Recent evolutionary radiation and host plant specialization in the Xylella fastidiosa subspecies native to the United States

Abstract

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 (ρ/θ = 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.

Fig 1

Distance tree based on the concatenated MLST and pilU sequence data showing the relationship of the 23 non-IHR X. fastidiosa subsp. multiplex STs, i.e., those with no evidence of IHR (110 isolates), to the 1 intermediate ST (2 isolates) and 8 IHR X. fastidiosa subsp. multiplex STs (31 isolates). The multiplex group (shown within the rectangle) was rooted using all published STs of X. fastidiosa subsp. fastidiosa (including those from Costa Rica [CR]), X. fastidiosa subsp. sandyi, and X. fastidiosa subsp. pauca (all from Brazil), with the ST of the type indicated (see references and 22). For X. fastidiosa subsp. multiplex, the number of isolates represented by each ST (i.e., xN indicates N isolates for N > 1) is shown, as are the bootstrap values >70%. *, ST22 was represented by three isolates with pilU allele 1 (as shown), plus one additional isolate carrying pilU allele 9 that differs by 1 bp, which maps almost identically on the tree but is not shown.

Fig 2

Maximum-likelihood phylogeny of X. fastidiosa subsp. multiplex independent of intersubspecific introgression, with outgroup representatives from subsp. fastidiosa (from the United States and Costa Rica [CR]) and subsp. sandyi (from the United States). The tree was rooted using representatives of subsp. pauca (not shown). The MLST sequence data used were from 110 nonrecombinant subsp. multiplex isolates (23 STs) from across the United States. For the groups of subsp. multiplex STs indicated, all isolate locations (by state) are shown, plus representative plants hosts (see the text for further detail of hosts). The number of replicates and bootstrap values are shown as in Fig. 1, plus some relevant estimates of evolutionary times. Kyrs, 1,000 years.

Fig 3

Minimum estimate of intrasubspecific recombination among the non-IHR subsp. multiplex STs making up CC1(thus excluding ST50, the only member of CC2) illustrated using the maximum-likelihood tree from Fig. 2. Using presumed ancestral allelic states of the seven MLST loci (A = leuA, C = petC, F = malF, G = cysG, H = holC, L = nuoL, and T = gltT), all points of allelic change in the tree are shown. STs that appear to have acquired an allele by recombination are identified by indicating the recombining allele followed by an exclamation mark (!). In the case of the cysG allele 19, the donor versus recipient ST cannot be identified (hence, there is a “?” showing both). All other allelic changes are due to single-base-pair substitutions.