Twitching experiments were performed at least three times independently for PD3 and PW with and without BSA and once for XFM with and without BSA.Several hypotheses have been proposed to explain the existence of natural competence in bacteria. One explanation is that starvation signals induce competence, and the incoming DNA serves as a nutrient source under poor nutrient conditions as demonstrated in H. influenzae , Pseudomonas stutzeri , and R. solanacearum . Based on the results with a minimal medium and a rich undefined medium , a previous study speculated that growth in a low-nutrient medium favors natural competence inX. fastidiosa. However, the results of this study with these two media and PD3, another undefined rich medium, demonstrated that growth in PD3 significantly increases the recombination frequency. This suggests that starvation is not necessary to induce competence in X. fastidiosa. Further investigations of the differences between PD3 and PW were performed by either removing or adding these components to/from one another. Initial screening with the components showed a pronounced effect of BSA on the number of recombinants recovered. Additional experiments confirmed that BSA significantly reduces the recombination frequency when present in PD3, PW, and XFM. Since both XFM and PW contain BSA, blueberry grow pot this may explain the lower recombination frequencies in these media. In a previous study, BSA had been found to reduce the surface attachment and twitching motility of X. fastidiosa .

In fact, natural competence and twitching motility are dependent on the activity of type IV pili in X. fastidiosa . Therefore, in this study the correlation between twitching movement and natural competence in different media was investigated. interestingly, PD3 allowed the highest fringe width, and the presence of BSA significantly reduced twitching motility in all three media. 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, 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 the MC_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 othernaturally 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. Extracellular DNA has been shown to enhance biofilm formation in X. fastidiosa . Moreover, Kung et al. also showed that a knockout mutant on a biosynthetic 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 , hydroponic bucket 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 . The recombination events observed in this study are based on horizontal acquisition of antibiotic resistance markers , 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.Since domestication efforts began in the early 1900s, high bush blueberry has rapidly become a high-value fruit crop worldwide. high bush blueberry, compared to hundreds of closely related blueberry species in the Ericaceae, is widely cultivated due to its adaptation to temperate climates, excellent fruit quality, yield, and composition of phytonutrients. As a result for the demand for fresh blueberries as a ”superfruit” , high bush blueberry production has increased 600% during the past three decades and steadily grown to a multi-billion dollar industry. In addition to its short domestication history, high bush blueberry is unique in being one of only three major commercially valuable fruit crops, accompanied by cranberry and the garden strawberry, with wild progenitor species native to North America. Blueberries have a single epidermal layer that expresses a rich profile of anthocyanins during ripening that, in combination with epicuticular wax, generates its characteristic ”powdery blue” color. The cuticular and epidermal layers contain nearly all of the phytonutrients in the fruit such as anthocyanins, proanthocyanidins, and flavonols. Previous studies on blueberry have reported that these groups of compounds may have diverse health-promoting properties, including controlling diabetes, improving cognitive function, and inhibiting tumor growth . With the growing awareness of the potential health benefits of blueberry and increasing consumer demand, a primary goal of the blueberry research community is to develop cultivars with improved antioxidant levels along with other important fruit quality traits. However, despite its economic importance and health benefit potential, breeding efforts to improve fruit quality traits in blueberry have been slow due, in large part, to the lack of genomic resources. A draft genome for a wild diploid species of blueberry was previously assembled. However, that draft genome consists of a large number of scaffolds , high percentage of gaps in a ∼393.16 Mb assembly, and, most importantly, does not reflect the genome complexity of the economically important and cultivated tetraploid high bush blueberry. Here, we present the first chromosome-scale genome assembly of tetraploid high bush blueberry. The haplotype-phased assembly consists of 48 pseudomolecules with ∼1.68 Gb of assembled sequence, ∼1.29% gaps, and an average of 32,140 protein coding genes per haplotype . A haplotype is the complete set of DNA within the nucleus of an individual that was inherited from one parent. We leveraged this genome to examine the origin of the polyploid event, gain insights into the underlying genetics of fruit development, and identify candidate genes involved in the biosynthesis of metabolites contributing to superior fruit quality. Furthermore, we examined gene expression patterns among the four haplotypes in high bush blueberry. This analysis uncovered the presence of spatial-temporal specific dominantly expressed sub genomes.