Bee-mediated delivery of bacteriophage for biocontrol of the cherry canker pathogen Pseudomonas syringae pv. syringae

Bee-mediated delivery of bacteriophage for biocontrol of the cherry canker pathogen Pseudomonas syringae pv. syringae | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Bee-mediated delivery of bacteriophage for biocontrol of the cherry canker pathogen Pseudomonas syringae pv. syringae Shannon F. Greer, Sneha Chakravorty, Kieran Cooney-Nutley, Dave Chandler, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7053094/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Bacteriophages, phages or viruses that specifically infect bacteria, have shown promise for the biocontrol of bacterial plant diseases. However, one of the main challenges of using phages in agricultural systems is their precision application, being able to deliver an effective dose to the site of bacterial infection. In this study, a series of artificial and real cherry flower experiments was conducted to test whether commercially managed bumblebees ( Bombus terrestris audax ) could deliver phage effective against the cherry canker pathogen Pseudomonas syringae pv. syringae ( Pss ). Freeze-dried phage powder was formulated with powdered-skimmed milk and when tested, was found to retain viability for seven days in artificial bee feed after storage at room temperature or under glasshouse conditions. In both artificial and cherry flower experiments, bees successfully transferred the formulated phage from their hive to up to 88% of flowers, resulting in significant reduction in Pss populations. Bees were also able to transfer phage between cherry flowers. The application of phages disrupted the cycle of Pss transmission by bees. These results highlight the potential of bee-mediated phage delivery as an effective biocontrol strategy against floral pathogens like Pss . Biological sciences/Biological techniques Biological sciences/Biotechnology Biological sciences/Microbiology Biological sciences/Plant sciences phage bumblebee Pseudomonas cherry floral pathogen biocontrol Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Bacteriophages (phages) are viruses that specifically infect bacteria and have been explored as an alternative to chemical and antibiotic treatments for the control of bacterial plant diseases (Grace et al., 2021 ; Greer et al., 2024 ). Phages effective against some bacterial plant pathogens have been identified (Ahern et al., 2014 ; Di Lallo et al., 2014 ; Nakayinga et al., 2021 ; Ke et al., 2024 ) and some have been characterised in vitro and in planta , like phages effective against the cherry canker pathogen Pseudomonas syringae pv. syringae ( Pss ) (Bultreys and Kałużna, 2010 ; Rabiey et al., 2020 ; Papp-Rupar et al., 2025 ). Pss can cause 75% losses in young cherry orchards and can infect all aerial parts of a plant including leaves, blossoms and fruit, causing huge economic loss to cherry fruit industry (Sundin et al., 1988; Hulin et al., 2018 ). One of the main challenges of using phage in agricultural systems is their application. Phages need to be delivered in an effective dose at the target site of infection in a cost effective way (Wagemans et al., 2022 ). Several application methods have been considered and include spray treatments, seed treatments and application through irrigation water (Holtappels et al., 2021 ). The use of pollinating insects as vectors for phage delivery has not been explored but has the potential to be a more precise and efficient application method; insect pollinators can deliver phage directly to the sites where they are needed most and at the right time, like the flowers and developing fruits, with minimal labour and requirements for application machinery. Insect pollinators play a crucial role in plant reproduction and food security (Saha et al., 2023 ), but they also interact with diverse microbial communities on plant surfaces, including beneficial symbionts and pathogenic bacteria. Some studies have demonstrated that bees can act as vectors for plant pathogens, including P. syringae , facilitating their transmission from contaminated flowers to healthy plants (Pattemore et al., 2014 ; Biosecurity - Department of Industry Tourism and Trade, 2020 ). This interaction is shaped by a complex network of ecological and behavioural factors, including floral morphology, bee foraging patterns, and plant-emitted volatile organic compounds (VOCs) (McFrederick and Rehan, 2019 ; Vaudo et al., 2024 ). VoCs play a pivotal role in mediating these interactions. Plants produce VOCs, including terpenoids, green leaf volatiles, and aromatic compounds, that serve as attractants or repellents to pollinators and herbivores. These VOCs influence pollinator visitation rates and shape the floral microbiome by selectively attracting or deterring specific microbial taxa (Junker and Tholl, 2013 ; Burkle and Runyon, 2019 ). Pathogens like P. syringae can also modulate host plant volatile profiles to enhance spread, potentially increasing floral attractiveness to pollinators promoting transmission (Rering et al., 2018 ). Additionally, bees emit cuticular hydrocarbons and microbial-derived volatiles, which may influence pathogen dispersal (Russell and McFrederick, 2022 ). Bee foraging behaviour is central to the dissemination of floral microbes and plant pathogens. One would expect aspects such as floral constancy (the tendency to visit the same flower species), grooming habits, and frequency of inter-plant movements to influence the potential for pathogen transmission. For example, bees often contact reproductive floral structures, inadvertently picking up and transferring microbial cells or viral particles such as bacteriophages between flowers (Graystock et al., 2020 ). Their grooming behaviour can redistribute microbes within the colony, while their capacity to forage over long distances and across diverse plant species increases the spatial scale of microbial dispersal (Durrer and Schmid-Hempel, 1994 ). The extent to which bees contribute to the dissemination of Pss and its corresponding phages remains largely unexplored, yet understanding this process could provide novel strategies for the delivery of phages for targeted disease control in flowering crops. If bees are shown to effectively transport phages alongside or independently of bacterial hosts, this natural delivery system could be harnessed to manage plant diseases in flowering crops in an ecologically sustainable manner. This study aimed to investigate whether the buff-tailed bumblebee, Bombus terrestris audax , (i) can transfer phage from hive to flowers, (ii) can transfer phage or Pss from flower to flower, and (iii) can deliver phage to reduce Pss populations on flowers. We first formulated and tested the viability of phage powders under various conditions and then, under controlled glasshouse conditions, monitored the movement of phage and Pss in artificial and cherry flowers settings. Results Bee-mediated phage and Pss transfer in artificial flowers Phage powder is stable in artificial bee feed The stability of phage powder in artificial bee feed was tested under 4 o C, lab (room temperature) and glasshouse conditions (20±2 o C). Feed spiked with phage powder was positive for phage at all time points and conditions tested, including the final time point T7 (7-days post-inoculation) with no significant loss in phage titre (Fig. S1). A GLM with negative log link was used to model phage powder viability (PFU ml -1 ) in artificial bee feed based on sampling timepoint and treatment (4 o C, lab and glasshouse), with goodness-of-fit statistics: Deviance/df = 0.009, Pearson Chi-squared/df = 0.009 and AIC =410.435. The Omnibus Test was not significant X 2 (11) = 0.646, p = 1.000, indicating the model provided was not a better fit to the data compared to the intercept only model. Timepoint (Wald X 2 (3) = 0.379, p = 0.944), treatment (Wald X 2 (2) = 0.068, p = 0.966) and interaction of these two variables (Wald X 2 (6) = 0.219, p = 1.00) did not significantly influence phage viability. Phage was transferred from hive to artificial flowers To determine if bees could carry phages from the hive to flower, an artificial flower setup was used. Phage powder was applied to a runway at the hive entrance and bees were released, ensuring contact with the powder prior to flower access. Samples were taken repeatedly from each feed. No, phage was detectable on flowers prior to bee release (T0). At T1, phages were detected in 12 of 24 flowers (50%) (Fig. 1A), with titers ranging from 10² to 10⁵ PFU ml -1 (Mdn=10 2.5 PFU ml -1 , n =12). However, phage was not detectable at T4 or T7, even in flowers that initially tested positive. All bees collected at T7 had detectable phage on their bodies, with titers ranging from 10² to 10⁴ PFU ml -1 (Mdn=10² PFU ml -1 , n =3). These findings suggest that phage persistence was transient and likely influenced by environmental factors, bee foraging behavior, and the absence of a bacterial host. Phage was not transferred between artificial flowers To determine whether bees could mediate phage transfer between flowers, the feed of six (25%) out of 24 artificial flowers were spiked with phage powder before bees were released from a clean hive (no phage). Samples were taken repeatedly from each feed. Only the spiked flowers tested positive for phage at T0 with titers between 10⁵ and 10⁶ PFU mL⁻¹ (Mdn=10 5.5 PFU mL⁻¹, n =6) and at T1 with all titers at 10⁶ PFU mL⁻¹ ( n =6) (Fig. 1B). As for the hive to flower experiment, phages were not detectable after T1 in the spiked flowers. No phages were detected in unspiked flowers at any time point. Phages were detected on 60% of bees at T7 at a titre of 10¹ to 10³ PFU ml -1 (Mdn= 10¹ PFU ml -1 , n =3), but no evidence of inter-flower phage transfer was observed. Pss was transferred between artificial flowers To determine whether bees could transfer Pss between flowers, the feed of six (25%) out of the 24 artificial flowers were spiked with Pss before bees were released from a clean hive (no phage). Samples were taken repeatedly from each feed. Bees did transfer Pss from spiked flowers to unspiked flowers (Fig. 1C). At T0, prior to bee release, all spiked flowers contained Pss at 10 4 to 10⁵ CFU ml -1 (Mdn=10 4.5 CFU mL⁻¹, n =6) and none of the unspiked flowers were positive for Pss . By T1, Pss was detected in 89% of unspiked flowers (10 4 -10 7 CFU mL⁻¹, Mdn=10 6 CFU mL, n =16⁻¹) increasing to 94% at T4 (10 4 -10 8 CFU mL⁻¹, Mdn=10 5 CFU mL⁻¹, n =17) and decreasing to 80% and T7 (10 5 -10 7 CFU mL⁻¹, Mdn=10 7 CFU mL⁻¹, n =8). Pss was detected in 100% of spiked flowers at T1 (10 10 -10 11 CFU mL⁻¹, Mdn=10 11 CFU mL⁻¹, n =6) but dropped to 50% at T4 (10 6 -10 9 CFU mL⁻¹, Mdn=10 8 CFU mL⁻¹, n =3) and 20% at T7 (10 9 CFU mL⁻¹, n =1). Pss was detected on all bees at T7 at 10 2 to 10 3 CFU ml -1 (Mdn= 10 2 CFU ml -1 , n =4). Transferred phage reduced Pss populations in artificial flowers To assess the impact of bee-transferred phage on Pss populations, artificial flowers were inoculated with Pss before bees were released from a hive through a runway containing phage powder ( Pss +phage) or from a clean hive with no phage ( Pss only). Samples were taken repeatedly from each feed. In the Pss +phage treatment group, phages were successfully transferred and detected in 88% of flowers at T1 (Fig. 2A) at titers of 10⁵ to 10⁸ PFU ml -1 (Mdn=10 6 PFU ml -1 , n =21), but as for previous experiments phage was not detectable after T1. Phage was not detected on flowers from the Pss only treatment group. A GLM with negative binomial log link was used to model Pss colony count (CFU ml -1 ) based on sampling timepoint and phage treatment, with goodness-of-fit statistics: Deviance/df = 0.282, Pearson Chi-squared/df = 0.134 and AIC =945.592. The Omnibus Test was significant X 2 (7) = 110.318, p <0.001, indicating the model provided a better fit to the data compared to the intercept only model. Phage treatment (Wald X 2 (1) = 9.143, p = 0.002), timepoint (Wald X 2 (3) = 9.024, p = 0.029) and interaction of these two variables (Wald X 2 (3) = 6.099, p = 0.047) significantly influenced colony count. Compared to flowers treated with Pss alone, those exposed to phage exhibited significant median reductions in colony count from 10⁹ to 10⁴ CFU ml -1 at T1 ( U = 0.500, p = <0.001), from 10¹⁰ to 10 4 CFU ml -1 at T3 ( U = 0.000, p = <0.001), and from 10 2 CFU ml -1 to complete clearance at T7 ( U = 144.000, p = <0.001) (Fig. 2B). This suggests that bee-mediated delivery of phage can effectively reduce Pss populations in an artificial setting. Phage was recovered from three of four bees tested at T7 from the Pss +phage treatment group at a titres of 10³–10⁵ PFU ml -1 (Mdn=10 4 PFU mL⁻¹, n =3) but not from bees from the Pss only treatment group. Conversely, Pss was detected on two of three bees tested from the Pss treatment only group at titres of 10⁸–10⁹ CFU ml -1 (Mdn=10 8.5 CFU mL⁻¹, n =2) but not from bees from the Pss +phage treatment group. This suggests that phage treatment can break the Pss -bee transmission cycle. Bee-mediated transfer of phage and Pss in cherry flowers Phages were transferred from hive to cherry flowers In a cherry flower setup, bees exposed to phage powder at the hive entrance were allowed to forage. Flowers were randomly sampled at each time point. At T1, phages were detected in 7 of 12 flowers tested (58%) (Fig. 3A) at a titer of 10¹–10³ PFU ml -1 (Mdn=10 3 PFU mL⁻¹, n =7). By T3 and T7, phage was no longer detectable on flowers, as for the artificial flower experiment, but was detectable on all bees at T7 at titers of 10³–10⁴ PFU ml -1 (Mdn=10 3 PFU mL⁻¹, n =3). As in the artificial system, limited phage persistence may reflect environmental degradation, bee foraging behavior and the absence of host bacteria. Phages were transferred between cherry flowers Unlike the artificial system, bee-mediated phage transfer between cherry flowers was observed. Flowers from six branches were spiked with phage powder at T0, bees were then released from a clean hive (no phage) and flowers from spiked and unspiked branches were randomly sampled at each time point. At T0, prior to bee release, all spiked flowers were positive for phage at tires of 10 4 to 10 7 PFU ml -1 (Mdn=10 6 PFU mL⁻¹, n =6) and none of the unspiked flowers were positive for phage. At T1, phage was detected on 8 out of 12 (67%) unspiked flowers (Fig. 3B) at very low titers of <10 1 PFU ml -1 but phage wasn’t detected on any unspiked flowers at T3 or T7. Phage was detected on 100% of spiked flowers at T1 (10 4 -10 5 PFU mL⁻¹, Mdn=10 4 PFU mL⁻¹, n =6), dropping to 50% at T3 (10 1 -10 4 PFU mL⁻¹, Mdn=10 2 PFU mL⁻¹, n =3), and at T7 (10 1 -10 7 CFU mL⁻¹, Mdn=10 6 PFU mL⁻¹, n =3). All bees were positive for phage at T7 at titers of 10²–10⁴ PFU ml -1 (Mdn=10 3 PFU mL⁻¹, n =4). Pss was not transferred between cherry flowers Unlike the artificial system, bee-mediated Pss transfer between cherry flowers was not observed. Flowers from 12 branches were inoculated with Pss at T0, bees were then released from a clean hive (no phage) and flowers from spiked and unspiked branches were randomly sampled at each time point. Pss was not detected on unspiked flowers at any time point (Fig. 3C). Pss was detected on 100% spiked flowers at T0 (10 4 -10 5 CFU ml -1 , Mdn=10 4 CFU mL⁻¹, n =12) and T1 (10 3 -10 7 CFU ml -1 , Mdn=10 4 CFU mL⁻¹, n =12) but reduced to 75% by T4 (10 3 –10 6 CFU ml -1 , Mdn=10 5 CFU mL⁻¹, n =9) and T7 (10 1 –10 5 CFU ml -1 , Mdn=10 4 CFU mL⁻¹, n =9) (Fig. 3C). Pss was only detected on one bee out of seven tested, at 10 7 CFU mL⁻¹. Transferred phage reduced Pss populations in cherry flowers To investigate bee-mediated phage control of Pss in real flowers, cherry flowers were inoculated with Pss before bees were released from a hive through a runway containing phage powder ( Pss +phage) or from a clean hive with no phage ( Pss only). Flowers were randomly sampled at each time point. In the Pss +phage treatment group, phages were successfully transferred and detected in 33% of flowers at T1 (Fig. 4A) at titers of 10 7 to 10⁸ PFU ml -1 (Mdn=10 7 PFU mL⁻¹, n =4), but as for previous experiments phage was not detected after T1. Phage was not detected on flowers from the Pss only treatment group. A GLM with negative binomial log link was used to model Pss colony count (CFU ml -1 ) in cherry flowers based on sampling timepoint and phage treatment, with goodness-of-fit statistics: Deviance/df = 0.521, Pearson Chi-squared/df = 0.258 and AIC =469.018. The Omnibus Test was not significant X 2 (7) = 5.150, p = 0.642, indicating the model did not provide a better fit to the data compared to the intercept only model. Phage treatment (Wald X 2 (1) = 0.669, p = 0.413), timepoint (Wald X 2 (3) = 4.413, p = 0.220) and interaction of these two variables (Wald X 2 (2) = 6.099, p = 0.047) were found not to significantly influence colony count. Although not significant, flowers from the Pss +phage treatment had reductions in median bacterial populations compared to the Pss only treatment group: from 10 4 to 10 CFU ml -1 at T1 ( U = 44.000 p = 0.094), from 10 5 to 10 4 CFU mL⁻¹ at T4 (U = 63.500, p = 0.607), and from 10 3.5 to 10 1 CFU mL⁻¹ at T7 (U = 54.500, p = 0.298)(Fig. 4B). This suggests that bee-mediated delivery of phage can reduce Pss populations in a real flower setting, but the effect is not as strong as in artificial flowers. Phage was recovered from all bees tested at T7 from the Pss +phage treatment group at 10³–10 4 PFU ml -1 (Mdn=10 3 PFU mL⁻¹, n =5) but not from bees from the Pss only treatment group. Conversely, Pss was detected on one of seven bees tested from the Pss treatment only group with a titre of 10 7 CFU ml -1 but not from any bees from the Pss +phage treatment group. This is a similar finding to the artificial flower experiment and further supports that phage treatment can break the Pss -bee transmission cycle. Discussion This study highlights the promising potential of bee-mediated phage delivery as an effective biocontrol strategy against floral pathogens such as Pss . It underscores the importance of continued investment in the development of stable phage formulations and the optimisation of bee vectoring techniques. Importantly, producers of commercial fruit crops routinely deploy managed bee colonies to enhance pollination and maximise yields, an existing agricultural practice that could be harnessed for the targeted delivery of therapeutic phages. This creates a timely and practical opportunity to integrate bee-mediated biocontrol into existing crop management systems, particularly in crops vulnerable to bacterial infection via the floral route. While the findings are encouraging, the study's small scale reflects inherent logistical constraints. Only a limited number of bees could be tested due to space restrictions in cage trials, as higher densities would not accurately replicate field conditions and might result in unrepresentative flower visitation rates. Also, the work was confined to a narrow flowering window, typically just a couple of weeks, which imposed additional time pressure on experimental design and execution. In this study, Pss phage MR6 powder was formulated using skimmed milk as a stabilizing matrix. Phage powder was stable, retaining infectivity for at least seven days post-synthesis in artificial bee feed under 4 o C, lab and glasshouse conditions. The observed stability of phages in bee feed aligns with previous reports on phage formulation using skimmed milk and other protein-based protectants (Balogh et al., 2003 ; Iriarte et al., 2007 ; Grace et al., 2021 ). Skimmed milk has been widely employed as a stabiliser for freeze-dried phages, offering protection against the environment (temperature and UV), enhancing shelf-life and facilitating application under field-relevant conditions (González-Menéndez et al., 2018 ; Wdowiak et al., 2022 ), thus making them more amenable for practical deployment of phage powders in glasshouse and field settings. This study demonstrated that bees can effectively transfer the formulated phage powder from their hive to both artificial flowers and cherry flowers. While pollinator-mediated dissemination of beneficial microorganisms such as Trichoderma harzianum and Bacillus subtilis for fungal pathogen control has been documented (Dedej et al., 2004 ; Maccagnani et al., 2005 ; Shafir et al., 2006 ), this study, to the best of our knowledge, provides the first evidence of phages being effectively vectored by bees for the biocontrol of bacterial phytopathogens. The efficiency in which bees could transfer phage from their hive to cherry flowers varied and was as high as 88% in artificial flowers and 58% in cherry flowers. This efficiency is remarkable considering bees were only exposed to phage powder once. A higher transfer efficiency in artificial flowers compared to cherry flowers is likely a result of the experimental design. For example, fewer artificial flowers were available to bees than real flowers giving them less choice, and the sucrose feeds may have incentivised bees to revisit artificial flowers, not a typical behaviour for bees in a real flower setting (Goulson et al., 1998 ). Repeat exposure to phage, by leaving the phage powder at the entrance/exit to the hive could improve transfer efficiency. In all phage transfer experiments from hive to artificial or cherry flowers, phages were not detectable after T1. Whilst bee-mediated phage delivery is promising, this finding also highlights limitations. This result could be due to environmental degradation of phages from UV light, desiccation, or absence of the bacterial host. This has been noted in field-based phage studies as a challenge for persistence (Iriarte et al., 2007 ; Papp-Rupar et al., 2025 ) and therefore, phage encapsulation methods and formulation additives may enhance phage longevity in future applications (Wdowiak et al., 2022 ). Yet, phage remained stable and detectable at T7 in sucrose feed not exposed to bees and phage was also detectable on bees at T7. So, bee foraging and feeding behaviour could also be playing a role in phage depletion after T1. The transfer of phage by bees between flowers was observed only in cherry flowers and not in the artificial flowers. Conversely, transfer of Pss by bees between flowers was observed only in artificial flowers and not cherry flowers. These differences suggest that movement of phage and Pss by bees could be influenced by different environmental and behavioural factors. For example, differences in flower structure, volatile emissions, or surface properties could influence bee foraging behaviour and microbial acquisition and deposition (Russell et al., 2019 ). In cherry flowers, bees may forage more intensively or make longer contact with reproductive floral organs, allowing phage acquisition and deposition. Whereas artificial flowers have less realistic floral cues which could alter bee grooming behaviour, resulting in reduced phage acquisition and/or deposition. In addition, the sucrose feed in the artificial flowers provides a more optimal environment for Pss survival and replication compared to cherry flower surfaces. It could be that higher concentrations of Pss in the feed facilitated acquisition and transfer by bees in an artificial setting, but this is not representative of what happens in real flowers. Importantly, phage delivery by bees reduced Pss populations in both artificial and cherry flowers, despite a limited persistence of detectable phage in flowers after T1. Our findings support the novelty and feasibility of bee-mediated phage biocontrol of floral pathogens such as Pss , building on prior research into pollinator-based delivery of biological agents (Dedej et al., 2004 ; Maccagnani et al., 2005 ; Shafir et al., 2006 ). The reduction in Pss populations following bee-mediated phage delivery is consistent with previous reports of phage efficacy against Pseudomonas spp. in planta (Buttimer et al., 2017 ; Rabiey et al., 2020 ; Papp-Rupar et al., 2025 ). In artificial flowers, there was a significant reduction in Pss populations at all time points. In comparison, there was a less apparent reduction in Pss in cherry flowers. The differences observed between artificial and cherry flower experiments could be down to several factors. For example, fewer cherry flowers were sampled at each time point due to flower availability, the proportion of flowers positive for phage were lower at T1 in the cherry flower experiment (33%) compared to the artificial experiment (88%). For the artificial flower experiments, samples were collected repeatedly from the same flowers. In contrast, different cherry flowers were collected at each time point in the real cherry flower experiments. Although the same concentration of Pss was used to inoculate both artificial and cherry flowers, the methods differed; cherry flowers were sprayed with Pss , while artificial flowers were directly spiked. It is possible that Pss took longer to fully establish in the cherry flowers, when sprayed, before phage application. Also, Pss or phage were in a more optimal environment in the sucrose feed compared to real flowers. The latter is supported by an increase in Pss population from T0 to T3 in the artificial flowers not treated with phage and a reduction in population between these timepoints for cherry flowers not treated with phage. In both artificial and cherry flower experiments there was reduction in Pss populations at time points where phage was not detectable on flowers. These findings suggest that initial phage activity may have reduced the bacterial population sufficiently to prevent detectable regrowth, or that low levels of phage, below the limit of detection, remained biologically active. Such residual phage activity could have continued to suppress the host population through sustained infection cycles, even in the absence of visible plaques. This phenomenon aligns with the concept of top-down control, in which higher trophic levels (phages) regulate the abundance and dynamics of lower trophic levels (bacteria). Even at low concentrations, phages can exert strong ecological pressure, limiting bacterial proliferation and maintaining population control. Similar observations have been reported in other microbial systems, where persistent phage activity, despite low detectable titres, was sufficient to influence bacterial community structure and dynamics (Abedon et al., 2001 ). In addition, Pss was not detected on bees when phages were present in both artificial and cherry flower biocontrol experiments, indicating that phage activity may interrupt the cycle of pathogen dissemination, avoiding the spread of the pathogen by bees (Pattemore et al., 2014 ; Biosecurity - Department of Industry Tourism and Trade, 2020 ). This dual function, reducing pathogen load and halting transmission, highlights the potential of phage-biocarrier systems to contribute to sustainable crop protection strategies through both suppression and containment of bacterial diseases. The study underscores the value of continued investment in developing stable phage formulations and optimising bee vectoring techniques to enhance disease management in flowering crops. Notably, this approach may offer practical relevance beyond the laboratory, particularly for other high-value crops vulnerable to floral pathogens, such as Erwinia amylovora (causing fire blight in apples and pears), Pseudomonas syringae pv. actinidiae ( Psa ) (causing bacterial canker in kiwifruit) and Acidovorax citrulli (causing bacterial fruit blotch in cucurbits like watermelon and melon)(Burdman and Walcott, 2012 ; McCann et al., 2013 ; Pedroncelli and Puopolo, 2024 ). In New Zealand, for instance, the 2010 outbreak of Psa devastated the kiwifruit industry, costing producers an estimated NZ $ 930 million over several years (Vanneste, 2017 ). Because kiwifruit yield is directly tied to pollination efficiency, and the crop is increasingly grown under netting (which does not deter bumblebee activity), bumblebees have become the preferred pollinators. This makes them a promising vehicle for targeted phage delivery in commercial settings. However, to realise the full potential of this technology, further research is needed to improve phage persistence in floral environments and to better understand the environmental and behavioural factors that influence phage transmission via pollinators. Ultimately, such work could unlock a novel, pollinator-compatible biocontrol method with direct relevance to crop protection strategies in high-value agricultural systems. Materials and methods Bacterial isolates, phage isolates and bee species Pss strain 9097, isolated from cankerous cherry ( Prunus avium ) wood, Warwickshire, UK in 2010 (Hulin et al., 2018), was cultured in King's Medium B (KB) at 28 o C with shaking at 200rpm overnight or plated on KB with 1.5% agar (KBA) (King et al., 1954) at 28 o C overnight. For long-term storage at -80°C, overnight bacterial liquid culture was combined with an equal volume of 40% glycerol. Pss -infecting phage (MR6) isolated from a cherry orchard in the UK and previously characterised by Rabiey et al. (2020) was used in this study. Phage was amplified by an enrichment assay (see below) and was diluted in phosphate buffered saline for short-term storage at 4 o C. For longer-term storage at -80 o C, phage diluted in PBS was combined with an equal volume of 40% glycerol. Mini hives (8.5 W x 8 D x 13 H cm) of a European native bumblebee species Bombus terrestris audax (Natupol Seeds, Koppert, distributed by Dragonfli, UK) were used. These hives contained ~15 worker bees, five males and a male brood and are specifically designed for use in small scale crop pollination in glasshouses, polytunnels, and allotments. The hives do not have a queen and have a lifespan of only 6 - 8 weeks. Production of phage powder Enrichment assay For large-scale propagation of phage, 2 ml of bacterial culture (OD 600 = 0.2, 2x10 8 CFU ml -1 ) was added to a 50 ml Falcon tube containing 23 ml of KB medium and incubated at 28°C with shaking at 200 rpm. After one hour, 1 ml of phage suspension in PBS (>1x10 8 PFU ml -1 ) and 50 µl of 1M CaCl₂ (Sigma-Aldrich) and 50 µl 1M MgCl₂ (Sigma-Aldrich) was added to the KB medium and incubated for an additional 5 hours with shaking, after which 250 µl of chloroform (Sigma-Aldrich) was added and incubated for another hour. The suspension was centrifuged at 4500 rpm for 45 minutes at room temperature to pellet the bacterial cells. The supernatant was carefully collected, avoiding disturbance of the pellet, and filtered through a sterile PES 0.22 µm filter (SLS select) into a fresh 50 ml Falcon tube. 24% polyethylene glycol 8000 (Fisher Bioreagents) was added to a final concentration of 8% w/v, and the mixture was incubated overnight at 4°C. Phages were then precipitated by centrifugation (4500 rpm, 45 min, 4°C), and the resulting phage pellet was resuspended in 5 ml of phosphate buffered saline (PBS). Phage stocks were titered by spot assay (see below) and stored at 4°C. Spot assay A bacterial lawn of Pss was prepared by mixing 100ul of Pss (OD 600 =0.2) with 5ml of molten but cooled soft KB agar (0.75%) containing cycloheximide (100mg L -1 ) and cephalexin (40mg L -1 ). This was then poured on to the surface of pre-prepared KB agar (1.5%) plates. Phage stocks were serial diluted (10 1 -10 12 ) in PBS and 3μl was spotted in duplicate onto the bacteriallawn. Plates were incubated at 28°C for 24 hours, after which clearing zones with a countable number of plaques were recorded to determine phage titre (PFU ml -1 ). Phage powder formulation and testing To prepare phage powder, 500mg (10% w/v) skimmed milk powder (Marvel) was added to 5ml phage diluted in PBS buffer (at 10 12-15 PFU ml -1 ), vortexed and then frozen at -80 o C for 1 hour. Then the mixture was freeze-dried for 24hours at -50 o C and 0.001mbar (Christ Alpha 1-2 LD plus, Sciquip) to yield ~500mg of phage powder. The resulting phage powder was subsequently titred by reconstituting 50mg of phage powder in 500μl PBS, serial diluted and spotted onto Pss lawns as described above. The stability of phage powder in artificial bee feed (20% sucrose solution) was then assessed under 4 o C, lab (room temperature) and glasshouse conditions (20±2 o C). A 10μl pipette tip was used to spike 3ml of bee feed in six individual wells of a flat-bottomed 24-well cell culture plate (Falcon) with phage powder. Presence and titre of phage was tested by spot assay at T0 (immediately after inoculation), T1 (1-day post-inoculation), T4 (4-day post-inoculation), and T7 (7-day post-inoculation). Bacterial counts To determine bacterial counts, bacterial cultures were serial diluted (10 1 -10 8 ) in PBS and 3μl was spotted in duplicate onto KB agar plates containing cycloheximide (100mg L -1 ) and cephalexin (40mg L -1 ). Plates were incubated at 28°C for 24-48 hours, after which colonies in spots with countable numbers were recorded to determine the colony-forming units (CFU ml -1 ). Bee-mediated transfer of phage and suppression of Pss on artificial and cherry flowers General experimental conditions All experiments were conducted under controlled glasshouse conditions (day and night temperature of 20±2 o C with natural lighting) using either artificial cherry-like flowers or real cherry flowers ( Prunus avium cv. Sweetheart) to assess the role of bees in phage dissemination and biocontrol of Pss . Experiments were performed in insect-proof polyethylene cages (Outsunny, 2 L× 2 H x 1 W meter, with two mesh windows) in two neighbouring glasshouse compartments (each 20m 2 ), one compartment was used for experiments using phage and the other as a phage-free control compartment. Artificial flower assays Artificial flowers (5cm Ø) were design to mimic cherry flowers, with five pink petals and five yellow spots representing anthers around a central hole. These were then mounted on to brown card, laminated and placed on a 24 well flat-bottomed cell culture plate (Falcon), with the hole in the centre of the flower overlapping with one well of the plate containing 3 ml of 20% sucrose feed. For each experiment, 24 artificial flowers were distributed evenly within the cage in two rows with 10cm space in between them. In all experiments, six bees were released into the cage from a hive through a runway (10 L x 3 Ø cm tubing) containing ~500mg of phage powder (produced from 5ml phage lysate) attached to the circular hole of hive entrance/exit, unless stated otherwise. After bee release the hive remained in situ, but the entrance was sealed and if present, the phage runway removed. Feed from each flower was repeatedly sampled at four timepoints: prior to bee release (T0), and at 1-, 3- or 4-, and 7-days post-release (T1, T3 or T4, T7) and tested for presence and titre of phage or Pss using spot assays. Bees were collected at T7, individually incubated in 1ml PBS for 1hr at 28 o C and 200rpm, and a spot assay was performed on the wash to determine phage or Pss titre. Cherry flower assays One-year-old dwarf cherry trees (Sweetheart on Gisela 5 rootstock, 1.5 m height, sourced from Chris Bowers & Sons, UK) were grown in 12-litre pots containing Levingtons M2 compost supplemented with Osmocote® (rate of 3g L -1 ) and arranged within insect-proof cages as described above. Experiments were carried out between March-April when the cherry trees were in bloom. Each tree had at least 3-4 branches and bore at least 20-30 open flowers during experimentation. Bees were released after phage exposure, and individual flowers were destructively sampled by cutting the stalk with sterilised scissors and tested at T0, T1, T3 or T4, and T7. Flowers were incubated individually in 1ml PBS for 1hr at 28 o C and 200rpm, and a spot assay was performed on the wash to determine phage or Pss titre. Bees were collected at T7 and tested in the same way. Experimental designs Phage transfer from hive to flowers To test whether bees can transfer phages from the hive to flowers, bees were released from the hive through a runway containing phage powder attached to the hive entrance/exit. At each timepoint, sucrose feed from artificial flowers (n = 25) or individual cherry flowers (n = 12) were sampled and tested for phage presence and titre. Phage transfer between flowers To test whether bees can move phage between flowers, the feed of a subset of artificial flowers (n = 6) was spiked with phage powder (as for 2.2.3) or cherry flowers on a subset of branches (n = 6) were each inoculated with ~3mg phage powder using a paintbrush, previously sterilised in 70% ethanol, at T0. Bees were then released from a clean hive (no phage powder). At each time point, inoculated feed or cherry flowers (n = 6) were sampled and tested for phage presence and titre alongside feed from uninoculated artificial flowers (n = 18) and individual cherry flowers (n = 12). Bacterial transfer between flowers To test whether bees could transfer Pss between flowers, the feed of a subset of artificial flowers (n = 6) was inoculated with Pss to a final concentration of 2x10 7 CFU ml -1 l, or cherry flowers on a subset of branches (n = 12) were spray inoculated using a hand operated mist sprayer with each flower receiving one mist spray (~15μl ) of Pss at 2x10 7 CFU ml -1 . Bees were then released from a clean hive (no phage powder). At each time point, inoculated feed (n = 6) or cherry flowers (n=12) were sampled and tested for Pss presence and count alongside feed from uninoculated artificial flowers (n =18) and individual cherry flowers (n = 12). Bee-mediated phage biocontrol of Pss To test the efficacy of phages delivered by bees in controlling Pss, artificial and cherry flowers were inoculated with Pss as described above. Two cages/conditions were compared: (i) bees released from a hive through a runway containing phage powder attached to the hive entrance/exit (treatment) and (ii) bees released from a clean hive without phage exposure (control). At each time point, feed from artificial flowers (n = 24) and cherry flower (n = 12) were sampled and tested for Pss and phage presence and titer/count. Statistical tests and software Statistical analyses were carried out in IBM® SPSS® Statistics version 28. Firstly, Shapiro-Wilk tests for normality were conducted on the data (Shapiro and Wilk, 1965). Generalized linear models (GLM) with negative binomial log link were then conducted (IBM SPSS Statistics, 2025) to determine main and interaction effects of timepoint and treatment (4 o C, lab, glasshouse) on phage powder viability in artificial bee feed and phage treatment and timepoint on Pss counts (CFU ml -1 ) in the artificial and cherry flower biocontrol experiments. This GLM model fitted the data best, when compared to a Poisson log link model, also suitable for modelling count data. To identify significant differences ( p < 0.05) in bacteria count between treatment groups within a timepoint post hoc Mann-Whitney U tests were conducted (Mann and Whitney, 1947). Figures were made in GraphPad prism 9 (Boston, Massachusetts USA, www.graphpad.com). Declarations Competing interests The authors declare no competing interests. Funding and Acknowledgments The authors acknowledge funding from Carus Animal Health Ltd and The University of Warwick start-up fund, supporting S.F.G. and M.R. The authors acknowledge the support of Horticultural Services at the University of Warwick for plant care. Author Contribution S.G., G.F., R.O., and M.R. conceived and contributed to experimental design. S.G., S. C., K.C-N., carried out experimental work. S.G., D.C., and M.R. performed statistical analysis. S.G. and M.R. wrote the manuscript. All authors contributed to interpretation of results, read and approved the final manuscript. Acknowledgement The authors acknowledge funding from Carus Animal Health Ltd and The University of Warwick start-up fund, supporting S.F.G. and M.R. The authors acknowledge the support of Horticultural Services at the University of Warwick for plant care. Data Availability All data have been included within the manuscript. References Abedon, S.T., Herschler, T.D., and Stopar, D. (2001). Bacteriophage latent-period evolution as a response to resource availability. Applied and Environmental Microbiology 67 , 4233-4241. 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Role of pollinators in plant reproduction and food security: A concise review. Research Journal of Agricultural Sciences 14 , 72-79. Shafir, S., Dag, A., Bilu, A., Abu-Toamy, M., and Elad, Y. (2006). Honey bee dispersal of the biocontrol agent Trichoderma harzianum T39: Effectiveness in suppressing Botrytis cinerea on strawberry under field conditions. European Journal of Plant Pathology 116 , 119-128. Shapiro, S.S., and Wilk, M.B. (1965). An analysis of variance test for normality (complete samples). Biometrika 52 , 591-611. Sundin, G.W., L., J.A., and Olson, B.D. (1988). Overwintering and population dynamics of Pseudomonas syringae pv. syringae and P . s . pv. morsprunorum on sweet and sour cherry trees. Canadian Journal of Plant Pathology 10 , 281-288. Vanneste, J.L. (2017). The scientific, economic, and social impacts of the New Zealand outbreak of bacterial canker of kiwifruit ( Pseudomonas syringae pv. actinidiae ). Annual Review of Phytopathology 55 , 377-399. Vaudo, A.D., Dyer, L.A., and Leonard, A.S. (2024). Pollen nutrition structures bee and plant community interactions. Proceedings of the National Academy of Sciences 121 , e2317228120. Wagemans, J., Holtappels, D., Vainio, E., Rabiey, M., Marzachì, C., Herrero, S., Ravanbakhsh, M., Tebbe, C.C., Ogliastro, M., Ayllón, M.A., and Turina, M. (2022). Going viral: Virus-based biological control agents for plant protection. Annual Review of Phytopathology 60 , 21-42. Wdowiak, M., Paczesny, J., and Raza, S. 2022. Enhancing the stability of bacteriophages using physical, chemical, and nano-based approaches: A review. Pharmaceutics [Online], 14. Additional Declarations No competing interests reported. Supplementary Files Supplementaryinformation.docx Supplementary information Figure S1. Detection and titre of phage in artificial bee feed ( n =6) at T0 (immediately after inoculation), T1 (1-day post-inoculation), T4 (4 -day post-inoculation), and T7 (7-day post-inoculation), under 4 o C, lab and glasshouse conditions (20±2 o C) after spiking with phage powder. Bars show the median PFU ml -1 and error bars represent the interquartile range. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7053094","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":483963506,"identity":"1aa2c59b-5d7c-4289-9129-b3dc2bf79ab0","order_by":0,"name":"Shannon F. 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Significant differences between treatment groups are denoted as **** - \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7053094/v1/d4f770ed871cf9e50afbc2dd.jpg"},{"id":86710170,"identity":"ae874df5-fb22-4a9c-a290-5a8569591571","added_by":"auto","created_at":"2025-07-14 18:24:43","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1399285,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBees transferred phage from hive to flower and flower to flower but did not transfer \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePss \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003efrom flower to flower in cherry flower experiments\u003c/strong\u003e. Proportion of flowers tested positive for phage or \u003cem\u003ePss \u003c/em\u003ein artificial flowers \u003cstrong\u003eA.\u003c/strong\u003ephage hive to flower, \u003cstrong\u003eB.\u003c/strong\u003e phage flower to flower and \u003cstrong\u003eC.\u003c/strong\u003e \u003cem\u003ePss \u003c/em\u003eflower to flower.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7053094/v1/829729be9646e9be732d35c7.jpg"},{"id":86710173,"identity":"c80b5c9d-8955-4a7b-9418-8a28ca1c2232","added_by":"auto","created_at":"2025-07-14 18:24:43","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":849743,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBee-mediated delivery of phage from the hive reduces \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePss \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003epopulations in\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003echerry flowers. A.\u003c/strong\u003e proportion of cherry flowers positive for phage delivered by bees and \u003cstrong\u003eB.\u003c/strong\u003e \u003cem\u003ePss\u003c/em\u003e colony counts (CFU per ml) in the absence and presence of phage.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7053094/v1/36e887046260e281d4ca5d18.jpg"},{"id":90564498,"identity":"d3ea6205-64d6-4bd9-ac6f-969ffaba6616","added_by":"auto","created_at":"2025-09-04 07:01:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5925263,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7053094/v1/1ef177d9-8ca1-4dcc-81aa-b1c7b8af47e6.pdf"},{"id":86711065,"identity":"53135ce8-ff34-4a48-a552-d79c2074216c","added_by":"auto","created_at":"2025-07-14 18:40:43","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":92512,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S1. Detection and titre of phage in artificial bee feed (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003en\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e=6) at T0\u003c/strong\u003e \u003cstrong\u003e(immediately after inoculation), T1 (1-day post-inoculation),\u003c/strong\u003e \u003cstrong\u003eT4 (4 -day post-inoculation), and T7 (7-day post-inoculation),\u003c/strong\u003e \u003cstrong\u003eunder 4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eC, lab and glasshouse conditions (20±2\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eC)\u003c/strong\u003e \u003cstrong\u003eafter spiking with phage powder.\u003c/strong\u003e\u0026nbsp; Bars show the median PFU ml\u003csup\u003e-1\u003c/sup\u003e and error bars represent the interquartile range.\u003c/p\u003e","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7053094/v1/e11134664bae98beb6c48d10.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bee-mediated delivery of bacteriophage for biocontrol of the cherry canker pathogen Pseudomonas syringae pv. syringae","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBacteriophages (phages) are viruses that specifically infect bacteria and have been explored as an alternative to chemical and antibiotic treatments for the control of bacterial plant diseases (Grace et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Greer et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Phages effective against some bacterial plant pathogens have been identified (Ahern et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Di Lallo et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Nakayinga et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ke et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and some have been characterised \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein planta\u003c/em\u003e, like phages effective against the cherry canker pathogen \u003cem\u003ePseudomonas syringae\u003c/em\u003e pv. \u003cem\u003esyringae\u003c/em\u003e (\u003cem\u003ePss\u003c/em\u003e) (Bultreys and Kałużna, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Rabiey et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Papp-Rupar et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). \u003cem\u003ePss\u003c/em\u003e can cause 75% losses in young cherry orchards and can infect all aerial parts of a plant including leaves, blossoms and fruit, causing huge economic loss to cherry fruit industry (Sundin et al., 1988; Hulin et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOne of the main challenges of using phage in agricultural systems is their application. Phages need to be delivered in an effective dose at the target site of infection in a cost effective way (Wagemans et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Several application methods have been considered and include spray treatments, seed treatments and application through irrigation water (Holtappels et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The use of pollinating insects as vectors for phage delivery has not been explored but has the potential to be a more precise and efficient application method; insect pollinators can deliver phage directly to the sites where they are needed most and at the right time, like the flowers and developing fruits, with minimal labour and requirements for application machinery.\u003c/p\u003e\u003cp\u003eInsect pollinators play a crucial role in plant reproduction and food security (Saha et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), but they also interact with diverse microbial communities on plant surfaces, including beneficial symbionts and pathogenic bacteria. Some studies have demonstrated that bees can act as vectors for plant pathogens, including \u003cem\u003eP. syringae\u003c/em\u003e, facilitating their transmission from contaminated flowers to healthy plants (Pattemore et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Biosecurity - Department of Industry Tourism and Trade, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This interaction is shaped by a complex network of ecological and behavioural factors, including floral morphology, bee foraging patterns, and plant-emitted volatile organic compounds (VOCs) (McFrederick and Rehan, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Vaudo et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). VoCs play a pivotal role in mediating these interactions. Plants produce VOCs, including terpenoids, green leaf volatiles, and aromatic compounds, that serve as attractants or repellents to pollinators and herbivores. These VOCs influence pollinator visitation rates and shape the floral microbiome by selectively attracting or deterring specific microbial taxa (Junker and Tholl, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Burkle and Runyon, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Pathogens like \u003cem\u003eP. syringae\u003c/em\u003e can also modulate host plant volatile profiles to enhance spread, potentially increasing floral attractiveness to pollinators promoting transmission (Rering et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Additionally, bees emit cuticular hydrocarbons and microbial-derived volatiles, which may influence pathogen dispersal (Russell and McFrederick, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Bee foraging behaviour is central to the dissemination of floral microbes and plant pathogens. One would expect aspects such as floral constancy (the tendency to visit the same flower species), grooming habits, and frequency of inter-plant movements to influence the potential for pathogen transmission. For example, bees often contact reproductive floral structures, inadvertently picking up and transferring microbial cells or viral particles such as bacteriophages between flowers (Graystock et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Their grooming behaviour can redistribute microbes within the colony, while their capacity to forage over long distances and across diverse plant species increases the spatial scale of microbial dispersal (Durrer and Schmid-Hempel, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). The extent to which bees contribute to the dissemination of \u003cem\u003ePss\u003c/em\u003e and its corresponding phages remains largely unexplored, yet understanding this process could provide novel strategies for the delivery of phages for targeted disease control in flowering crops. If bees are shown to effectively transport phages alongside or independently of bacterial hosts, this natural delivery system could be harnessed to manage plant diseases in flowering crops in an ecologically sustainable manner.\u003c/p\u003e\u003cp\u003eThis study aimed to investigate whether the buff-tailed bumblebee, \u003cem\u003eBombus terrestris audax\u003c/em\u003e, (i) can transfer phage from hive to flowers, (ii) can transfer phage or \u003cem\u003ePss\u003c/em\u003e from flower to flower, and (iii) can deliver phage to reduce \u003cem\u003ePss\u003c/em\u003e populations on flowers. We first formulated and tested the viability of phage powders under various conditions and then, under controlled glasshouse conditions, monitored the movement of phage and \u003cem\u003ePss\u003c/em\u003e in artificial and cherry flowers settings.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eBee-mediated phage and \u003cem\u003ePss\u003c/em\u003e transfer in artificial flowers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhage powder is stable in artificial bee feed\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe stability of phage powder in artificial bee feed was tested under 4\u003csup\u003eo\u003c/sup\u003eC, lab (room temperature) and glasshouse conditions (20±2\u003csup\u003eo\u003c/sup\u003eC). Feed spiked with phage powder was positive for phage at all time points and conditions tested, including the final time point T7 (7-days post-inoculation) with no significant loss in phage titre (Fig. S1). A GLM with negative log link was used to model phage powder viability (PFU ml\u003csup\u003e-1\u003c/sup\u003e) in artificial bee feed based on sampling timepoint and treatment (4\u003csup\u003eo\u003c/sup\u003eC, lab and glasshouse), with goodness-of-fit statistics: Deviance/df = 0.009, Pearson Chi-squared/df = 0.009 and AIC =410.435. The Omnibus Test was not significant \u003cem\u003eX\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e(11) = 0.646, \u003cem\u003ep\u003c/em\u003e = 1.000, indicating the model provided was not a better fit to the data compared to the intercept only model. Timepoint (Wald \u003cem\u003eX\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e (3) = 0.379, \u003cem\u003ep\u003c/em\u003e = 0.944), treatment (Wald \u003cem\u003eX\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e (2) = 0.068, \u003cem\u003ep\u003c/em\u003e = 0.966) and interaction of these two variables (Wald \u003cem\u003eX\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e (6) = 0.219, \u003cem\u003ep\u003c/em\u003e = 1.00) did not significantly influence phage viability.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhage was transferred from hive to artificial flowers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine if bees could carry phages from the hive to flower, an artificial flower setup was used. Phage powder was applied to a runway at the hive entrance and bees were released, ensuring contact with the powder prior to flower access. Samples were taken repeatedly from each feed. No, phage was detectable on flowers prior to bee release (T0). At T1, phages were detected in 12 of 24 flowers (50%) (Fig. 1A), with titers ranging from 10² to 10⁵ PFU ml\u003csup\u003e-1\u003c/sup\u003e (Mdn=10\u003csup\u003e2.5\u0026nbsp;\u003c/sup\u003ePFU ml\u003csup\u003e-1\u003c/sup\u003e,\u0026nbsp;\u003cem\u003en\u003c/em\u003e=12). However, phage was not detectable at T4 or T7, even in flowers that initially tested positive. All bees collected at T7 had detectable phage on their bodies, with titers ranging from 10² to 10⁴ PFU ml\u003csup\u003e-1\u003c/sup\u003e (Mdn=10² PFU ml\u003csup\u003e-1\u003c/sup\u003e, \u003cem\u003en\u003c/em\u003e=3). These findings suggest that phage persistence was transient and likely influenced by environmental factors, bee foraging behavior, and the absence of a bacterial host.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhage was not transferred between artificial flowers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether bees could mediate phage transfer between flowers, the feed of six (25%) out of 24 artificial flowers were spiked with phage powder before bees were released from a clean hive (no phage). Samples were taken repeatedly from each feed. Only the spiked flowers tested positive for phage at T0 with titers between 10⁵ and 10⁶ PFU mL⁻¹ (Mdn=10\u003csup\u003e5.5\u0026nbsp;\u003c/sup\u003ePFU mL⁻¹, \u003cem\u003en\u003c/em\u003e=6) and at T1 with all titers at 10⁶ PFU mL⁻¹ (\u003cem\u003en\u003c/em\u003e=6) (Fig. 1B). As for the hive to flower experiment, phages were not detectable after T1 in the spiked flowers. \u0026nbsp;No phages were detected in unspiked flowers at any time point. Phages were detected on 60% of bees at T7 at a titre of 10¹ to 10³ PFU ml\u003csup\u003e-1\u003c/sup\u003e (Mdn= 10¹ PFU ml\u003csup\u003e-1\u003c/sup\u003e, \u003cem\u003en\u003c/em\u003e=3), but no evidence of inter-flower phage transfer was observed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePss\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;was transferred between artificial flowers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether bees could transfer \u003cem\u003ePss\u0026nbsp;\u003c/em\u003ebetween flowers, the feed of six (25%) out of the 24 artificial flowers were spiked with \u003cem\u003ePss\u003c/em\u003e before bees were released from a clean hive (no phage). Samples were taken repeatedly from each feed. Bees did transfer \u003cem\u003ePss\u003c/em\u003e from spiked flowers to unspiked flowers (Fig. 1C). At T0, prior to bee release, all spiked flowers contained \u003cem\u003ePss\u003c/em\u003e at 10\u003csup\u003e4\u003c/sup\u003e to 10⁵ CFU ml\u003csup\u003e-1\u003c/sup\u003e (Mdn=10\u003csup\u003e4.5\u0026nbsp;\u003c/sup\u003eCFU mL⁻¹, \u003cem\u003en\u003c/em\u003e=6) and none of the unspiked flowers were positive for \u003cem\u003ePss\u003c/em\u003e. By T1, \u003cem\u003ePss\u0026nbsp;\u003c/em\u003ewas detected in 89% of unspiked flowers (10\u003csup\u003e4\u003c/sup\u003e-10\u003csup\u003e7\u003c/sup\u003e CFU mL⁻¹, Mdn=10\u003csup\u003e6\u003c/sup\u003e CFU mL,\u003cem\u003e\u0026nbsp;n\u003c/em\u003e=16⁻¹) increasing to 94% at T4 (10\u003csup\u003e4\u003c/sup\u003e-10\u003csup\u003e8\u003c/sup\u003e CFU mL⁻¹, Mdn=10\u003csup\u003e5\u003c/sup\u003e CFU mL⁻¹, \u003cem\u003en\u003c/em\u003e=17) and decreasing to 80% and T7 (10\u003csup\u003e5\u003c/sup\u003e-10\u003csup\u003e7\u003c/sup\u003e CFU mL⁻¹, Mdn=10\u003csup\u003e7\u003c/sup\u003e CFU mL⁻¹, \u003cem\u003en\u003c/em\u003e=8). \u003cem\u003ePss\u003c/em\u003e was detected in 100% of spiked flowers at T1 (10\u003csup\u003e10\u003c/sup\u003e-10\u003csup\u003e11\u003c/sup\u003e CFU mL⁻¹, Mdn=10\u003csup\u003e11\u003c/sup\u003e CFU mL⁻¹, \u003cem\u003en\u003c/em\u003e=6) but dropped to 50% at T4 (10\u003csup\u003e6\u003c/sup\u003e-10\u003csup\u003e9\u003c/sup\u003e CFU mL⁻¹, Mdn=10\u003csup\u003e8\u003c/sup\u003e CFU mL⁻¹, \u003cem\u003en\u003c/em\u003e=3) and 20% at T7 (10\u003csup\u003e9\u003c/sup\u003e CFU mL⁻¹, \u003cem\u003en\u003c/em\u003e=1). \u003cem\u003ePss\u003c/em\u003e was detected on all bees at T7 at 10\u003csup\u003e2\u003c/sup\u003e to 10\u003csup\u003e3\u003c/sup\u003e CFU ml\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003e(Mdn= 10\u003csup\u003e2\u0026nbsp;\u003c/sup\u003eCFU ml\u003csup\u003e-1\u003c/sup\u003e, \u003cem\u003en\u003c/em\u003e=4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransferred phage reduced \u003cem\u003ePss\u003c/em\u003e populations in artificial flowers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the impact of bee-transferred phage on \u003cem\u003ePss\u003c/em\u003e populations, artificial flowers were inoculated with \u003cem\u003ePss\u003c/em\u003e before bees were released\u0026nbsp;from a hive through a runway containing phage powder (\u003cem\u003ePss\u003c/em\u003e+phage) or from a clean hive with no phage (\u003cem\u003ePss\u0026nbsp;\u003c/em\u003eonly). Samples were taken repeatedly from each feed. In the \u003cem\u003ePss\u003c/em\u003e+phage treatment group, phages were successfully transferred and detected in 88% of flowers at T1 (Fig. 2A) at titers of 10⁵ to 10⁸ PFU ml\u003csup\u003e-1\u003c/sup\u003e (Mdn=10\u003csup\u003e6\u003c/sup\u003e PFU ml\u003csup\u003e-1\u003c/sup\u003e, \u003cem\u003en\u003c/em\u003e=21), but as for previous experiments phage was not detectable after T1. Phage was not detected on flowers from the \u003cem\u003ePss\u0026nbsp;\u003c/em\u003eonly treatment group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA GLM with negative binomial log link was used to model \u003cem\u003ePss\u003c/em\u003e colony count (CFU ml\u003csup\u003e-1\u003c/sup\u003e) based on sampling timepoint and phage treatment, with goodness-of-fit statistics: Deviance/df = 0.282, Pearson Chi-squared/df = 0.134 and AIC =945.592. The Omnibus Test was significant \u003cem\u003eX\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e(7) = 110.318, \u003cem\u003ep\u003c/em\u003e \u0026lt;0.001, indicating the model provided a better fit to the data compared to the intercept only model. Phage treatment (Wald \u003cem\u003eX\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e (1) = 9.143, \u003cem\u003ep\u003c/em\u003e = 0.002), timepoint (Wald \u003cem\u003eX\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e (3) = 9.024, \u003cem\u003ep\u003c/em\u003e = 0.029) and interaction of these two variables (Wald \u003cem\u003eX\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e (3) = 6.099, \u003cem\u003ep\u003c/em\u003e = 0.047) significantly influenced colony count. Compared to flowers treated with\u003cem\u003e\u0026nbsp;Pss\u003c/em\u003e alone, those exposed to phage exhibited significant median reductions in colony count from 10⁹ to 10⁴ CFU ml\u003csup\u003e-1\u003c/sup\u003e at T1 (\u003cem\u003eU\u0026nbsp;\u003c/em\u003e= 0.500, \u003cem\u003ep\u003c/em\u003e = \u0026lt;0.001), from 10¹⁰ to 10\u003csup\u003e4\u0026nbsp;\u003c/sup\u003eCFU ml\u003csup\u003e-1\u003c/sup\u003e at T3 (\u003cem\u003eU\u0026nbsp;\u003c/em\u003e= 0.000, \u0026nbsp;\u003cem\u003ep\u003c/em\u003e = \u0026lt;0.001), and from 10\u003csup\u003e2\u003c/sup\u003e CFU ml\u003csup\u003e-1\u003c/sup\u003e to complete clearance at T7 (\u003cem\u003eU\u0026nbsp;\u003c/em\u003e= 144.000, \u003cem\u003ep\u003c/em\u003e = \u0026lt;0.001) (Fig. 2B). This suggests that bee-mediated delivery of phage can effectively reduce \u003cem\u003ePss\u0026nbsp;\u003c/em\u003epopulations in an artificial setting.\u003c/p\u003e\n\u003cp\u003ePhage was recovered from three of four bees tested at T7 from the \u003cem\u003ePss\u003c/em\u003e+phage treatment group at a titres of 10³–10⁵ PFU ml\u003csup\u003e-1\u003c/sup\u003e (Mdn=10\u003csup\u003e4\u003c/sup\u003e PFU mL⁻¹, \u003cem\u003en\u003c/em\u003e=3) but not from bees from the \u003cem\u003ePss\u0026nbsp;\u003c/em\u003eonly treatment group. Conversely, \u003cem\u003ePss\u003c/em\u003e was detected on two of three bees tested from the \u003cem\u003ePss\u003c/em\u003e treatment only group at titres of 10⁸–10⁹ CFU ml\u003csup\u003e-1\u003c/sup\u003e (Mdn=10\u003csup\u003e8.5\u003c/sup\u003e CFU mL⁻¹, \u003cem\u003en\u003c/em\u003e=2) but not from bees from the \u003cem\u003ePss\u003c/em\u003e+phage treatment group. This suggests that phage treatment can break the \u003cem\u003ePss\u003c/em\u003e-bee transmission cycle.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBee-mediated transfer of phage and\u003cem\u003e\u0026nbsp;Pss\u003c/em\u003e in cherry flowers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhages were transferred from hive to cherry flowers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn a cherry flower setup, bees exposed to phage powder at the hive entrance were allowed to forage. Flowers were randomly sampled at each time point. At T1, phages were detected in 7 of 12 flowers tested (58%) (Fig. 3A) at a titer of 10¹–10³ PFU ml\u003csup\u003e-1\u003c/sup\u003e (Mdn=10\u003csup\u003e3\u003c/sup\u003e PFU mL⁻¹, \u003cem\u003en\u003c/em\u003e=7). By T3 and T7, phage was no longer detectable on flowers, as for the artificial flower experiment, but was detectable on all bees at T7 at titers of 10³–10⁴ PFU ml\u003csup\u003e-1\u003c/sup\u003e (Mdn=10\u003csup\u003e3\u003c/sup\u003e PFU mL⁻¹, \u003cem\u003en\u003c/em\u003e=3). As in the artificial system, limited phage persistence may reflect environmental degradation, bee foraging behavior and the absence of host bacteria.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhages were transferred between cherry flowers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnlike the artificial system, bee-mediated phage transfer between cherry flowers was observed. Flowers from six branches were spiked with phage powder at T0, bees were then released from a clean hive (no phage) and flowers from spiked and unspiked branches were randomly sampled at each time point. At T0, prior to bee release, all spiked flowers were positive for phage at tires of 10\u003csup\u003e4\u003c/sup\u003e to 10\u003csup\u003e7\u003c/sup\u003e PFU ml\u003csup\u003e-1\u003c/sup\u003e (Mdn=10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003ePFU mL⁻¹, \u003cem\u003en\u003c/em\u003e=6) and none of the unspiked flowers were positive for phage. \u0026nbsp;At T1, phage was detected on 8 out of 12 (67%) unspiked flowers (Fig. 3B) at very low titers of \u0026lt;10\u003csup\u003e1\u003c/sup\u003e PFU ml\u003csup\u003e-1\u003c/sup\u003e but phage wasn’t detected on any unspiked flowers at T3 or T7. Phage was detected on 100% of spiked flowers at T1 (10\u003csup\u003e4\u003c/sup\u003e-10\u003csup\u003e5\u003c/sup\u003e PFU mL⁻¹, Mdn=10\u003csup\u003e4\u003c/sup\u003e PFU mL⁻¹, \u003cem\u003en\u003c/em\u003e=6), dropping to 50% at T3 (10\u003csup\u003e1\u003c/sup\u003e-10\u003csup\u003e4\u003c/sup\u003e PFU mL⁻¹, Mdn=10\u003csup\u003e2\u003c/sup\u003e PFU mL⁻¹,\u003cem\u003e\u0026nbsp;n\u003c/em\u003e=3), and at T7 (10\u003csup\u003e1\u003c/sup\u003e-10\u003csup\u003e7\u003c/sup\u003e CFU mL⁻¹, Mdn=10\u003csup\u003e6\u003c/sup\u003e PFU mL⁻¹, \u003cem\u003en\u003c/em\u003e=3). All bees were positive for phage at T7 at titers of 10²–10⁴ PFU ml\u003csup\u003e-1\u003c/sup\u003e (Mdn=10\u003csup\u003e3\u003c/sup\u003e PFU mL⁻¹, \u003cem\u003en\u003c/em\u003e=4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePss\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;was not transferred between cherry flowers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnlike the artificial system, bee-mediated \u003cem\u003ePss\u0026nbsp;\u003c/em\u003etransfer between cherry flowers was not observed. Flowers from 12 branches were inoculated with \u003cem\u003ePss\u003c/em\u003e at T0, bees were then released from a clean hive (no phage) and flowers from spiked and unspiked branches were randomly sampled at each time point. \u003cem\u003ePss\u0026nbsp;\u003c/em\u003ewas not detected on unspiked flowers at any time point (Fig. 3C). \u003cem\u003ePss\u0026nbsp;\u003c/em\u003ewas detected on 100% spiked flowers at T0 (10\u003csup\u003e4\u003c/sup\u003e-10\u003csup\u003e5\u003c/sup\u003e CFU ml\u003csup\u003e-1\u003c/sup\u003e, Mdn=10\u003csup\u003e4\u003c/sup\u003e CFU mL⁻¹, \u003cem\u003en\u003c/em\u003e=12) and T1 (10\u003csup\u003e3\u003c/sup\u003e-10\u003csup\u003e7\u003c/sup\u003e CFU ml\u003csup\u003e-1\u003c/sup\u003e,\u0026nbsp;Mdn=10\u003csup\u003e4\u003c/sup\u003e CFU mL⁻¹, \u003cem\u003en\u003c/em\u003e=12) but reduced to 75% by T4 (10\u003csup\u003e3\u003c/sup\u003e–10\u003csup\u003e6\u003c/sup\u003e CFU ml\u003csup\u003e-1\u003c/sup\u003e,\u0026nbsp;Mdn=10\u003csup\u003e5\u003c/sup\u003e CFU mL⁻¹, \u003cem\u003en\u003c/em\u003e=9) and T7 (10\u003csup\u003e1\u003c/sup\u003e–10\u003csup\u003e5\u003c/sup\u003e CFU ml\u003csup\u003e-1\u003c/sup\u003e,\u0026nbsp;Mdn=10\u003csup\u003e4\u003c/sup\u003e CFU mL⁻¹, \u003cem\u003en\u003c/em\u003e=9) (Fig. 3C). \u003cem\u003ePss\u003c/em\u003e was only detected on one bee out of seven tested, at 10\u003csup\u003e7\u003c/sup\u003e CFU mL⁻¹.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransferred phage reduced \u003cem\u003ePss\u0026nbsp;\u003c/em\u003epopulations in cherry flowers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate bee-mediated phage control of \u003cem\u003ePss\u003c/em\u003e in real flowers, cherry flowers were inoculated with \u003cem\u003ePss\u003c/em\u003e before bees were released\u0026nbsp;from a hive through a runway containing phage powder (\u003cem\u003ePss\u003c/em\u003e+phage) or from a clean hive with no phage (\u003cem\u003ePss\u0026nbsp;\u003c/em\u003eonly). Flowers were randomly sampled at each time point. In the \u003cem\u003ePss\u003c/em\u003e+phage treatment group, phages were successfully transferred and detected in 33% of flowers at T1 (Fig. 4A) at titers of 10\u003csup\u003e7\u003c/sup\u003e to 10⁸ PFU ml\u003csup\u003e-1\u003c/sup\u003e (Mdn=10\u003csup\u003e7\u003c/sup\u003e PFU mL⁻¹, \u003cem\u003en\u003c/em\u003e=4), but as for previous experiments phage was not detected after T1. Phage was not detected on flowers from the \u003cem\u003ePss\u0026nbsp;\u003c/em\u003eonly treatment group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA GLM with negative binomial log link was used to model \u003cem\u003ePss\u003c/em\u003e colony count (CFU ml\u003csup\u003e-1\u003c/sup\u003e) in cherry flowers based on sampling timepoint and phage treatment, with goodness-of-fit statistics: Deviance/df = 0.521, Pearson Chi-squared/df = 0.258 and AIC =469.018. The Omnibus Test was not significant \u003cem\u003eX\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e(7) = 5.150, \u003cem\u003ep\u003c/em\u003e = 0.642, indicating the model did not provide a better fit to the data compared to the intercept only model. Phage treatment (Wald \u003cem\u003eX\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e (1) = 0.669, \u003cem\u003ep\u003c/em\u003e = 0.413), timepoint (Wald \u003cem\u003eX\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e (3) = 4.413, \u003cem\u003ep\u003c/em\u003e = 0.220) and interaction of these two variables (Wald \u003cem\u003eX\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e (2) = 6.099, \u003cem\u003ep\u003c/em\u003e = 0.047) were found not to significantly influence colony count. Although not significant, flowers from the \u003cem\u003ePss\u003c/em\u003e+phage treatment had \u0026nbsp;reductions in median bacterial populations compared to the \u003cem\u003ePss\u003c/em\u003e only treatment group: from 10\u003csup\u003e4\u003c/sup\u003e to 10 CFU ml\u003csup\u003e-1\u003c/sup\u003e at T1 (\u003cem\u003eU\u003c/em\u003e = 44.000 \u003cem\u003ep\u003c/em\u003e = 0.094), from 10\u003csup\u003e5\u003c/sup\u003e to 10\u003csup\u003e4\u003c/sup\u003e CFU mL⁻¹ at T4 (U = 63.500, \u003cem\u003ep\u003c/em\u003e = 0.607), and from 10\u003csup\u003e3.5\u003c/sup\u003e to 10\u003csup\u003e1\u003c/sup\u003e CFU mL⁻¹ at T7 (U = 54.500, \u003cem\u003ep\u003c/em\u003e = 0.298)(Fig. 4B). This suggests that bee-mediated delivery of phage can reduce \u003cem\u003ePss\u003c/em\u003e populations in a real flower setting, but the effect is not as strong as in artificial flowers.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePhage was recovered from all bees tested at T7 from the \u003cem\u003ePss\u003c/em\u003e+phage treatment group at 10³–10\u003csup\u003e4\u003c/sup\u003e PFU ml\u003csup\u003e-1\u003c/sup\u003e (Mdn=10\u003csup\u003e3\u003c/sup\u003e PFU mL⁻¹, \u003cem\u003en\u003c/em\u003e=5) but not from bees from the \u003cem\u003ePss\u0026nbsp;\u003c/em\u003eonly treatment group. Conversely, \u003cem\u003ePss\u003c/em\u003e was detected on one of seven bees tested from the \u003cem\u003ePss\u003c/em\u003e treatment only group with a titre of 10\u003csup\u003e7\u003c/sup\u003e CFU ml\u003csup\u003e-1\u003c/sup\u003e but not from any bees from the \u003cem\u003ePss\u003c/em\u003e+phage treatment group. This is a similar finding to the artificial flower experiment and further supports that phage treatment can break the \u003cem\u003ePss\u003c/em\u003e-bee transmission cycle.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study highlights the promising potential of bee-mediated phage delivery as an effective biocontrol strategy against floral pathogens such as \u003cem\u003ePss\u003c/em\u003e. It underscores the importance of continued investment in the development of stable phage formulations and the optimisation of bee vectoring techniques. Importantly, producers of commercial fruit crops routinely deploy managed bee colonies to enhance pollination and maximise yields, an existing agricultural practice that could be harnessed for the targeted delivery of therapeutic phages. This creates a timely and practical opportunity to integrate bee-mediated biocontrol into existing crop management systems, particularly in crops vulnerable to bacterial infection via the floral route.\u003c/p\u003e\u003cp\u003eWhile the findings are encouraging, the study's small scale reflects inherent logistical constraints. Only a limited number of bees could be tested due to space restrictions in cage trials, as higher densities would not accurately replicate field conditions and might result in unrepresentative flower visitation rates. Also, the work was confined to a narrow flowering window, typically just a couple of weeks, which imposed additional time pressure on experimental design and execution.\u003c/p\u003e\u003cp\u003eIn this study, \u003cem\u003ePss\u003c/em\u003e phage MR6 powder was formulated using skimmed milk as a stabilizing matrix. Phage powder was stable, retaining infectivity for at least seven days post-synthesis in artificial bee feed under 4\u003csup\u003eo\u003c/sup\u003eC, lab and glasshouse conditions. The observed stability of phages in bee feed aligns with previous reports on phage formulation using skimmed milk and other protein-based protectants (Balogh et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Iriarte et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Grace et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Skimmed milk has been widely employed as a stabiliser for freeze-dried phages, offering protection against the environment (temperature and UV), enhancing shelf-life and facilitating application under field-relevant conditions (Gonz\u0026aacute;lez-Men\u0026eacute;ndez et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wdowiak et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), thus making them more amenable for practical deployment of phage powders in glasshouse and field settings.\u003c/p\u003e\u003cp\u003eThis study demonstrated that bees can effectively transfer the formulated phage powder from their hive to both artificial flowers and cherry flowers. While pollinator-mediated dissemination of beneficial microorganisms such as \u003cem\u003eTrichoderma harzianum\u003c/em\u003e and \u003cem\u003eBacillus subtilis\u003c/em\u003e for fungal pathogen control has been documented (Dedej et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Maccagnani et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Shafir et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), this study, to the best of our knowledge, provides the first evidence of phages being effectively vectored by bees for the biocontrol of bacterial phytopathogens.\u003c/p\u003e\u003cp\u003eThe efficiency in which bees could transfer phage from their hive to cherry flowers varied and was as high as 88% in artificial flowers and 58% in cherry flowers. This efficiency is remarkable considering bees were only exposed to phage powder once. A higher transfer efficiency in artificial flowers compared to cherry flowers is likely a result of the experimental design. For example, fewer artificial flowers were available to bees than real flowers giving them less choice, and the sucrose feeds may have incentivised bees to revisit artificial flowers, not a typical behaviour for bees in a real flower setting (Goulson et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Repeat exposure to phage, by leaving the phage powder at the entrance/exit to the hive could improve transfer efficiency.\u003c/p\u003e\u003cp\u003eIn all phage transfer experiments from hive to artificial or cherry flowers, phages were not detectable after T1. Whilst bee-mediated phage delivery is promising, this finding also highlights limitations. This result could be due to environmental degradation of phages from UV light, desiccation, or absence of the bacterial host. This has been noted in field-based phage studies as a challenge for persistence (Iriarte et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Papp-Rupar et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) and therefore, phage encapsulation methods and formulation additives may enhance phage longevity in future applications (Wdowiak et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Yet, phage remained stable and detectable at T7 in sucrose feed not exposed to bees and phage was also detectable on bees at T7. So, bee foraging and feeding behaviour could also be playing a role in phage depletion after T1.\u003c/p\u003e\u003cp\u003eThe transfer of phage by bees between flowers was observed only in cherry flowers and not in the artificial flowers. Conversely, transfer of \u003cem\u003ePss\u003c/em\u003e by bees between flowers was observed only in artificial flowers and not cherry flowers. These differences suggest that movement of phage and \u003cem\u003ePss\u003c/em\u003e by bees could be influenced by different environmental and behavioural factors. For example, differences in flower structure, volatile emissions, or surface properties could influence bee foraging behaviour and microbial acquisition and deposition (Russell et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In cherry flowers, bees may forage more intensively or make longer contact with reproductive floral organs, allowing phage acquisition and deposition. Whereas artificial flowers have less realistic floral cues which could alter bee grooming behaviour, resulting in reduced phage acquisition and/or deposition. In addition, the sucrose feed in the artificial flowers provides a more optimal environment for \u003cem\u003ePss\u003c/em\u003e survival and replication compared to cherry flower surfaces. It could be that higher concentrations of \u003cem\u003ePss\u003c/em\u003e in the feed facilitated acquisition and transfer by bees in an artificial setting, but this is not representative of what happens in real flowers.\u003c/p\u003e\u003cp\u003eImportantly, phage delivery by bees reduced \u003cem\u003ePss\u003c/em\u003e populations in both artificial and cherry flowers, despite a limited persistence of detectable phage in flowers after T1. Our findings support the novelty and feasibility of bee-mediated phage biocontrol of floral pathogens such as \u003cem\u003ePss\u003c/em\u003e, building on prior research into pollinator-based delivery of biological agents (Dedej et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Maccagnani et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Shafir et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The reduction in \u003cem\u003ePss\u003c/em\u003e populations following bee-mediated phage delivery is consistent with previous reports of phage efficacy against \u003cem\u003ePseudomonas\u003c/em\u003e spp. \u003cem\u003ein planta\u003c/em\u003e (Buttimer et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Rabiey et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Papp-Rupar et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In artificial flowers, there was a significant reduction in \u003cem\u003ePss\u003c/em\u003e populations at all time points. In comparison, there was a less apparent reduction in \u003cem\u003ePss\u003c/em\u003e in cherry flowers. The differences observed between artificial and cherry flower experiments could be down to several factors. For example, fewer cherry flowers were sampled at each time point due to flower availability, the proportion of flowers positive for phage were lower at T1 in the cherry flower experiment (33%) compared to the artificial experiment (88%). For the artificial flower experiments, samples were collected repeatedly from the same flowers. In contrast, different cherry flowers were collected at each time point in the real cherry flower experiments. Although the same concentration of \u003cem\u003ePss\u003c/em\u003e was used to inoculate both artificial and cherry flowers, the methods differed; cherry flowers were sprayed with \u003cem\u003ePss\u003c/em\u003e, while artificial flowers were directly spiked. It is possible that \u003cem\u003ePss\u003c/em\u003e took longer to fully establish in the cherry flowers, when sprayed, before phage application. Also, \u003cem\u003ePss\u003c/em\u003e or phage were in a more optimal environment in the sucrose feed compared to real flowers. The latter is supported by an increase in \u003cem\u003ePss\u003c/em\u003e population from T0 to T3 in the artificial flowers not treated with phage and a reduction in population between these timepoints for cherry flowers not treated with phage.\u003c/p\u003e\u003cp\u003eIn both artificial and cherry flower experiments there was reduction in \u003cem\u003ePss\u003c/em\u003e populations at time points where phage was not detectable on flowers. These findings suggest that initial phage activity may have reduced the bacterial population sufficiently to prevent detectable regrowth, or that low levels of phage, below the limit of detection, remained biologically active. Such residual phage activity could have continued to suppress the host population through sustained infection cycles, even in the absence of visible plaques. This phenomenon aligns with the concept of top-down control, in which higher trophic levels (phages) regulate the abundance and dynamics of lower trophic levels (bacteria). Even at low concentrations, phages can exert strong ecological pressure, limiting bacterial proliferation and maintaining population control. Similar observations have been reported in other microbial systems, where persistent phage activity, despite low detectable titres, was sufficient to influence bacterial community structure and dynamics (Abedon et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn addition, \u003cem\u003ePss\u003c/em\u003e was not detected on bees when phages were present in both artificial and cherry flower biocontrol experiments, indicating that phage activity may interrupt the cycle of pathogen dissemination, avoiding the spread of the pathogen by bees (Pattemore et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Biosecurity - Department of Industry Tourism and Trade, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This dual function, reducing pathogen load and halting transmission, highlights the potential of phage-biocarrier systems to contribute to sustainable crop protection strategies through both suppression and containment of bacterial diseases.\u003c/p\u003e\u003cp\u003eThe study underscores the value of continued investment in developing stable phage formulations and optimising bee vectoring techniques to enhance disease management in flowering crops. Notably, this approach may offer practical relevance beyond the laboratory, particularly for other high-value crops vulnerable to floral pathogens, such as \u003cem\u003eErwinia amylovora\u003c/em\u003e (causing fire blight in apples and pears), \u003cem\u003ePseudomonas syringae pv. actinidiae\u003c/em\u003e (\u003cem\u003ePsa\u003c/em\u003e) (causing bacterial canker in kiwifruit) and \u003cem\u003eAcidovorax citrulli\u003c/em\u003e (causing bacterial fruit blotch in cucurbits like watermelon and melon)(Burdman and Walcott, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; McCann et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Pedroncelli and Puopolo, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In New Zealand, for instance, the 2010 outbreak of \u003cem\u003ePsa\u003c/em\u003e devastated the kiwifruit industry, costing producers an estimated NZ\u003cspan\u003e$\u003c/span\u003e930\u0026nbsp;million over several years (Vanneste, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Because kiwifruit yield is directly tied to pollination efficiency, and the crop is increasingly grown under netting (which does not deter bumblebee activity), bumblebees have become the preferred pollinators. This makes them a promising vehicle for targeted phage delivery in commercial settings.\u003c/p\u003e\u003cp\u003eHowever, to realise the full potential of this technology, further research is needed to improve phage persistence in floral environments and to better understand the environmental and behavioural factors that influence phage transmission via pollinators. Ultimately, such work could unlock a novel, pollinator-compatible biocontrol method with direct relevance to crop protection strategies in high-value agricultural systems.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eBacterial isolates, phage isolates and bee species\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePss\u003c/em\u003e strain 9097, isolated from cankerous cherry (\u003cem\u003ePrunus avium\u003c/em\u003e) wood, Warwickshire, UK in 2010 (Hulin et al., 2018), was cultured in King\u0026apos;s Medium B (KB) at 28\u003csup\u003eo\u003c/sup\u003eC with shaking at 200rpm overnight or plated on KB with 1.5% agar (KBA) (King et al., 1954) at 28\u003csup\u003eo\u003c/sup\u003eC overnight.\u0026nbsp;For long-term storage at -80\u0026deg;C, overnight bacterial liquid culture was combined with an equal volume of 40% glycerol.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePss\u003c/em\u003e-infecting phage (MR6) isolated from a cherry orchard in the UK and previously characterised by Rabiey et al. (2020) was used in this study. Phage was amplified by an enrichment assay (see below) and was diluted in phosphate buffered saline for short-term storage at 4\u003csup\u003eo\u003c/sup\u003eC. For longer-term storage at -80\u003csup\u003eo\u003c/sup\u003eC, phage diluted in PBS was combined with an equal volume of 40% glycerol. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMini hives (8.5 W x 8 D x 13 H cm) of a European native bumblebee species \u003cem\u003eBombus terrestris audax\u003c/em\u003e (Natupol Seeds, Koppert, distributed by Dragonfli, UK) were used. These hives contained ~15 worker bees, five males and a male brood and are specifically designed for use in small scale crop pollination in glasshouses, polytunnels, and allotments. The hives do not have a queen and have a lifespan of only 6 - 8 weeks.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProduction of phage powder\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnrichment assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor large-scale propagation of phage, 2 ml of bacterial culture (OD\u003csub\u003e600\u003c/sub\u003e = 0.2, 2x10\u003csup\u003e8\u0026nbsp;\u003c/sup\u003eCFU ml\u003csup\u003e-1\u003c/sup\u003e) was added to a 50 ml Falcon tube containing 23 ml of KB medium and incubated at 28\u0026deg;C with shaking at 200 rpm. After one hour, 1 ml of phage suspension in PBS (\u0026gt;1x10\u003csup\u003e8\u003c/sup\u003e PFU ml\u003csup\u003e-1\u003c/sup\u003e) and 50 \u0026micro;l of 1M CaCl₂ (Sigma-Aldrich) and 50 \u0026micro;l 1M MgCl₂ (Sigma-Aldrich) was added to the KB medium and incubated for an additional 5 hours with shaking, after which 250 \u0026micro;l of chloroform (Sigma-Aldrich) was added and incubated for another hour. The suspension was centrifuged at 4500 rpm for 45 minutes at room temperature to pellet the bacterial cells. The supernatant was carefully collected, avoiding disturbance of the pellet, and filtered through a sterile PES 0.22 \u0026micro;m filter (SLS select) into a fresh 50 ml Falcon tube. 24% polyethylene glycol 8000 (Fisher Bioreagents) was added to a final concentration of 8% w/v, and the mixture was incubated overnight at 4\u0026deg;C. Phages were then precipitated by centrifugation (4500 rpm, 45 min, 4\u0026deg;C), and the resulting phage pellet was resuspended in 5 ml of phosphate buffered saline (PBS). Phage stocks were titered by spot assay (see below) and stored at 4\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSpot assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA bacterial lawn of \u003cem\u003ePss\u0026nbsp;\u003c/em\u003ewas prepared by mixing 100ul of \u003cem\u003ePss\u003c/em\u003e (OD\u003csub\u003e600\u003c/sub\u003e=0.2) with 5ml of molten but cooled soft KB agar (0.75%) containing\u0026nbsp;cycloheximide (100mg L\u003csup\u003e-1\u003c/sup\u003e) and cephalexin (40mg L\u003csup\u003e-1\u003c/sup\u003e). This was then poured on to the surface of pre-prepared KB agar (1.5%) plates. Phage stocks were serial diluted (10\u003csup\u003e1\u003c/sup\u003e-10\u003csup\u003e12\u003c/sup\u003e) in PBS and 3\u0026mu;l was spotted in duplicate onto the bacteriallawn. Plates were incubated at 28\u0026deg;C for 24 hours, after which clearing zones with a countable number of plaques were recorded to determine phage titre (PFU ml\u003csup\u003e-1\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhage powder formulation and testing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo prepare phage powder, 500mg (10% w/v) skimmed milk powder (Marvel) was added to 5ml phage diluted in PBS buffer (at 10\u003csup\u003e12-15\u003c/sup\u003e PFU ml\u003csup\u003e-1\u003c/sup\u003e), vortexed and then frozen at -80\u003csup\u003eo\u003c/sup\u003eC for 1 hour. Then the mixture was freeze-dried for 24hours at -50\u003csup\u003eo\u003c/sup\u003eC and 0.001mbar (Christ Alpha 1-2 LD plus, Sciquip) to yield ~500mg of phage powder. The resulting phage powder was subsequently titred by reconstituting 50mg of phage powder in 500\u0026mu;l PBS, serial diluted and spotted onto \u003cem\u003ePss\u003c/em\u003e lawns as described above.\u003c/p\u003e\n\u003cp\u003eThe stability of phage powder in artificial bee feed (20% sucrose solution) was then assessed under 4\u003csup\u003eo\u003c/sup\u003eC, lab (room temperature) and glasshouse conditions (20\u0026plusmn;2\u003csup\u003eo\u003c/sup\u003eC). \u0026nbsp;A 10\u0026mu;l pipette tip was used to spike 3ml of bee feed in six individual wells of a flat-bottomed 24-well cell culture plate (Falcon) with phage powder. Presence and titre of phage was tested by spot assay at T0 (immediately after inoculation), T1 (1-day post-inoculation), T4 (4-day post-inoculation), and T7 (7-day post-inoculation).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBacterial counts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine bacterial counts, bacterial cultures were serial diluted (10\u003csup\u003e1\u003c/sup\u003e-10\u003csup\u003e8\u003c/sup\u003e) in PBS and 3\u0026mu;l was spotted in duplicate onto KB agar plates containing\u0026nbsp;cycloheximide (100mg L\u003csup\u003e-1\u003c/sup\u003e) and cephalexin (40mg L\u003csup\u003e-1\u003c/sup\u003e). Plates were incubated at 28\u0026deg;C for 24-48 hours, after which colonies in spots with countable numbers were recorded to determine the colony-forming units (CFU ml\u003csup\u003e-1\u003c/sup\u003e).\u003c/p\u003e\n\u003ch3\u003e\u003cstrong\u003eBee-mediated transfer of phage and suppression of\u0026nbsp;\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePss\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;on artificial and cherry flowers\u003c/strong\u003e\u003c/h3\u003e\n\u003ch4\u003e\u003cstrong\u003eGeneral experimental conditions\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eAll experiments were conducted under controlled glasshouse conditions (day and night temperature of 20\u0026plusmn;2\u003csup\u003eo\u003c/sup\u003eC with natural lighting) using either artificial cherry-like flowers or real cherry flowers (\u003cem\u003ePrunus avium\u003c/em\u003e cv. Sweetheart) to assess the role of bees in phage dissemination and biocontrol of \u003cem\u003ePss\u003c/em\u003e. Experiments were performed in insect-proof polyethylene cages (Outsunny, 2 L\u0026times; 2 H x 1 W meter, with two mesh windows) in two neighbouring glasshouse compartments (each 20m\u003csup\u003e2\u003c/sup\u003e), one compartment was used for experiments using phage and the other as a phage-free control compartment.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eArtificial flower assays\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eArtificial flowers (5cm \u0026Oslash;) were design to mimic cherry flowers, with five pink petals and five yellow spots representing anthers around a central hole. These were then mounted on to brown card, laminated and placed on a 24 well flat-bottomed cell culture plate (Falcon), with the hole in the centre of the flower overlapping with one well of the plate containing 3 ml of 20% sucrose feed. For each experiment, 24 artificial flowers were distributed evenly within the cage in two rows with 10cm space in between them. In all experiments, six bees were released into the cage from a hive through a runway (10 L x 3 \u0026Oslash; cm tubing) containing ~500mg of phage powder (produced from 5ml phage lysate) attached to the circular hole of hive entrance/exit, unless stated otherwise. After bee release the hive remained in situ, but the entrance was sealed and if present, the phage runway removed. Feed from each flower was repeatedly sampled at four timepoints: prior to bee release (T0), and at 1-, 3- or 4-, and 7-days post-release (T1, T3 or T4, T7) and tested for presence and titre of phage or \u003cem\u003ePss\u0026nbsp;\u003c/em\u003eusing spot assays. Bees were collected at T7, individually incubated in 1ml PBS for 1hr at 28\u003csup\u003eo\u003c/sup\u003eC and 200rpm, and a spot assay was performed on the wash to determine phage or \u003cem\u003ePss\u003c/em\u003e titre.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eCherry flower assays\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eOne-year-old dwarf cherry trees (Sweetheart on Gisela 5 rootstock, 1.5 m height, sourced from Chris Bowers \u0026amp; Sons, UK) were grown in 12-litre pots containing Levingtons M2 compost supplemented with Osmocote\u0026reg; (rate of 3g L\u003csup\u003e-1\u003c/sup\u003e) and arranged within insect-proof cages as described above. Experiments were carried out between March-April when the cherry trees were in bloom. Each tree had at least 3-4 branches and bore at least 20-30 open flowers during experimentation. Bees were released after phage exposure, and individual flowers were destructively sampled by cutting the stalk with sterilised scissors and tested at T0, T1, T3 or T4, and T7. Flowers were incubated individually in 1ml PBS for 1hr at 28\u003csup\u003eo\u003c/sup\u003eC and 200rpm, and a spot assay was performed on the wash to determine phage or \u003cem\u003ePss\u003c/em\u003e titre. Bees were collected at T7 and tested in the same way.\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eExperimental designs\u003c/strong\u003e\u003c/h4\u003e\n\u003ch4\u003e\u003cstrong\u003ePhage transfer from hive to flowers\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003eTo test whether bees can transfer phages from the hive to flowers, bees were released from the hive through a runway containing phage powder attached to the hive entrance/exit. At each timepoint, sucrose feed from artificial flowers (n = 25) or individual cherry flowers (n = 12) were sampled and tested for phage presence and titre.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ePhage transfer between flowers\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo test whether bees can move phage between flowers, the feed of a subset of artificial flowers (n = 6) was spiked with phage powder (as for 2.2.3) or cherry flowers on a subset of branches (n = 6) were each inoculated with ~3mg phage powder using a paintbrush, previously sterilised in 70% ethanol, at T0. Bees were then released from a clean hive (no phage powder). At each time point, inoculated feed or cherry flowers (n = 6) were sampled and tested for phage presence and titre alongside feed from uninoculated artificial flowers (n = 18) and individual cherry flowers (n = 12).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBacterial transfer between flowers\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo test whether bees could transfer \u003cem\u003ePss\u003c/em\u003e between flowers, the feed of a subset of artificial flowers (n = 6) was inoculated with \u003cem\u003ePss\u003c/em\u003e to a final concentration of 2x10\u003csup\u003e7\u003c/sup\u003e CFU ml\u003csup\u003e-1\u003c/sup\u003el, or cherry flowers on a subset of branches (n = 12) were spray inoculated using a hand operated mist sprayer with each flower receiving \u0026nbsp;one mist spray (~15\u0026mu;l ) of \u003cem\u003ePss\u0026nbsp;\u003c/em\u003eat 2x10\u003csup\u003e7\u0026nbsp;\u003c/sup\u003eCFU ml\u003csup\u003e-1\u003c/sup\u003e. Bees were then released from a clean hive (no phage powder). At each time point, inoculated feed (n = 6) or cherry flowers (n=12) were sampled and tested for \u003cem\u003ePss\u003c/em\u003e presence and count alongside feed from uninoculated artificial flowers (n =18) and individual cherry flowers (n = 12).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBee-mediated phage biocontrol of Pss\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo test the efficacy of phages delivered by bees in controlling \u003cem\u003ePss,\u003c/em\u003e artificial and cherry flowers were inoculated with \u003cem\u003ePss\u003c/em\u003e as described above. Two cages/conditions were compared: (i) bees released from a hive through a runway containing phage powder attached to the hive entrance/exit (treatment) and (ii) bees released from a clean hive without phage exposure (control). At each time point, feed from artificial flowers (n = 24) and cherry flower (n = 12) were sampled and tested for \u003cem\u003ePss\u003c/em\u003e and phage presence and titer/count.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical tests and software\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were carried out in IBM\u0026reg; SPSS\u0026reg; Statistics version 28. Firstly, Shapiro-Wilk tests for normality were conducted on the data (Shapiro and Wilk, 1965).\u0026nbsp;Generalized linear models (GLM) with negative binomial log link were then conducted\u0026nbsp;(IBM SPSS Statistics, 2025)\u0026nbsp;to determine main and interaction effects of timepoint and treatment (4\u003csup\u003eo\u003c/sup\u003eC, lab, glasshouse) on phage powder viability in artificial bee feed and phage treatment and timepoint on \u003cem\u003ePss\u003c/em\u003e counts (CFU ml\u003csup\u003e-1\u003c/sup\u003e) in the artificial and cherry flower biocontrol experiments.\u0026nbsp;This GLM model fitted the data best, when compared to a Poisson log link model, also suitable for modelling count data.\u0026nbsp;To identify significant differences (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in bacteria count between treatment groups within a timepoint post hoc Mann-Whitney U tests were conducted (Mann and Whitney, 1947). Figures were made in GraphPad prism 9 (Boston, Massachusetts USA, www.graphpad.com).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eand Acknowledgments\u003c/p\u003e\u003cp\u003eThe authors acknowledge funding from Carus Animal Health Ltd and The University of Warwick start-up fund, supporting S.F.G. and M.R. The authors acknowledge the support of Horticultural Services at the University of Warwick for plant care.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eS.G., G.F., R.O., and M.R. conceived and contributed to experimental design. S.G., S. C., K.C-N., carried out experimental work. S.G., D.C., and M.R. performed statistical analysis. S.G. and M.R. wrote the manuscript. All authors contributed to interpretation of results, read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors acknowledge funding from Carus Animal Health Ltd and The University of Warwick start-up fund, supporting S.F.G. and M.R. The authors acknowledge the support of Horticultural Services at the University of Warwick for plant care.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data have been included within the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbedon, S.T., Herschler, T.D., and Stopar, D. (2001). Bacteriophage latent-period evolution as a response to resource availability. \u003cem\u003eApplied and Environmental Microbiology\u003c/em\u003e 67\u003cstrong\u003e,\u003c/strong\u003e 4233-4241.\u003c/li\u003e\n\u003cli\u003eAhern, S.J., Das, M., Bhowmick, T.S., Young, R., and Gonzalez, C.F. (2014). 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Enhancing the stability of bacteriophages using physical, chemical, and nano-based approaches: A review. \u003cem\u003ePharmaceutics \u003c/em\u003e[Online], 14.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"phage, bumblebee, Pseudomonas, cherry floral pathogen, biocontrol","lastPublishedDoi":"10.21203/rs.3.rs-7053094/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7053094/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBacteriophages, phages or viruses that specifically infect bacteria, have shown promise for the biocontrol of bacterial plant diseases. However, one of the main challenges of using phages in agricultural systems is their precision application, being able to deliver an effective dose to the site of bacterial infection. In this study, a series of artificial and real cherry flower experiments was conducted to test whether commercially managed bumblebees (\u003cem\u003eBombus terrestris audax\u003c/em\u003e) could deliver phage effective against the cherry canker pathogen \u003cem\u003ePseudomonas syringae\u003c/em\u003e pv. \u003cem\u003esyringae\u003c/em\u003e (\u003cem\u003ePss\u003c/em\u003e). Freeze-dried phage powder was formulated with powdered-skimmed milk and when tested, was found to retain viability for seven days in artificial bee feed after storage at room temperature or under glasshouse conditions. In both artificial and cherry flower experiments, bees successfully transferred the formulated phage from their hive to up to 88% of flowers, resulting in significant reduction in \u003cem\u003ePss\u003c/em\u003e populations. Bees were also able to transfer phage between cherry flowers. The application of phages disrupted the cycle of \u003cem\u003ePss\u003c/em\u003e transmission by bees. These results highlight the potential of bee-mediated phage delivery as an effective biocontrol strategy against floral pathogens like \u003cem\u003ePss\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Bee-mediated delivery of bacteriophage for biocontrol of the cherry canker pathogen Pseudomonas syringae pv. syringae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-14 18:24:38","doi":"10.21203/rs.3.rs-7053094/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fd59184d-b65a-4519-97bb-e70bc58d4591","owner":[],"postedDate":"July 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":51388764,"name":"Biological sciences/Biological techniques"},{"id":51388765,"name":"Biological sciences/Biotechnology"},{"id":51388766,"name":"Biological sciences/Microbiology"},{"id":51388767,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2025-09-04T06:53:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-14 18:24:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7053094","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7053094","identity":"rs-7053094","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}