Moreover, recombination could also occur at genomic regions other than the antibiotic insertion site, as the whole genomic DNA was used as donor to generate these recombinants. However, due to the bias of the method used here that selected recombinants acquiring antibiotic resistance, recombination that occurred at other regions in the genome may not have been present in the recombinants selected for assessment in this study. In addition, only the minimum size of recombination events could be estimated based on existing polymorphisms. Nonetheless, by targeting various genomic regions, it was confirmed that recombination occurred at multiple regions. Future studies by optimizing the selection procedures for recombinants in the context of pathogenicity to plants could reveal changes in virulence due to IHR in X. fastidiosa. Interestingly, IHR was bidirectional, meaning that both subspecies could act as both donor and recipient for one another. The evidence from field observation of X. fastidiosa disease emergence in new plant species and the detection of IHR in strains isolated from these infections by MLST/MLSA and from confirmation of natural competence in habitats mimicking natural environments to the experimental validation of IHR suggests that IHR is occurring in nature and may have broader evolutionary implications in X. fastidiosa disease dynamics.
In conclusion, X. fastidiosa strains showed extensive natural competence abilities and the recombination potential differed among strains. Moreover, plastic plant pot intersubspecific recombination occurred readily between X. fastidiosa subsp. fastidiosa and subsp. multiplex strains. These results emphasize the importance of quarantine measures to limit the introduction of novel genotypes of X. fastidiosa in areas with pre-existing infection. Moreover, measures to isolate host plants of different subspecies may be required to prevent mixed infections, minimizing the risk of generating novel and virulent genotypes of X. fastidiosa by recombination.Plasmids pAX1.Cm and pKLN61 were used from previous studies. Plasmids pMSRA-Km and pMOPB-Km were prepared as described earlier and are being characterized for another study in our laboratory . Briefly, about 800 bp long upstream and downstream fragments flanking open reading frames of methionine S-S-oxide reductase and outer membrane protein , respectively, were PCR amplified from the Temecula1 genomic DNA. The upstream and downstream fragments were digested using Asci restriction enzyme , were ligated, and were cloned into pJET1.2/blunt cloning vector, and a kanamycin-resistant cassette was inserted between the two fragments. All the plasmids were transformed into E. coli EAM1competent cells that express X. fastidiosa DNA methylase . Plasmids were prepared from the overnight cultures of EAM1 using an extraction kit , and concentration was adjusted to100 ng/µl . Aliquots were stored at _20 C until use. Natural competence assays were performed in PD3 agar plates.
The recipient strains were adjusted to OD600 of 0.25 in PD3 broth. Ten microliters of this suspension were spotted onto PD3 agar plates and 1 µg of plasmid in a 10-µl volume was added to the spots. Following incubation at 28 C for about 3 days , spots were suspended in 1 ml of PD3 and serial dilutions were plated in the respective antibiotic PW plates in triplicates, depending on which antibiotic cassette each plasmid carried, and PW plates without antibiotics. After 2 to 3 weeks of incubation at 28 C, CFUs were enumerated for recombinants and total viable cells , followed by calculation of recombination frequency as the ratio of the number of recombinants to total viable cells. For a given experiment, at least three repetitions were performed per strain, and the experiments had two to six biological replicates. For each strain, spots without the addition of plasmids were included as controls for every experiment. Genomic incorporation of the antibiotic-resistant marker from the donor plasmid was confirmed by PCR as previously described .To compare flanking region DNA sequence homology among recipient X. fastidiosa strains with respect to each donor plasmid, up- and downstream flanking regions of the antibiotic insertion sites of plasmids pAX1.Cm, pKLN61, pMSRA-Km, and pMOPB-Km were obtained. Up-and downstream sequences homologous to each plasmid region were obtained from the genomes of strains WM1-1, Temecula1, Temecula1*, BB08-1, AlmaEM3, BBI64 , and EB92-1 . The genomes were sequenced using Illumina Miseq and PacBio sequencing systems, and resulting reads after quality trimming were mapped to the Temecula1 reference genome, using the Geneious map to reference algorithm . The two up-and downstream sequences were concatenated, were aligned using the Muscle Multiple Sequence Alignment tool in Geneious, and percent identity between each of the donor plasmids and recipient strains was determined.Commerce via global trade and transport provides a mechanism for introduction of invasive species to new territories, extending pest habitats outside of their native regions . Invasive species threaten biodiversity, habitat, nutritious food, clean water, resilient environments, sustainable economies, and human health . Agricultural production systems are continuously challenged by invasive species that attack high-value crops, thereby significantly hampering the ability of food industries to maintain profitability . The geographic range of agricultural crops provides the potential for invasive species to colonize regions on a global scale .
Factors that aid expansion include short life cycle, fast growth rate, high plasticity, and resiliency to a wide range of environmental conditions . Such factors are drivers of rapid evolutionary change, population increase, and global colonization . Practitioners and stakeholders should aim to implement new strategies to manage such new invasive species in agricultural production . Drosophila suzukii Matsumura is an invasive species native to Southeast Asia. Passive transportation is the main reason of the dispersal of this species . It was first detected in North America and Europe in 2008 , and later in South America in 2013 , and Northern Africa in 2017 . The long-serrated ovipositor of D. suzukii enables it to oviposit inside fresh fruit, which creates a challenging management problem . Emerged larvae burrow within fruit pulp rendering fruit unmarketable . When D. suzukii became established in the U.S. during 2008, the total annual revenue losses for the West Coast berry and cherry industries were estimated at over $500 million . Currently the situation is not changed in term of economic impact . This particular insect is challenging to manage due to its high dispersal potential, ability to survive and adapt to harsh environmental conditions, and ability to attack a wide host range. For these reasons, D. suzukii is a key pest of these fruit industries worldwide. In the last decade, conventional insecticide uses on affected crops significantly increased to manage D. suzukii fruit damage. Typically used insecticides include spinosyns, pyrethroids, and organophosphates . Intensive use of insecticides poses a tremendous risk to non-target organisms such as pollinators, natural enemies, and humans . In addition, frequent insecticide applications likely resulted in resistance development . These factors require development of an IPM program that includes alternatives to conventional insecticides for managing D. suzukii. Non-insecticidal control methods including cladding, irrigation, netting, mulching, pruning, monitoring and mass trapping have been implemented against D. suzukii . While each method provides some relief to D. suzukii pressure,nursery pots they provide limited reductions in crop damage . Behavioral control of D. suzukii on susceptible fruit indicated promise for industry adoption. The food-grade gum possesses tactile and odorant cues resulting in reduced egg infestation. The food grade gum makes use of physical properties to mimic fruit, resulting in D. suzukii laying their eggs in a soft gel-like substrate, instead of the fruit itself. The food grade gum is a mixture of food-grade ingredients which is highly attractive to D. suzukii and competes with the ripening fruit throughout the season . To the best of our knowledge, the food-grade gum modifies various D. suzukii behaviors, ultimately resulting in a significant decrease in fruit damage. The product diverts D. suzukii away from ripening fruit, which results in significant retention of the pest, keeping it away from fruit. Third, the food-grade gum acts as an egg sink. Since the D. suzukii eggs laid in this medium cannot develop, this translates in a substantial reduction of the pest population growth . The aim of this work was to determine the potential of the food grade gum to reduce D. suzukii damage in large-scale commercial open-field and screen house fruit production units on blueberry, cherry, raspberry, blackberry, and wine grape. The hypothesis was that food-grade gum would reduce D. suzukii damage in small fruit, tree fruit and grapes under semi-field and small-scale field conditions.
These studies were conducted during 2019 and 2020 in California and Oregon in the western United States.In all field trials, GUM dispensers were placed at least 27 meters away from untreated control plots to minimize volatile plume interaction between treatments. In the current study, cotton pads were used to apply ~1.8 g of GUM on each dispenser at the rate of 124 dispensers per hectare under commercial production conditions . Cotton pads were placed directly on the ground close to irrigation drippers to provide adequate daily moisture. Earlier work illustrated that dispensers have a field longevity of 21 days and for this reason, dispensers were therefore deployed 1 to 4 times depending on the duration of crop ripening and susceptibility. In three trials , egg laying data were collected in buffer plots that were located between UTC and GUM plots to determine the active range of released volatiles beyond treated areas. This design was implemented based on the assumption that volatiles from treatment plots may be blown or diffuse beyond treatment plots. Berries were brought to the laboratory to determine number of eggs in fruit for each of the plots using a dissecting microscope. All soft or damaged fruits were excluded when assessing presence of eggs. In some cases, at first fruit color, laboratory-reared D. suzukii flies were released in each plot with the intent to create a relatively even pest pressure in all plots. Colonies of D. suzukii used in field studies consisted of seasonally collected wild adults from multiple field sites in the Willamette Valley, Oregon, and Oxnard, California. Collected adults were released into plastic cages and reared at 24°Cand 70% relative humidity, with a 16:8 h photoperiod before being released in the respective field trials. Flies were constantly provided with water and artificial diet that served as both a food source and an oviposition medium. Before their use in experiments, all flies were allowed to mate for 8 d in mixed-sex cages. A replicated field trial on drip-irrigated Pinot noir wine grape was conducted in Yamhill County, Oregon, USA from 10 to 18 October 2019 on ~2.6 hectares. Vines were spaced at 1.5 by 5 m, and trellised on a standard four wire trellis system, supporting a ~2 m canopy. Rows were oriented along a north-south direction on an east facing slope. Three treatments , were included with ~0.056 ha plots. No pesticides were applied during the experimental period. Here, there were 28 GUM and buffer plots each and 18 UTC plots. GUM dispensers were applied on 10 October and ten berries were collected from each plot on this date. Sampling dates were 11, 14, and 18 October 2019.Trials were conducted in a commercial sweet cherry orchard located at the Mid-Columbia Agricultural Research and Extension Center , Hood River, Oregon, USA. A 1.12-hectare orchard was divided into twelve plots . UTC, buffer, and GUM plots were replicated four times. The GUM dispensers were deployed on day 0 . No insecticides were applied to the orchard for the duration of the experiment. Here, an additional 200 mated 8- to 12-day-old D. suzukii were released in the center of each plot on a weekly basis on 23 June, and 1, 8, and 15 July 2020 . Data were collected for 35 days from 16 June through 22 July 2019. Because of relatively large canopy size of cherry trees compared to the other crops, ten cherries were collected from the lower , middle , and upper portions of the central two trees in each plot weekly.Trials were conducted in 1.8 hectare of high bush blueberry plants . The experiment ran from 6 October to 15 October 2020. There were three treatment levels i.e., UTC , buffer , and GUM. The GUM plots were located directly next to the buffer, followed by UTC plots of equal size. Plots were each ~0.05 hectares . Spinosad was applied on 6 October on the UTC and buffer areas. Insecticide application and GUM deployment occurred only on 6 October. On 8, 10, 13 and 15 October, one fruit sample consisting of 10 berries was collected from each of the 36 plots.