Twitching motility in XFM was lower than in either PD3 or PW as poor growth in XFM resulted in smaller colony sizes. Still the fringe widths of colonies in XFM without BSA were bigger than in XFM with BSA. Most of the colonies spotted in XFM and XFM-BSA showed very little or no visible growth. This can be expected as XFM is a nutrient-limited minimal medium. Moreover, the pglA-KmR mutant that did not show twitching movement was not competent when tested with heat-killed NS1-CmR mutant and plasmid DNA as the donor. These results of concomitant decreases in natural competence and twitching motility in BSA-supplemented media and non-competency of twitch minus strain suggest that twitching motility is correlated with natural competence in X. fastidiosa. Natural competence in other Gramnegative bacteria is mediated by type IV pili-like structures . In light of the effect of BSA on twitching, it remains to be determined if BSA only alters movement or biogenesis of type IV pili. Our results with different growth settings showed that the recombination frequency is significantly higher in the MC_in fraction than in the MC_out fraction. The MC_in environment closely mimics xylem vessels and the insect foregut with respect to continuous liquid flow, adhesion of cells on channel walls in a fashion similar to adhesion of cells on xylem vessels and the insect foregut,growing strawberries vertically and formation of biofilms. This environment is conducive for both biofilm formation and twitching motility as demonstrated in previous studies .
Moreover, expression levels of some of the type IV pili genes were shown to be increased in the MC_in environment compared to those under the other growth conditions , implying that activity of type IV pili is increased in this system, which may explain the higher rates of recombination in the MC_in fraction. The MC_out environment, on the other hand, consists mostly of planktonic cells and some detached biofilm fraction from MC_in, which is washed away with the liquid flow. The differences in recombination frequencies in these two environments suggest that the continuous media flow condition of the xylem vessels and growth in biofilm may increase the chances of recombination. Batch cultures in tubes also allowed recombination but at a lower rate than the continuous flow environment of MC_in and surface-attached condition of solid agar plates. A previous study also showed that growth in solid plates increases recombination compared to the growth in the liquid culture tubes .Recombinantsin theMC_outfraction were recovered when profuse biofilm growth was observed in the MC_in fraction with many recombinants formed. It is possible that the recombinants recovered in the MC_out fraction are due to detachment and washing away of portions of biofilms from the MC_in fractions, supporting the proposition that biofilm formation induces competence. Biofilm formation and quorum sensing signals have been shown to induce natural competence in other naturally competent bacteria such as Vibrio cholerae, Acinetobacter sp. , and Streptococcus mutans . Biofilms, in addition to having dense populations of cells, contain elevated amounts of extracellular DNA , which can be used for transformation by competent cells.
Moreover, Kung et al. also showed that a knockout mutant on a bio-synthetic gene for diffusible signaling factor , a cell-cell communication signal in X. fastidiosa, had a reduced rate of recombination, implying that a cell-cell communication signal also may be involved in regulating natural competence in X. fastidiosa. MC experiments with grapevine sap provide a closer resemblance to the natural habitat than MCs with the artificial culture medium. Previously, we have shown that the biofilm structure in grapevine sap is more similar to the natural biofilm than are the aggregates observed in synthetic medium inside MCs . The experiments with amendments of sap in the MCs detected natural competence, providing an indication that natural competence occurs in the xylem vessels of host plants and possibly in the insect vectors. Although the results with pure sap experiments were not reproducible due to inconsistent growth of one of the strains used, recombinants were recovered once with pure Chardonnay sap as the medium. Recombinants were readily recovered with the 50% sap in PD3 for both tolerant and susceptible varieties. Maintenance of competence with the addition of xylem sap indicates that sap components support DNA acquisition and transformation. Natural competence occurring in environments resembling natural habitats also have been demonstrated in other naturally competent bacteria such as P. stutzeri and V.cholerae, in which artificial medium resembling natural soil extract and natural growth substrate , respectively, induced competence. In R. solanacearum, another xylem-colonizing plant pathogen, natural competence has been demonstrated in planta , and the recipient strains were shown to have increased virulence, acquiring DNA regions as long as 40 kb from donor strains.
Findings from competence experiments with grapevine sap and the MCs suggest that when two different strains are established together in the xylem vessels or in the vector foregut, recombination is possible. Noteworthy is the fact that in the experiments reported here, recombination was higher with sap from a tolerant grapevine variety, where infection by X. fastidiosa is symptomless. Coinfection by two genetically different isolates together in the same plant has been documented before , and there are reports of artificial mixed infection of a vector and of a single vector being able to transmit all four subspecies of X. fastidiosa . Moreover, it was shown that isolates from two different subspecies can cause disease in a single host . Hence, the possibility that two different X. fastidiosa strains may encounter one another and exchange DNA, as shown by MLST analyses, exists in nature. Donor DNA may be derived from dead cells or may be secreted by a type IV secretion system, as shown in N. gonorrhoeae . Moreover, the experiment with heat-killed donor cells suggests that recombination is possible if homologous DNA fragments are present in the environment. Although the majority of recombination events will not be beneficial to the recipient cell, some may have adaptive advantages and increased virulence, among other phenotypes under selective pressure. For example, the relatively recent emergence of citrus variegated chlorosis and coffee leaf scorch in South America is proposed to be due to intersubspecific recombination between a X. fastidiosa subsp. multiplex donor and an unidentified native recipient based on MLST . In addition, strains that are classi- fied in the newly proposed subspecies, Xylella fastidiosa subsp. morus, that infects mulberry, have been suggested to be generated by recombination between an X. fastidiosa subsp. fastidiosa donor and an X. fastidiosa subsp. multiplex recipient . A similar mechanism may have resulted in strains that infect blueberry and blackberry . The recombination events observed in this study are based on horizontal acquisition of antibiotic resistance markers ,drainage planter pot which represent a small fraction of the genome of X. fastidiosa. Since the natural competence experiments were performed under conditions without any selective pressure, recombination events should be expected to have occurred at other regions of the genome as well but were not detected due to the experimental approach used here. Under the simplistic assumption that gene exchange occurs randomly throughout the genome and with similar frequencies at all loci, the recombination frequencies reported in this study for one locus could be as much as 2.5 103 higher, considering the size of the X. fastidiosa genome . In summary, X. fastidiosa is naturally competent with a high rate of recombination when cultured under the liquid flow conditions of the MC system, which mimics plant xylem vessels and the insect vector foregut. Natural competence in the MCs was maintained even when the medium was supplemented with grapevine xylem sap, suggesting that the natural habitat of X. fastidiosa supports natural competence. Moreover, habitats and media that favored increased biofilm growth and increased twitching motility showed increased rates of recombination. This study advances the characterization of the phenomenon of natural competence in X. fastidiosa that needs to be further studied to understand the evolution and adaptation of this important plant pathogen.Over the past decade, Drosophila suzukii , also known as the spotted-wing drosophila or the Asian vinegar fly, has become an incredibly invasive pest species and a threat to softskinned fruit agricultural production worldwide . Unlike the large majority of Drosophilidae , which preferentially breed in decaying plant material, female D. suzukii possess a serrated ovipositor, enabling them to lay eggs in fresh ripening soft-skinned fruits .
First described in Japan as an agricultural pest of cherries, D.suzukii was primarily distributed across East Asia until researchers found wild specimens in Hawaii in 1980 . In 2008, D. suzukii was detected in California, and by 2009 was widespread across the Western US coast . In the Eastern United States, D. suzukii first appeared in Florida in 2009 , before again rapidly spreading across the entire east coast within a few years. Meanwhile in Europe, D. suzukii was first detected in Spain and Italy in 2008 and rapidly spread across Europe, appearing in France, Switzerland, Austria, Germany, and Belgium by 2012. Subsequently, D. suzukii arrived in South America when it was detected in Brazil in 2013 , Argentina in 2014 , and Chile in 2015 . Its rapid spread across continents suggests that human transportation is likely a major factor, as eggs laid in fresh fruit are difficult to detect before shipment. Once established in a new continent, D. suzukii rapidly disperse to neighboring regions, aided by its ability to adapt to a wide range of climates through phenotypic plasticity . In the Western US coastal states alone, estimated economic losses were as high as 511 million dollars per year, assuming a 20% average yield loss . Thus, there is much interest in understanding the patterns of migration and origin of these invasive populations, as these data can be used to inform shipping and quarantine policies and to identify routes of entry. Previous research on the population genomics of D. suzukii was performed using a relatively small number of molecular markers. Adrion et al. used six X-linked gene fragments from flies collected across the world, and detected signals of differentiation between European, Asian, and US populations. However, they found no evidence of differentiation within the 12 US populations sampled, possibly due to the limited power provided from a small number of markers. A follow-up study using 25 microsatellite loci of samples collected between 2013 and 2015 greatly improved estimations of migration patterns worldwide; the authors found evidence for multiple invasion events from Asia into Europe and the United States as well as an East–West differentiation in the seven populations sampled in the continental United States . However, using microsatellites alone may miss more subtle signals of population structure compared with genome-wide datasets, as increasing the number of independent loci genotyped increases accuracy of population parameter estimates, even when the number of biological samples is low . With the advent of affordable whole-genome sequencing , it has become feasible to sequence hundreds of individuals to study population genomics, enabling improved inference of population structure using hundreds of thousands to millions of single nucleotide polymorphism markers . A study of D. suzukii in Hawaii used double digest restriction-site-associated DNA sequencing to identify several thousand SNPs and observed population structure between islands . However, a comprehensive survey of D. suzukii in the continental United States using a large number of SNPs enabled by WGS has not been conducted. In this study, we leverage the power of WGS to individually sequence hundreds of D. suzukii samples to determine whether U.S. populations are stratified along a north-south cline corresponding to varying winter climates, as well as to detect whether migration is freely occurring between the Eastern and Western United States. In addition, we include several populations from Asia, Europe, and Brazil to determine frequency and source of international migrations and compare genetic diversity between invasive and native populations. We expect these analyses and the large sequencing dataset will be of value in developing policies and furthering research into mitigating the harmful effects of D. suzukii worldwide.We received either flash-frozen or ethanol-preserved samples of D. suzukii for genomic analysis. Japanese samples were obtained from the Ehime Japanese Stock Center . Hawaiian samples were taken from a small laboratory population maintained in vials that was established in 2009 from wild-caught samples in Oahu, Hawaii. All other samples were field-collected. Ethanol-preserved samples were re-hydrated in 100-lL water prior to DNA extraction.