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, nft channel 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 , 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.To identify data sets suitable to address our research questions, we performed a search in the ISI Web of Science and SCOPUS . To minimise potential publication bias and to maximise the number of relevant data sets we also searched for unpublished data by contacting potential data holders through researcher networks. Data sets had to meet the following requirements to be included in the analysis: pollination and/or pest control services in crops were measured in both crop fields adjacent to floral plantings and control fields without planting; the replication at the field level was ≥ six fields per study . We contacted data holders fulfilling these requirements and requested primary data on plant species richness of plantings, time since establishment, landscape context and crop yield in addition to measured pollination and pest control services. Overall, we analysed data from 35 studies. We here define a study as a dataset collected by the same group of researchers for a particular crop species and ecosystem service in a particular region during one or several sampling years.
We collected 18 pest control service and 17 pollination service studies, representing a total of 868 service-site-year combinations across 529 sites . In eight of these studies both crop pollination and pest control services were measured .As different studies used different methods and measures to quantify pollination services, pest control services and crop yield, we standardised data prior to statistical analysis using z-scores . The use of z-scores has clear advantages compared with other transformations or standardisation approaches because average z-scores follow a normal distribution, and the variability present in the raw data is not constrained as in other indices that are bound between 0 and 1 . Pollination services were measured as seed set , fruit set , pollen deposition rate and, in one study, flower visitation rate . If available, differences in pollination service measures of open pollinated flowers and flowers from which pollinators were excluded were analysed. Measures of pest control services were quantified as pest parasitism , pest predation , population growth or crop damage by pests or pest densities . Whenever possible, the pest control index based on population growth proposed by Gardiner et al. was calculated and analysed . Note that standardised values of pest density and crop damage were multiplied by 1 because lower values of these measures reflect an increased pest control service . Crop yield was only considered for the analysis if a direct measure of final crop yield was available. Too few studies assessed crop quality which was therefore not considered further. Yield was measured as crop mass or number of fruits produced per unit area. Due to a lack of studies measuring crop yield in fields with and without adjacent hedgerows, the analysis of crop yield focused on effects of flower strips. Crop yield measures were available from a total of 11 flower strip studies and 194 fields .Flower strips are here defined as strips or other areas of planted wild native and/or non-native flowering herbaceous plants. Hedgerows are defined as areas of linear shape planted with native and/or non-native at least partly flowering woody plants and typically also herbaceous flowering plants. For hedgerows, information about the exact time since establishment and number of plant species was not available for most studies. The analyses of these drivers therefore focus on flower strip effects on pollination and pest control services. Information on plant species richness was available in 12 out of 18 pest control studies and 10 out of 17 pollination studies. Whenever available, the species richness of flowering plants was used. Otherwise, for some flower strip studies, the number of sown, potentially flowering plant species was used. Time since establishment of flower strips, that is the time span between seeding or planting and data sampling, was available for all studies ranging from 3 to 122 months. The proportional cover of arable crops was available and analysed as a proxy for landscape simplification in 11 pest control and 12 pollination studies. Proportional cover of arable crops was calculated in circular sectors of 1 km radius around focal crops, or 750 m or 500 m radius .We used a mixed effect-modelling approach to address our research questions. In all models, hydroponic nft study was included as a random intercept to account for the hierarchical structure of the data with field measures nested within study. To assess whether flower strips and hedgerows enhanced pollination and pest control services in adjacent crops linear mixed-effect models with planting were separately fitted for flower strips and hedgerows for the response variables pollination service and pest control service. To test how the effects on service provisioning change with distance from plantings and with landscape simplification these explanatory variables and their interactions with the fixed effects described earlier were included in the models. Exploratory analyses showed that neither distance nor landscape simplification effects differed between flower strips and hedgerows; that is no significant interactive effects of planting type with any of the tested fixed effects.
We therefore pooled flower strip and hedgerow data in the final models, excluding planting type and its two or three-way interactions as fixed effects. In addition to linear relationships we tested for an exponential decline of measured response variables from the border of the field by fitting log10 in the linear mixed-effect models described earlier. In this case, field nested within study was included as a random effect. To test the intermediate landscape complexity hypothesis, we tested for linear as well as hump-shaped relationships between landscape context, and its interaction with local floral plantings by fitting landscape variables as a quadratic fixed predictor in the models described earlier . To present the ranges covered by the agricultural landscape gradients, we did not standardise measures of landscape simplification within studies . To examine how pollination and pest control service provisioning relates to flower strip plant diversity and time since establishment plant species richness and log10 were included as fixed effects in models with study as a random effect. Plant species richness and time since establishment of flower strips were not correlated . Only 10 studies measured services in several years since establishment , and we included only data from the last sampling year. To assess how the presence of plantings affected the agronomic yield of adjacent crops , we fitted a linear mixed-effect model with the same fixed and random structure as described for question 1, but with crop yield as the response variable. Statistical analyses for different models and response variables differed in sample sizes as not all studies measured crop yield in addition to pollination or pest control services . In all models we initially included planting area as a co-variate in an explorative analysis, but removed it in the final models, as it did not explain variation in any of the models and did not improve model fit . Effect sizes provided in the text and figures are model estimates of z-transformed response variables. For statistical inference of fixed effects we used log-likelihood ratio tests recommended for testing significant effects of a priori selected parameters relevant to the hypotheses . For all models, assumptions were checked according to the graphical validation procedures recommended by Zuur et al. . All statistical analyses were performed in R version 3.5.2 using the R-package lme4 .The provisioning of pest control services in crop fields adjacent to flower strips was enhanced by 16% on average compared to fields without flower strips. On average, pest control services were also increased in crops adjacent to hedgerows, but effects were more variable and overall not statistically significant . Pest control services declined exponentially with distance from the field edge, but the slopes of the distance functions between fields with and without adjacent floral plantings did not differ . Crop pollination effects were more variable across studies and overall not significantly different between crops with or without adjacent floral planting across all studies and within- field distances . However, effects of distance to field edge differed for fields with floral plantings compared with control fields . Pollination services were increased near floral plantings and decreased exponentially with increasing distance from plantings, while no such effect of distance to field edge was detected for control fields . The fitted distance curves for fields with or without floral plantings intersected at 43 m .Crop pollination services, but not pest control services, tended to increase with flowering plant species richness of the adjacent flower strip . Crop pollination services also tended to increase with time since establishment of the adjacent flower strip, but showed a positive saturating relationship .Pollination services increased by 27% in 2 year old strips compared with the youngest plantings , while the additional predicted increase from 2 to 4 years or older strips was approximately 5% on average . Pest control services in crops adjacent to flower strips did not increase with flower strip age .Our quantitative synthesis demonstrates a generally positive effect of flower strips on pest control services but these effects did not consistently translate into higher yields. Although in most cases beneficial effects of plantings were also found for crop pollination services, effects on crop pollination and final crop yield were variable and overall not significant. The effect of wildflower strips on pollination services increased with age and species-richness suggesting that the quality of such plantings plays a pivotal role in effective service provision. Moreover, crop pollination declined with increasing distance to floral plantings .