The physical properties of different cultivars did not seem to affect the fly’s oviposition. The percentage of eggs that developed to adults decreased with the increasing egg density per gram of fruit probably due to intra-specific competition, and this was further confirmed by manipulating the egg density and using the same ‘Bing’ cherry cultivar as the tested host. Females preferred larger fruit for oviposition, which is consistent with the density-dependent survival as the large fruit support higher numbers of fly larvae per fruit. It is well known that many fruit flies employ a variety of fruit characters to assess host quality and tend to be more attracted to larger fruits. Female D. suzukii appears to be able to assess host quality based on fruit size, and this behavior would likely increase foraging efficiency per unit time. Though we recovered very low numbers of D. suzukii from damaged citrus fruits, our laboratory study showed the fly can oviposit into and develop from freshly damaged or rotting navel oranges. Kaçar et al. showed that D. suzukii overwinter in citrus, surviving 3–4 months when fresh oranges were provided as adult food or ovipositional medium, and field-emerged adults from soil-buried pupae could produce and oviposit viable eggs on halved mandarin fruit. Thus, citrus fruit likely play an important role as reservoirs in sustaining the fly populations during San Joaquin Valley winter seasons,square black flower bucket wholesale and in the spring, those populations may migrate into early season crops, such as cherries. We did not observe grape infestation in our field collections, and our laboratory trials showed a low survival rate of D. suzukii offspring on grapes when compared to other fruits .
The oviposition susceptibility and offspring survival could vary among varieties or cultivars due to the variations in skin hardness and chemical properties. For example, Ioriatti et al. demonstrated that oviposition increased consistently as the skin hardness of the grape decreased. Chemical properties, such as sugar content and acidity levels, may play a role in host susceptibility. In the current study, we found that although table grapes had a tougher skin than raisin or wine grape cultivars tested , females were able to lay eggs into all three types of grapes, often through the fruit surface or near the petiole . The sugar levels of all tested grapes were either equal to or considerably higher than other fruits tested. We also found that tartaric acid concentration negatively affected the fly’s developmental performance. Still, about 20% eggs successfully developed to adults in the diet mixed with the highest tartaric acid, whereas only 4.5% eggs developed from the wine grape cultivar tested. Overall, our results are consistent with other reported studies that grapes are not good reproductive hosts for D. suzukii.California’s San Joaquin Valley is one of the world’s most important fruit production regions, with a diverse agricultural landscape that can consist of a mosaic of cultivated and unmanaged host fruit crops. Such diverse landscapes result in the inevitable presence of D. suzukii populations that represent a difficult challenge for the management of this polyphagous pest.
We showed that only the early seasonal fruits, such as cherries, seem to be at greatest risk to D. suzukiiMany of other later seasonal fruits are not as vulnerable to this pest, because either their intact skin reduces oviposition, they ripen during a period of low D. suzukii abundance, or their flesh has chemical attributes that retard survival. However, some of these alternative hosts—such as citrus and damaged, unharvested stone fruit—may act as shelters for overwintering populations and provide sources for early populations moving into the more susceptible crops. Consequently, area-wide management strategies may need to consider fruit sanitation to lessen overwintering populations, suppressing fall and winter populations by releasing natural enemies, and reducing pest pressure in susceptible crops through ‘border-sprays’ and/or ‘mass trapping’ to kill adults before they move into the vulnerable crop. Alternative and sustainable area-wide management strategies such as biological control are highly desirable to naturally regulate the fly population, especially in uncultivated habitats. An understanding of the temporal and spatial dynamics of the fly populations would be of aid in the optimal timing of the future release of biological control agents to reduce the source populations in the agricultural landscape. Previous studies on natural competence in X. fastidiosa were based on a few strains from a single subspecies , although recombination among strains of other subspecies has been described . On testing natural competence in 13 different strains, almost ubiquitous natural competence ability was detected. The frequency of recombination varied among strains, even for a single genomic region , as in other naturally competent bacteria . Flanking region DNA similarity of the strains was not correlated with the recombination frequency, but most of the strains within a subspecies had identical flanking regions.
A clearer understanding on the rate of recombination and homology between recombining DNA could have been obtained by using donor DNA containing a different level of similarity with the recipient strain at a given recombination region. However, this was not performed in this study. Even if differences in recombination frequency, especially between strains of different subspecies, could be due to differences in homology between donor and recipient DNA sequences or differences in growth rates that showed positive correlation with recombination frequency, these parameters did not explain the non-competency of two strains that had average growth and similar sequence homology compared with the competent strains. Also, since growth of the strains was measured by OD and strains appeared to differ in the rate of cell-to-cell attachment, growth values could have been biased, especially for the strains that showed high rates of precipitation . This was not further investigated, as it was beyond the scope of this study. On further testing of other biological traits, twitching motility was significantly correlated with recombination frequency. Strain WM1-1 had the highest recombination frequency and showed highest motility among strains, while the two non-competent strains were nonmotile. Positive correlation between recombination frequency and twitching motility was also suggested in our previous study using different media components . Since components of type IV pili are involved in both natural competence and twitching motility in several naturally competent gram-negative bacteria , including X. fastidiosa ,plastic square flower bucket the activity of type IV pili could govern both of these phenomena. On further analysis of competence and pili genes, defective pili genes were detected in the noncompetent strains. One of the defective proteins detected was PilQ , a member of the secret in family that forms the secret in pore of the outer membrane and is involved in type IV pili biogenesis and importing extracellular DNA into the periplasmic space . Previous studies in X. fastidiosa have demonstrated that pilQ mutants are nonmotile and noncompetent . Hence, we predict that the insertion in the pilQ coding region is responsible for the lack of twitching and natural competence, as BBI64 is unable to secrete the type IV pili. The lack of type IV pili was confirmed by TEM imaging. Motility has been described as a major virulence trait for X. fastidiosa . BBI64 has no motility and WM1-1 has the highest motility in this study. Consistent with the critical role of twitching in virulence, BBI64 had reduced virulence while WM1-1 was highly virulent . A further observation supporting the correlation between twitching and natural competence was the fact that the Fetzer strain showed recombination in this study, while a mutant in the polygalacturonase gene pglA of this strain did not . On closer examination, Fetzer is motile while the pglA mutant is not . Sequence data showed that the pglA mutant had an insertion in pilM, a type IV pili biogenesis gene that was shown to be involved in twitching motility of Acidovorax avenae in a previous study , most probably causing the lack of movement in this strain. Additional factors could be involved in causing differences in natural competence of X. fastidiosa strains. X. fastidiosa genomes contain high levels of phage and phage-like regions , and natural competence could be a mechanism to help cells eliminate new integration of these regions by recombining the homologous DNA without phage sequences, as suggested by a recent study . Other studies have reported restriction-modification systems limiting transformation frequency .
In this study, although all donor plasmids were extracted from an Escherichia coli strain expressing X. fastidiosa DNA methyl transferase , it is possible that different strains, especially from different genetic backgrounds, possess different forms of R-M systems, which could lead to differences in the amount of DNA available for recombination, causing differences in recombination frequency. In this regard, a previous study has reported the inability of a plasmid isolated from a citrus-infecting strain to transform a grape strain , suggesting existence of specific recognition mechanisms to differentiate DNA from self or foreign sources. Sequence analysis and annotation of the X. fastidiosa Temecula1 genome predicts at least four different types of R-M systems . Future studies focused on these specific topics could explain the differences in recombination frequencies observed among X. fastidiosa strains.Differences in recombination frequencies based on genomic positions was previously reported in Ralstonia solanacearum , with positions containing recombination hot spots showing the highest frequency . In this study, higher recombination frequency was observed for pKLN61, a plasmid that recombines in the region of rpfF gene, a diffusible signaling factor involved in cell-to-cell communication of X. fastidiosa , compared with pAX1.Cm, which recombines at a neutral site , and pMOPB-Km and pMSRA-Km, which recombine at regions whose functions are being characterized. Differences in the length of the homologous flanking region and nonhomologous insert have been found to influence recombination frequency in a previous study . However, the upstream and downstream flanking region length was higher in pAX1.Cm , pMOPB-Km , and pMSRA-Km than for pKLN61 . The length of the nonhomologous insert between the homologous flanking regions was similar and the size of the plasmids is also comparable . Moreover, flanking region DNA sequence identity between the donor plasmids and recipient strains at these positions was also similar . This suggests that the difference in recombination frequency at different genomic position is not associated with the characteristics of plasmid regions, and it remains to be determined if this difference holds any evolutionary significance. Natural competence has been proposed to bring adaptive changes to the recipient bacteria, such as repair of damaged DNA and generation of genetic diversity that can lead to adaptation . For the generationof adapted strains, recombining regions should come from a more successful and genetically distinct donor . This could be possible when closely related but genetically different strains of a same species coexist in a single habitat. Detection of IHR in X. fastidiosa by MLST/MLSA studies supported this possibility. In fact, these studies proposed IHR leading to plant host shift of X. fastidiosa in citrus , mulberry , and blueberry . Moreover, mixed infection of the two subspecies have been suggested by previous studies. For example, almond leaf scorch strains isolated from the same orchard were found to be genetically different and were grouped into two different subspecies, i.e., subsp. fastidiosa and multiplex . Infection of a plum tree showing leaf scorch symptoms by subsp. multiplex and subsp. pauca strains was also reported in a recent study . Results of this and previous studies demonstrated that certain plants serve as hosts for strains from multiple subspecies. In addition, vectors of X. fastidiosa are found to be distributed worldwide in both temperate and tropical climates and, unlike with plant hosts, exhibit no specificity for pathogen genotype . In fact, a species of the vector was able to transmit four subspecies of X. fastidiosa . All these observations suggest that strains belonging to different subspecies may coexist within the same habitat , providing opportunities for recombination. Although IHR was detected between subspecies when whole genomes of the donor and recipient were mixed, recombinants did not differ significantly with the parent in virulence phenotypes, suggesting that recombination did not bring phenotypic changes. On analyzing the flanking homologous region of recombination, 0.7- to 4-kb regions were detected to have recombined, but the size could be greater, as up to 80 kb has been demonstrated to recombine by natural competence in R. solanacearum, with the recombinant strain showing increased virulence .