The possible benefits remain largely speculative, since adaptive consequences of recombination between close relatives are difficult to detect. Adaptive shifts may be important in maintenance of transformation , and there is some evidence that homologous recombination may be important in promoting a number of adaptive responses. These include maintaining variability of surface proteins in animal pathogens to avoid host defenses , transfer of virulence genes in plant-pathogenic bacteria , and evolution of new taxa by, for example, facilitating adaptation to novel hosts . Homologous recombination can most easily be studied when genetic transfer has occurred between genetically distinct but closely related taxa. Under such circumstances, it may be possible to detect not only recombinant events but also sources of incorporated DNA and, potentially, to identify adaptive consequences of the exchange. This scenario is found in the plant-pathogenic bacterium Xylella fastidiosa, black flower buckets where intersubspecific homologous recombination is well documented .
X. fastidiosa infects xylem vessels of a wide range of plant host species in the Americas . X. fastidiosa has been divided into four subspecies, three of which are found in the United States . These groups are genetically distinct, with values of DNADNA hybridization between them of less than 70% , sequence differences of 2% or more at synonymous sites , and distinct 16S rRNA gene and 16S-23S rRNA gene spacer sequences . These differences reflect estimated divergence times of more than 15,000 years . Furthermore, each subspecies has a distinct and largely nonoverlapping set of plant hosts : in the UnitedStates, X. fastidiosa subsp. fastidiosa causes Pierce’s disease of grape, X. fastidiosa subsp. sandyi causes oleander leaf scorch, and X. fastidiosa subsp. multiplex causes leaf scorch disease on a range of trees, including oak, elm, and peach. In South America, X. fastidiosa subsp. pauca infects citrus and coffee . X. fastidiosa is competent for transformation , and some isolates carry conjugative plasmids , so sympatry of subspecies potentially creates conditions conducive for both the occurrence and detection of IHR. Sympatry of X. fastidiosa subspecies appears to be relatively recent: while X. fastidiosa subsp. multiplex is probably native to the United States, there is compelling evidence that the other two subspecies found in the United States were introduced . X. fastidiosa subsp. sandyi has been known in the United States for only about 30 years, while X. fastidiosa subsp. fastidiosa has presumably been present since the first known outbreak of Pierce’s disease ca. 130 years ago.
Furthermore, it appears that a similar situation exists in South America. While X. fastidiosa subsp. pauca is native to South America, there is evidence of the introduction of a second subspecies into Argentina and/or Brazil causing plum leaf scald, first observed in 1935 . Analysis of sequences indeed demonstrated large-scale recombination of X. fastidiosa subsp. fastidiosa sequences into X. fastidiosa subsp. multiplex in the United States , and, in Brazil, there has been substantial recombination into X. fastidiosa subsp. pauca of sequences from a distinct taxon, tentatively identified as X. fastidiosa subsp. multiplex . However, large-scale introgression is not the rule. Analyses of genomes of U.S. isolates of X. fastidiosa subsp. fastidiosa show very limited introgression of X. fastidiosa subsp. multiplex ; moreover, large-scale introgression into X. fastidiosa subsp. multiplex is restricted to a well defined set of genotypes, suggesting that it may have been initiated by very few events . The majority of X. fastidiosa subsp. multiplex isolates show little evidence of IHR, and the data available suggest that even intrasubspecific recombination is limited . Thus, the picture emerging in X. fastidiosa is one of limited successful homologous recombination on a short time scale, with bursts of large-scale exchange occurring very infrequently. This raises the possibility that, by substantially increasing the available genetic variability, these large-scale events facilitate rapid evolutionary change that can result in colonization of new plant hosts.
This scenario has been proposed as the mechanism underpinning the invasion of blueberry by recombinant forms of X. fastidiosa subsp. multiplex and, more speculatively, the infection of citrus and coffee by X. fastidiosa subsp. pauca in Brazil . Another candidate for this scenario is the form of X. fastidiosa infecting mulberry, a form that does not appear to fit within the framework of the four subspecies so far identified. Kostka et al. first observed the disease of mulberry leaf scorch in the Washington, DC, area on the native red mulberry , and further study revealed infected trees along the east coast as far north as New York City, NY. Since that time, MLS has been observed in Nebraska and also in California on the introduced white mulberry . Previous genetic assessment showed that, although the 16S rRNA gene sequence of mulberry isolates is consistent with that of X. fastidiosa subsp. fastidiosa, based on analyses of randomly amplified polymorphic DNAs and 16S-23S rRNA gene spacer sequences, these types cluster as a distinct group . Here we used multilocus sequence typing to evaluate the genetic relationship of the mulberry isolates to the 4 subspecies and to establish its hybrid ancestry via IHR, supporting the hypothesis that IHR facilitates host shifts. We show that this ancestry is shared with the recombinant group of X. fastidiosa subsp. multiplex; however, the recombinant group has largely introgressed into X. fastidiosa subsp. multiplex, and we propose that the continued genetic distinctiveness of the mulberry type merits recognition as a new subspecies, X. fastidiosa subsp. morus.Isolates used. The study examined DNA sequences from 20 isolates from mulberry, Morus spp. , plus 1 isolate from heavenly bamboo, Nandina domestica, known to be genetically similar . These 21 isolates, referred to as a group as the mulberry isolates, included examples from California, Kentucky, and Washington, DC . They were all typed using the MLST scheme developed for X. fastidiosa and using 7 housekeeping loci . A sequence from one additional locus, the cell surface gene pilU, was also obtained. Genetic relationships of the isolates within X. fastidiosa. The DNA sequences were compared to previously published MLST and pilU data from 352 isolates of X. fastidiosa, defining 65 sequence types , where an ST is a unique genotype based on the MLST, as follows: 110 isolates ofX. fastidiosa subsp. fastidiosa , 21 isolates of X. fastidiosa subsp. sandyi , 143 isolates ofX. fastidiosa subsp.multiplex , and 78 isolates of X. fastidiosa subsp. pauca from Brazil . To show the genetic relationship of the isolates from mulberry to all previously published STs, we created a distance tree using a concatenation of the 8 sequenced loci for all of the known STs using the PHYLIP programs DNADIST and NEIGHBOR . In the analysis, the two known indels were given weights equivalent to 1 and 3 transversions, respectively. A distance tree was used since the IHR involved in the history of X. fastidiosa made a phylogenetic tree inappropriate; however, a maximum-likelihood phylogenetic tree was evaluated for completeness and comparison. Using maximum parsimony, we tested the hypothesis that the STs defined by the mulberry isolates were related to the STs of the recombinant-group X. fastidiosa subsp. multiplex though an ancestral introgression of X. fastidiosa subsp. multiplex into X. fastidiosa subsp. fastidiosa, with subsequent divergence due to additional X. fastidiosa subsp. multiplex introgression. We created a maximum-parsimony tree using the PARS program in PHYLIP, with each allele as a character.
Since our hypothesis emphasized a single ancestral X. fastidiosa subsp. fastidiosa strain, french flower bucket with potentially multiple X. fastidiosa subsp. multiplex donors, we weighted the loci that included alleles containingX. fastidiosa subsp. fastidiosa sequence more than those loci identified as having only X. fastidiosa subsp. multiplex sequence. We used a 2-fold weighting scheme; higher values produced identical results. The collection of equally parsimonious trees was reduced by applying the assumption that all nonrecombinant X. fastidiosa subsp. fastidiosa alleles absent from the current U.S. population of X. fastidiosa subsp. fastidiosa had been vertically transmitted through the tree. We evaluated the origins of all alleles found in the mulberry isolates using two tests. For chimeric alleles, we tested for recombination breakpoints using the targeted introgression test . In most cases, the alleles were not chimeric and their ancestry was obvious; however, in each case a ratio test was used to support this conclusion .We examined the plausibility of this hypothesis through parsimony analysis, using alleles as characters, and the result is broadly supportive of the idea of a common origin of the mulberry-type and recombinant-group STs . The initial analysis produced 7 equally parsimonious trees, but by assuming that the four X. fastidiosa subsp. fastidiosa alleles inconsistent with the current U.S. strains were themselves ancestral , the total was reduced to 2 trees. The only difference between these two trees involved the position of the clade corresponding to ST27, ST28, and ST40. In one tree , the required recombination transfer of cysG allele 18 is minimized to one event ; alternatively, in the second tree, the clade branches from the ST58 lineage, which removes the necessity of a recombination transfer of leuA allele 6 , which is an X. fastidiosa subsp. multiplex allele unique to the recombinant group. Consistent with the common-origin hypothesis, parsimony requires very little postorigin modification within X. fastidiosa subsp. morus. Specifically, it requires only the basal acquisition of cysG allele 18, derived by recombination from presumed ancestral cysG allele 12 . All other allelic changes are single base substitutions. The genesis of the recombinant group is more complex, consistent with the conclusions of Nunney et al. and with the assumption of a history of continued introgression. Thus, the data suggest that the recombinant group has undergone sufficient additional recombination with X. fastidiosa subsp. multiplex that it has ceased to be a separate taxon. On the other hand, the mulberry isolates show no evidence of such introgression and thus have remained a distinct taxon meriting subspecific status. To this point, there is no evidence of intermediate genotypes that bridge the genetic space that now exists between the recombinant group and X. fastidiosa subsp. morus . Nunney et al. previously proposed that IHR-generated genetic variation facilitated invasion of new hosts, based on the observation that all isolates from blueberry were recombinant-group X. fastidiosa subsp. multiplex. This hypothesis is further supported by the invasion of mulberry by the chimeric X. fastidiosa subsp. morus. These examples raise three additional points of support. First, given the long-term geographical association of the native X. fastidiosa subsp. multiplex with these 3 native host plants, the failure to infect them suggests that the genetic variation required for successful invasion had been absent from the native subspecies. Second, contact of these plant hosts with two newly introduced subspecies has failed to lead to infection of these plants; in all known cases of natural infection, these hosts were infected only by STs that had undergone large-scale IHR. Third, in each case, the STs found on these hosts show very little variation: blackberry, 1 ST; blueberry, 2 STs; and mulberry, 4 STs. This lack of within-host variation is consistent with host plants imposing strong host-specific selection on the bacterial genome. The data also suggest that host specificity is not determined by the lateral gene transfer of novel genetic material, since this would not impose the observed constraint on the genome. In addition, a similar pattern has been found in X. fastidiosa subsp. pauca in Brazil : evidence of large-scale IHR, combined with very limited genetic variation. From a sample of 55 citrus and 23 coffee isolates, only five STs were observed, with 85% of the citrus isolates having the same ST. The data from X. fastidiosa show that massive recombination can occur between subspecies. We see this in the creation of X. fastidiosa subsp. morus, and a similar event may have been involved in the genesis of the X. fastidiosa subsp. pauca strain that infects citrus and coffee in South America . But how did this happen? It has been established that conjugative plasmids can occur in X. fastidiosa , including a candidate found in the mulberry type . Furthermore, high rates of transformation have been observed in the laboratory . Which of these processes is involved in large-scale genomic exchange is not known. These data raise a second issue: how, given the clear potential for genetic exchange, X. fastidiosa subsp. morus and also the ancestral X. fastidiosa subsp. multiplex and X. fastidiosa subsp. fastidiosa strains have not introgressed into an ill-defined network of isolates.