspecific genetic distances calculated with RAPD and AFLP data.