High rates of homologous recombination (HR) in the bacterial plant pathogen Xylella fastidiosa have been previously detected. This study aimed to determine the extent and explore the ecological significance of HR in the genomes of recombinants experimentally generated by natural transformation and wild-type isolates. Both sets of strains displayed widespread HR and similar average size of recombined fragments consisting of random events (2-10 kb) of inter-and intrasubspecific recombination. A significantly higher proportion and greater lengths (>10 kb, maximum 31.5 kb) of recombined fragments were observed in subsp. morus and in strains isolated in Europe from intercepted coffee plants shipped from the Americas. Such highly recombinant strains pose a serious risk of emergence of novel variants, as genetically distinct and formerly geographically isolated genotypes are brought in close proximity by global trade. Recently recombined regions in wild-type strains included genes involved in regulation and signaling, host colonization, nutrient acquisition, and host evasion, all fundamental traits for X. fastidiosa ecology. Identification of four recombinant loci shared between wild-type and experimentally generated recombinants suggests potential hotspots of recombination in this naturally competent pathogen. These findings provide insights into evolutionary forces possibly affecting the adaptive potential to colonize the host environments of X. fastidiosa.

Standard aquaculture generates large-scale pollution and strains water resources. In aquaculture using zero discharge systems (ZDS), highly efficient fish growth and water recycling are combined. The wastewater stream is directed through compartments in which beneficial microbial activities induced by creating suitable environmental conditions remove biological and chemical pollutants, alleviating both problems. Bacterial predators, preying on bacterial populations in the ZDS, may affect their diversity, composition and functional redundancy, yet in-depth understanding of this phenomenon is lacking. The dynamics of populations belonging to the obligate predators Bdellovibrio and like organisms (BALOs) were analyzed in freshwater and saline ZDS over a 7-month period using QPCR targeting the Bdellovibrionaceae, and the Bacteriovorax and Bacteriolyticum genera in the Bacteriovoracaeae. Both families co-existed in ZDS compartments, constituting 0.13-1.4% of total Bacteria. Relative predator abundance varied according to the environmental conditions prevailing in different compartments, most notably salinity. Strikingly, the Bdellovibrionaceae, hitherto only retrieved from freshwater and soil, also populated the saline system. In addition to the detected BALOs, other potential predators were highly abundant, especially from the Myxococcales. Among the general bacterial population, Flavobacteria, Bacteroidetes, Fusobacteriaceae and unclassified Bacteria dominated a well mixed but seasonally fluctuating diverse community of up to 238 operational taxonomic units, as revealed by 16S rRNA gene sequencing.

TitleNatural competence rates are variable among xylella fastidiosa strains and homologous recombination occurs in vitro between subspecies fastidiosa and multiplex Xylella fastidiosa, an etiological agent of emerging crop diseases around the world, is naturally competent for the uptake of DNA from the environment that is incorporated into its genome by homologous recombination. Homologous recombination between subspecies of X. fastidiosa was inferred by in silico studies and was hypothesized to cause disease emergence. However, no experimental data are available on the degree to which X. fastidiosa strains are capable of competence and whether recombination can be experimentally demonstrated between subspecies. Here, using X. fastidiosa strains from different subspecies, natural competence in 11 of 13 strains was confirmed with plasmids containing antibiotic markers flanked by homologous regions and, in three of five strains, with dead bacterial cells used as source of donor DNA. Recombination frequency differed among strains and was correlated to growth rate and twitching motility. Moreover, intersubspecific recombination occurred readily between strains of subsp. fastidiosa and multiplex, as demonstrated by movement of antibiotic resistance and green fluorescent protein from donor to recipient cells and confirmed by DNA sequencing of the flanking arms of recombinant strains. Results demonstrate that natural competence is widespread among X. fastidiosa strains and could have an impact in pathogen adaptation and disease development.

Xylella fastidiosa is a xylem-limited bacterium that is the causal agent of emerging diseases in a number of economically important crops. Genetic diversity studies have demonstrated homologous recombination occurring among X. fastidiosa strains, which has been proposed to contribute to host plant shifts. Moreover, experimental evidence confirmed that X. fastidiosa is naturally competent for recombination in vitro. Here, as an approximation of natural habitats (plant xylem vessels and insect mouthparts), recombination was studied in microfluidic chambers (MCs) filled with media amended with grapevine xylem sap. First, different media were screened for recombination in solid agar plates using a pair of X. fastidiosa strains that were previously reported to recombine in coculture. The highest frequency of recombination was obtained with PD3 medium, compared to those with the other two media (X. fastidiosa medium [XFM] and periwinkle wilt [PW] medium) used in previous studies. Dissection of the media components led to the identification of bovine serum albumin as an inhibitor of recombination that was correlated to its previously known effect on inhibition of twitching motility. When recombination was performed in liquid culture, the frequencies were significantly higher under flow conditions (MCs) than under batch conditions (test tubes). The recombination frequencies in MCs and agar plates were not significantly different from each other. Grapevine xylem sap from both susceptible and tolerant varieties allowed high recombination frequency in MCs when mixed with PD3. These results suggest that X. fastidiosa has the ability to be naturally competent in the natural growth environment of liquid flow, and this phenomenon could have implications in X. fastidiosa environmental adaptation. IMPORTANCEXylella fastidiosa is a plant pathogen that lives inside xylem vessels (where water and nutrients are transported inside the plant) and the mouthparts of insect vectors. This bacterium causes emerging diseases in various crops worldwide, including recent outbreaks in Europe. The mechanisms by which this bacterium adapts to new hosts is not understood, but it was previously shown that it is naturally competent, meaning that it can take up DNA from the environment and incorporate it into its genome (recombination). In this study, we show that the frequency of recombination is highest when the bacterium is grown under flow conditions in microfluidic chambers modeled after its natural habitats, and recombination was still high when the medium was amended with grapevine sap. Our results suggest that this bacterium is able to recombine when growing inside plants or insects, and this can be a mechanism of adaptation of this pathogen that causes incurable diseases. N atural competence is a phenomenon that allows bacteria to take up DNA segments from the environment and incorporate them into the genome via homologous recombination (1). Natural competence was first demonstrated in Streptococcus pneumoniae in 1928 by Frederick G...