bioRxiv preprint doi: https://doi.org/10.1101/797738; this version posted October 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Insects as phyllosphere microbiome engineers: effects of aphids on a plant pathogen. 1 Melanie R. Smee*, Imperio Real-Ramirez and Tory A. Hendry. 2 Department of Microbiology, Cornell University, Ithaca, NY 14853, USA 3 *Correspondence: 4 Melanie R. Smee; [email protected] 5 6 Keywords – Aphids; environmental bacteria; honeydew; plant-microbe-insect interactions; plant 7 pathogens; Pseudomonas syringae. 8 9 Word Count = 6226 (inc. in-text citations) 10 Running title: Insects influence epiphytic bacteria 11 Manuscript type: Article 12 Supplementary files: “AphidEffects_SOM.pdf” 13 14 1 bioRxiv preprint doi: https://doi.org/10.1101/797738; this version posted October 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 15 Abstract – 200 words. 16 Insect herbivores are common in the phyllosphere, the above-ground parts of plants, and encounter 17 diverse plant-associated bacteria there, yet how these organisms interact remains largely unknown. 18 Strains of the bacterium Pseudomonas syringae grow well epiphytically and have been shown to 19 grow within and kill hemipteran insects like the pea aphid, Acyrthosiphon pisum. Aphids are 20 hypothesized to be an alternative host for these epiphytic bacteria but it is unclear if aphids provide 21 fitness benefits to these bacterial pathogens. To determine if epiphytic bacteria could be adapted 22 for infecting aphids, we characterized 21 strains of P. syringae for epiphytic ability and virulence 23 to pea aphids and found that the two traits were positively correlated. For a subset of strains, we 24 tested if the bacteria derived a fitness benefit from the presence of aphids. Some strains benefited 25 significantly, with up to 18.9% higher population densities when aphids were present, and lower 26 starting population density was predictive of higher benefit from aphid presence. However, further 27 investigation found that honeydew, the sugary waste product of aphids, and not growth in aphids, 28 increased P. syringae growth on leaves. This suggests that aphids may be important microbiome 29 engineers in the phyllosphere, but evolutionarily dead-ends for epiphytic bacteria. 30 31 32 33 34 2 bioRxiv preprint doi: https://doi.org/10.1101/797738; this version posted October 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 35 1 Introduction 36 The phyllosphere, consisting of all the aerial parts of plants, is a complex and dynamic 37 environment (Lindow and Brandl 2003; Vorholt 2012; Farré-Armengol et al. 2016; Laforest- 38 Lapointe et al. 2017; Saleem et al. 2017). Estimated at having approximately double the global 39 surface area of the land, the upper and lower leaf surfaces are an important, yet heterogeneous, 40 environment (Vorholt 2012). Leaf surfaces provide the stage for a multitude of interactions 41 between plants, animals and microbes. Work on these interactions shows co-evolutionary 42 dynamics between microbes and hosts (Koskella and Brockhurst 2014; Flórez et al. 2017), but 43 many such systems, especially tripartite interactions, are understudied despite their potential 44 importance in diverse systems. Some species of plant-associated bacteria form epiphytic 45 populations on leaf surfaces, with densities averaging 105 - 107 cells/cm2 (Beattie and Lindow 46 1995; Andrews and Harris 2000; Hirano and Upper 2000; Monier and Lindow 2004). For 47 phytopathogens, such high densities can lead to an increased chance of infection (Hirano and 48 Upper 2000), and epiphytic bacteria likely influence plants in other ways as well (Hornschuh et al. 49 2002; Abanda-Nkpwatt et al. 2006). At a local scale, communities of epiphytic bacteria are often 50 dominated by a few bacterial taxa (Bulgarelli et al. 2013), yet at a broader scale, even on a single 51 plant, bacterial communities can be diverse and variable (Kembel et al. 2014; Meaden et al. 2016). 52 Hence, it can be difficult to explain observed bacterial distributions. 53 Attempts to uncover what generates and maintains microbial diversity in the phyllosphere have 54 explored various abiotic and biotic factors. Frequently changing, and often extreme, temperatures, 55 humidity levels, radiation, and limited nutrients all combine to produce a challenging habitat 56 (Hirano and Upper 2000), as well as competition from other microbial species (Wilson and Lindow 57 1994), and even interactions with bacteriophages (Morella et al. 2018). Animals can also have an 3 bioRxiv preprint doi: https://doi.org/10.1101/797738; this version posted October 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 58 impact on the distribution of microbes in the phyllosphere, such as vectoring of pathogens on crops 59 (Perilla-Henao and Casteel 2016). Although phyllosphere microbes are known to impact animal 60 hosts (Leroy et al. 2011a; Kupferschmied et al. 2013; Mason et al. 2014; Farré-Armengol and 61 Junker 2019), few studies have considered the potential ecological or evolutionary implications of 62 interactions with animals for the microbe (Biere and Bennett 2013). To further complicate matters, 63 tripartite interactions in the phyllosphere often involve plant-mediated traits, which may be 64 difficult to discern from direct interactions (Stout et al. 2005; Humphrey et al. 2014). For instance, 65 herbivores can affect the growth of microorganisms on forest trees (Stadler and Müller 2000), and 66 in particular, leaves damaged by lepidopteran larvae host a greater diversity of bacteria, or a change 67 in bacterial community composition (Müller et al. 2003). However, it is not known if this effect is 68 plant-mediated. 69 The pea aphid, Acyrthosiphon pisum, is a major agricultural pest and acts as both potential host 70 and vector for some strains of Pseudomonas syringae, a ubiquitous plant-associated bacterium and 71 phytopathogen (Stavrinides et al. 2009; Smee et al. 2017). Along with a common host plant, Vicia 72 faba, they are a tractable system for exploring questions about tripartite interactions in the 73 phyllosphere. P. syringae is a diverse species complex in the Gammaproteobacteria, which can 74 differ greatly in their phenotypes despite being very phylogenetically close (Baltrus et al. 2012). 75 Strains vary dramatically in their ability to colonize plants epiphytically, but also in their virulence 76 to both their plant host and to A. pisum. Some strains are able to cause the death of up to 83% of 77 aphids in a 72 hour period (Smee et al. 2017). The variation in virulence to A. pisum across 12 78 strains of P. syringae was found to be correlated with epiphytic ability of the strains (Smee et al. 79 2017). Yet it is unclear why this correlation exists, and why virulence to hemipteran insects would 80 be adaptive for epiphytic bacteria. Several strains of P. syringae replicate to high densities (106 to 4 bioRxiv preprint doi: https://doi.org/10.1101/797738; this version posted October 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 81 108 CFUs per aphid) within aphid hosts and are also found in the honeydew of aphids infected 82 with at least one pathovar, P. syringae pv. syringae B728a (from here on referred to as PsyB728a) 83 and deposited onto plants (Stavrinides et al. 2009; and unpublished data). Perhaps the ability of 84 bacteria to grow on leaf surfaces may better allow them to infect transient insects visiting plants, 85 but in that case has high virulence to insects been selected for in epiphytic strains? Do such strains 86 experience a trade-off between virulence and dispersal, where growing inside an aphid for longer 87 could allow for more dispersal? To address these evolutionary questions, we must first understand 88 if the fitness of epiphytic bacteria is indeed influenced by interactions with insect hosts. 89 Firstly, we undertook a robust test of the hypothesis that epiphytic ability and virulence to aphids 90 are correlated, using a total of 21 P. syringae strains. Secondly, we determined whether this 91 correlation may be adaptive by testing if P. syringae benefits from using aphids as hosts, by 92 comparing epiphytic bacterial densities on plants with and without aphids. We lastly explored what 93 traits may influence this benefit, in the absence of plant-mediated effects, by separating fitness 94 benefits from growth inside insects and the benefits derived from changes that insects create in the 95 phyllosphere, such as deposition of aphid honeydew, a sugar-rich waste product. 96 97 98 99 100 5 bioRxiv preprint doi: https://doi.org/10.1101/797738; this version posted October 8, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 101 2 Materials and Methods 102 2.1 Insect and bacterial cultures 103 The pea aphid, A. pisum, reproduces parthenogenetically under summer conditions, so lab 104 populations were kept on fava bean (V. faba) plants at 20°C and a light:dark 16:8 hour cycle. We 105 used A. pisum clone CWR09/18 collected by Angela Douglas in Freeville, NY in 2009, which does 106 not harbor any bacterial symbionts aside from the obligate symbiont Buchnera aphidicola 107 (Macdonald et al. 2012). Aphids were age-controlled on V. faba for all experiments so that all 108 individuals were 5-6 days old at the start of each assay.