The IHR and intermediate types together were considered to define the “recombinant” group of X. fastidiosa subsp. multiplex isolates . Most were observed more than once, and 5 were found in two different U.S. states or districts . The analysis of Nunney et al. was focused on the evolution and host range ofX. fastidiosa subsp. multiplex. For this purpose, it was necessary to identify and exclude isolates whose recent evolution was influenced by inter sub-specific recombination. As such, once the 23 non-IHR STswere identified, there was no further analysis of the remaining recombinant group STs. In particular, no evidence was presented for classifying some alleles as atypical of X. fastidiosa subsp. multiplex beyond the observation that they were never found in the non-IHR group . Nunney et al. did observe one intriguing pattern when they compared their results to those of Parker et al. . Of the 143 isolates, 13 were also used in the study by Parker et al. , in which typing was based on a different set of 9 loci. Unexpectedly, these 13 isolates maintained the same grouping with the IHR and non-IHR types corresponding, respectively, to the clade A and clade B groupings . This highly statistically significant concordance strongly suggested that IHR is not distributed randomly across all X. fastidiosa subsp. multiplex isolates but instead is restricted to a small subset, while the remainder is little influenced by IHR. However, Parker et al. failed to find evidence of inter sub-specific recombination within any of the X. fastidiosa subsp. multiplex isolates, despite applying a series of 9 tests designed to detect recombination contained within the RDP4 program and the PHI program . This result presented a strong argument against our hypothesis that clade A members cluster because they are recombinant types carrying alleles derived from IHR . Here we reexamined the sequence data obtained in their study by using the more sensitive introgression test to determine if their tests missed evidence of IHR and, if so, whether it was confined to clade A.

In particular,plastic planters bulk what could be concluded about the origin of the group given the observation from 2 independent studies that the members appear to form a well-defined cluster of genotypes? Third, we used the sequence data to examine the hypothesis that the introgressed DNA was from X. fastidiosa subsp. fastidiosa, the subspecies that causes Pierce’s disease. X. fastidiosa subsp. fastidiosa is native to Central America, and all known isolates in the United States and northern Mexico can be traced back to a single introduced genotype . IHR would be of limited interest if it simply randomized the genetic differences among the subspecies but had a minimal effect on pathogenesis. For this reason, we were particularly interested in documenting any possible invasion of new plant hosts associated with IHR. The hypothesis is that IHR creates a range of novel genotypes that are far more variable than can arise from a lineage diversifying through point mutations, and this diversity facilitates adaptive evolution of a kind not possible for a clonal lineage. This kind of probabilistic evolutionary hypothesis can rarely be directly proven based on an individual case; however, it makes predictions that, if generally supported, would cause the hypothesis to be accepted. In the case of X. fastidiosa, compelling evidence supporting the hypothesis would be the invasion of a new native host plant that is uniquely associated with IHR. Our data support this hypothesis: in X. fastidiosa subsp. multiplex, IHR is indeed associated with the invasion of at least 2 new native plant hosts, blueberry and blackberry.To investigate inter sub-specific homologous recombination , we analyzed 31 isolates previously identified as IHR-type and 2 isolates previously identified as intermediate-type X. fastidiosa subsp. multiplex , based on sequence of the 7 housekeeping loci used in the MLST scheme defined by Yuan et al. plus a region of the pilUgene. Together, these 33 isolates made up the recombinant group. Details regarding the isolation and typing of the 33 isolates were provided by Nunney et al. , and a summary of salient features is provided in Table S1 in the supplemental material in that article. All sequences used have previously been published and are available both in GenBank and the MLST website . To detect IHR, we employed a modified version of the introgression test developed by Nunney et al. .

In its original form, the test compares a set of target sequences, some of which may have been involved in IHR, to a set of potential donor sequences. Each variable site is classified as F, a fixed difference between the target sequences and the donor sequences, or P, a polymorphic site within the target sequences where at least one variant base is shared with the donor set. A significant shift in the ratio of F to P marks a recombination break point. In the modified version of the test, the targeted introgression test, the target sequence is known a prioriand is compared to two references, the donor group, D , and the ancestral group, A . The minimum number of nucleotide differences between the target and the two references defines a ratio of D to A equivalent to the ratio of F to P and can be tested in the same way . In some cases, there is no break point because the whole locus appears to be an introgressed sequence . Although the signal of introgression across the entire sequenced region may be clear, it is valuable to have a statistical test that documents the strength of the signal. In this case, the null expectation is the ratio that reflects the pairwise differences between the donor and ancestral group versus the pairwise differences within the ancestral group . We used this ratio to define the expectation of the D/A ratio for a chi-square test of complete introgression. Gene diversity and distance trees were calculated using MEGA5 , and the maximum parsimony tree was created using the PARS program in Phylip . Distance trees and the maximum parsimony tree were used rather than other methods, given the known occurrence of inter sub-specific recombination in the data. Clonal Frame was used to provide an independent estimate of the relative importance of recombination versus mutation in the recombinant group.Analysis of the recombinant group ofX. fastidiosa subsp. multiplex showed three important results. First, inter sub-specific recombination was shown to have occurred in 50% of 8 loci scattered throughout the genome that were chosen independently of the data . Second, it was shown that the donor of the introgressed sequence was X. fastidiosa subsp. fastidiosa, a subspecies introduced from Central America into the United States as a single strain .

However,collection pot the introgressed sequence at two of the loci did not come from any of the X. fastidiosa subsp. fastidiosa genotypes that have been found in the United States. This result suggests that another introduction of X. fastidiosa subsp. fastidiosa must have occurred, an introduction that resulted in successful IHR, after which the donor genotype seems to have disappeared. This involvement of an unexpected X. fastidiosa subsp. fastidiosa strain supports the hypothesis that the members of the recombinant group share a single ancestral IHR event. Third, the hypothesis that IHR has facilitated a shift to new hosts is strongly supported by the example of blueberry, where 10 isolates have been typed and potentially supported by the example of blackberry . A link between the shift to a novel plant host and homologous recombination has not been previously identified. Of course, the direct causation of this link can never be proved without knowledge of the genetic changes driving this shift. It can always be argued that the link is fortuitous and that one or more point mutations in the nonrecombined X. fastidiosa subsp. multiplex genome are causal in the host shift. Arguing against this possibility are 2 additional pieces of information. First, both blueberry and blackberry are native to the United States, so if only a simple genetic change was required to infect these species, why did the native nonrecombinant X. fastidiosa subsp. multiplex apparently never acquire these changes? Second, a similar but even more extensive mixing of the genomes of X. fastidiosa subspp. fastidiosa and multiplex is found in the only form of X. fastidiosa that infects another U.S. native plant, mulberry . Furthermore, in other bacterial species, it has been demonstrated that recombination can drive rapid evolution, both in the laboratory and, in the case of Helicobacter pylori, in mice . Similarly, McCarthy et al. concluded that lineages of Campylobacter jejuni in chickens versus cattle and sheep were able to shift host type, because rapid adaptation was facilitated by recombination with the resident host population. In the study by Nunney et al. , it was shown that the recombinant genotypes formed a well-defined group , demonstrating that inter sub-specific homologous recombination was not randomly distributed across the X. fastidiosa subsp. multiplex isolates. This work was based on a survey of 143X. fastidiosa subsp. multiplex isolates using just 8 loci. There were 33 isolates that showed some evidence of IHR in at least 1 locus: all but 2 showed statistically significant evidence in at least 2 loci, while the remaining 110 showed no such evidence . The generality of this discrete group of recombinant forms was supported by our analysis presented here of the sequence data from 9 more loci sequenced by Parker et al. . These loci divided isolates into 2 groups that appeared to correspond to the recombinant and non-IHR groups, respectively , even though Parker et al. found no evidence of IHR. Upon reanalysis, we found statistically significant IHR in 6 of the 9 loci in the clade A data but no evidence of IHR in the clade B data. Clade A included 6 isolates that we had typed in the present study, and each of these showed evidence of IHR in 4 or 5 of the additional 9 loci. Thus, in two independent samplings that together examined 17 loci, there was clear evidence of substantial genome wide IHR in the recombinant group isolates, amounting to 50% of the genes showing IHR across the MLST locis plus the pilU locus .

The average was higher when based on the loci sequenced by Parker et al. ; however, this was probably biased upwards by the manner in which the loci were chosen . None of the IHR events in 6 of the 9 loci identified using the targeted introgression test, or in the case of complete introgression, a chi-square test, were detected by Parker et al. using PHI and the 9 tests implemented in RDP . This failure of the standard tests of recombination to detect IHR was previously noted by Nunney et al. , motivating the development of their introgression test. We examined the hypothesis that the recombinant group STs were derived from a single IHR event involving a X. fastidiosa subsp. multiplex recipient and an X. fastidiosa subsp. fastidiosa donor. The distribution of allelic differences among the recombinant STs was consistent with them all being derived from a single initial event, but a small number of other inter sub-specific and intrasubspecific recombination events would also be needed . More importantly, the genotypes seen in the recombinant group can be accounted for entirely, or very nearly so, based on a single X. fastidiosa subsp. fastidiosa donor genotype. For example, the substantial variation in cysGcan all be accounted for by an ancestral introgression of X. fastidiosa subsp. fastidiosa allele 12 followed by subsequent intrasubspecific recombination of X. fastidiosa subsp. multiplex sequence to form the other two alleles . In contrast, variation at pilU could be accounted for by a second donor contributing the X. fastidiosa subsp. fastidiosa pilU9 allele, but it could also have arisen by a single mutation in pilU1 unique to the recombinant group. A possible single X. fastidiosa subsp. multiplex recipient genotype was also identified . This genotype is consistent with a known ST: setting cysG to allele 3 makes the recipient identical to ST45, which was sampled from the states of California, Kentucky, and Texas . Elsewhere, we consider a slightly different hypothesis regarding the origin of the recombinant group in which the donor and recipient subspecies are reversed—i.e., that it was derived from a single IHR event, but involving an X. fastidiosa subsp. multiplex donor and an X. fastidiosa subsp. fastidiosa recipient; however, apart from the role reversal, the conclusions are unaltered .