Using a modified multilocus sequence typing (MLST) scheme for the bacterial plant pathogen Xylella fastidiosa based on the same seven housekeeping genes employed in a previously published MLST, we studied the genetic diversity of two subspecies, X. fastidiosa subsp. fastidiosa and X. fastidiosa subsp. sandyi, which cause Pierce's disease and oleander leaf scorch, respectively. Typing of 85 U.S. isolates (plus one from northern Mexico) of X. fastidiosa subsp. fastidiosa from 15 different plant hosts and 21 isolates of X. fastidiosa subsp. sandyi from 4 different hosts in California and Texas supported their subspecific status. Analysis using the MLST genes plus one cell-surface gene showed no significant genetic differentiation based on geography or host plant within either subspecies. Two cases of homologous recombination (with X. fastidiosa subsp. multiplex, the third U.S. subspecies) were detected in X. fastidiosa subsp. fastidiosa. Excluding recombination, MLST site polymorphism in X. fastidiosa subsp. fastidiosa (0.048%) and X. fastidiosa subsp. sandyi (0.000%) was substantially lower than in X. fastidiosa subsp. multiplex (0.240%), consistent with the hypothesis that X. fastidiosa subspp. fastidiosa and sandyi were introduced into the United States (probably just prior to 1880 and 1980, respectively). Using whole-genome analysis, we showed that MLST is more effective at genetic discrimination at the specific and subspecific level than other typing methods applied to X. fastidiosa. Moreover, MLST is the only technique effective in detecting recombination.

b Documenting the role of novel mutation versus homologous recombination in bacterial evolution, and especially in the invasion of new hosts, is central to understanding the long-term dynamics of pathogenic bacteria. We used multilocus sequence typing (MLST) to study this issue in Xylella fastidiosa subsp. pauca from Brazil, a bacterium causing citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS). All 55 citrus isolates typed (plus one coffee isolate) defined three similar sequence types (STs) dominated by ST11 (85%), while the remaining 22 coffee isolates defined two STs, mainly ST16 (74%). This low level of variation masked unusually large allelic differences (>1% divergence with no intermediates) at five loci (leuA, petC, malF, cysG, and holC). We developed an introgression test to detect whether these large differences were due to introgression via homologous recombination from another X. fastidiosa subspecies. Using additional sequencing around these loci, we established that the seven randomly chosen MLST targets contained seven regions of introgression totaling 2,172 bp of 4,161 bp (52%), only 409 bp (10%) of which were detected by other recombination tests. This high level of introgression suggests the hypothesis that X. fastidiosa subsp. pauca became pathogenic on citrus and coffee (crops cultivated in Brazil for several hundred years) only recently after it gained genetic variation via intersubspecific recombination, facilitating a switch from native hosts. A candidate donor is the subspecies infecting plum in the region since 1935 (possibly X. fastidiosa subsp. multiplex). This hypothesis predicts that nonrecombinant native X. fastidiosa subsp. pauca (not yet isolated) does not cause disease in citrus or coffee.

The bacterial pathogen, Xylella fastidiosa, infects many plant species in the Americas, making it a good model for investigating the genetics of host adaptation. We used multilocus sequence typing (MLST) to identify isolates of the native U.S. subsp. multiplex that were largely unaffected by intersubspecific homologous recombination (IHR) and to investigate how their evolutionary history influences plant host specialization. We identified 110 "non-IHR" isolates, 2 minimally recombinant "intermediate" ones (including the subspecific type), and 31 with extensive IHR. The non-IHR and intermediate isolates defined 23 sequence types (STs) which we used to identify 22 plant hosts (73% trees) characteristic of the subspecies. Except for almond, subsp. multiplex showed no host overlap with the introduced subspecies (subspecies fastidiosa and sandyi). MLST sequences revealed that subsp. multiplex underwent recent radiation (<25% of subspecies age) which included only limited intrasubspecific recombination (/ ‫؍‬ 0.02); only one isolated lineage (ST50 from ash) was older. A total of 20 of the STs grouped into three loose phylogenetic clusters distinguished by nonoverlapping hosts (excepting purple leaf plum): "almond," "peach," and "oak" types. These host differences were not geographical, since all three types also occurred in California. ST designation was a good indicator of host specialization. ST09, widespread in the southeastern United States, only infected oak species, and all peach isolates were ST10 (from California, Florida, and Georgia). Only ST23 had a broad host range. Hosts of related genotypes were sometimes related, but often host groupings crossed plant family or even order, suggesting that phylogenetically plastic features of hosts affect bacterial pathogenicity.T he genetic typing of bacterial isolates is an increasingly important tool for understanding the epidemiology of pathogenic bacteria. It permits us to track biogeographical patterns, host specificity, and the evolutionary changes occurring within taxa. To achieve these goals, one of the most useful and widely used typing methodologies is multilocus sequence typing (MLST), introduced in 1998 (1, 2). The spread of this methodology has been rapid in the study of human bacterial pathogens; however, MLST schemes are equally important in the study of plant pathogens (3), and schemes for plant bacteria are beginning to become established, e.g., Pseudomonas syringae (4), Xylella fastidiosa (5, 6), fruit tree phytoplasmas (7), Ralstonia solanacearum (8), Xanthomonas spp. (9), and Acidovorax citrulli (10).The genetic typing of plant pathogens provides a critical first step in answering the important question of what genetic factors determine host specificity. Genome comparison among species and pathovars of Xanthomonas suggest specificity is determined by a complex set of genetic differences involving both gene content and gene sequence (11). Of these two kinds of difference, content and sequence, gene content differences are more easily studied, since it...

Invasive diseases present an increasing problem worldwide; however, genomic techniques are now available to investigate the timing and geographical origin of such introductions. We employed genomic techniques to demonstrate that the bacterial pathogen causing Pierce's disease of grapevine (PD) is not native to the US as previously assumed, but descended from a single genotype introduced from Central America. PD has posed a serious threat to the US wine industry ever since its first outbreak in Anaheim, California in the 1880s and continues to inhibit grape cultivation in a large area of the country. It is caused by infection of xylem vessels by the bacterium Xylella fastidiosa subsp. fastidiosa, a genetically distinct subspecies at least 15,000 years old. We present five independent kinds of evidence that strongly support our invasion hypothesis: 1) a genome-wide lack of genetic variability in X. fastidiosa subsp. fastidiosa found in the US, consistent with a recent common ancestor; 2) evidence for historical allopatry of the North American subspecies X. fastidiosa subsp. multiplex and X. fastidiosa subsp. fastidiosa; 3) evidence that X. fastidiosa subsp. fastidiosa evolved in a more tropical climate than X. fastidiosa subsp. multiplex; 4) much greater genetic variability in the proposed source population in Central America, variation within which the US genotypes are phylogenetically nested; and 5) the circumstantial evidence of importation of known hosts (coffee plants) from Central America directly into southern California just prior to the first known outbreak of the disease. The lack of genetic variation in X. fastidiosa subsp. fastidiosa in the US suggests that preventing additional introductions is important since new genetic variation may undermine PD control measures, or may lead to infection of other crop plants through the creation of novel genotypes via inter-subspecific recombination. In general, geographically mixing of previously isolated subspecies should be avoided.

cHomologous recombination plays an important role in the structuring of genetic variation of many bacteria; however, its importance in adaptive evolution is not well established. We investigated the association of intersubspecific homologous recombination (IHR) with the shift to a novel host (mulberry) by the plant-pathogenic bacterium Xylella fastidiosa. Mulberry leaf scorch was identified about 25 years ago in native red mulberry in the eastern United States and has spread to introduced white mulberry in California. Comparing a sequence of 8 genes (4,706 bp) from 21 mulberry-type isolates to published data (352 isolates representing all subspecies), we confirmed previous indications that the mulberry isolates define a group distinct from the 4 subspecies, and we propose naming the taxon X. fastidiosa subsp. morus. The ancestry of its gene sequences was mixed, with 4 derived from X. fastidiosa subsp. fastidiosa (introduced from Central America), 3 from X. fastidiosa subsp. multiplex (considered native to the United States), and 1 chimeric, demonstrating that this group originated by large-scale IHR. The very low withintype genetic variation (0.08% site polymorphism), plus the apparent inability of native X. fastidiosa subsp. multiplex to infect mulberry, suggests that this host shift was achieved after strong selection acted on genetic variants created by IHR. Sequence data indicate that a single ancestral IHR event gave rise not only to X. fastidiosa subsp. morus but also to the X. fastidiosa subsp. multiplex recombinant group which infects several hosts but is the only type naturally infecting blueberry, thus implicating this IHR in the invasion of at least two novel native hosts, mulberry and blueberry.