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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 ; honeydew; plant-microbe-insect interactions; plant

7 pathogens; 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”

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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 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.

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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

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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 , 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

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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.

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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.

109 All strains of P. syringae used in the current study displayed resistance to the antibiotic rifampicin,

110 apart from one, pv. syringae B301D. Therefore, all bacterial cultures were grown and kept on

111 King’s B (KB) media with added rifampicin (50 ng/μl) apart from pv. syringae B301D which did

112 not have rifampicin added. Culture plates were kept incubated at 27°C and overnight cultures were

113 grown in an incubator shaker set at 28°C and 220 rpm.

114 2.2 In vitro pathogenicity assays

115 Oral infection assays were used to test the virulence of a total of 21 strains of P. syringae (Table

116 1). These strains represent the phylogenetic breadth of plant pathogenic strains commonly included

117 within the species complex P. syringae (phylogroups 1, 2, 3 and 5 are included), with the highest

118 representation within phylogroup 2, which has previously been found to include highly epiphytic

119 and insect virulent strains (Smee et al. 2017). We note that some strains are considered distinct

120 species in some classifications (Sarkar and Guttman 2004; Bull et al. 2011; Borschinger et al.

121 2016). Additionally, some of the included strains are environmental isolates from water sources or

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122 are thought to be non-pathogenic to plants. All isolates were provided by David Baltrus and

123 references for origins of strains are given in Table 1.

124 Table 1 – A summary of P. syringae strains used in the current study. MLST phylogroup designation and

125 original source are given, with GenBank accession numbers for genome sequences used in phylogenetic

126 tree construction (Borschinger et al. 2016). Those strains for which data are already reported by Smee et al.

127 (2017) are highlighted in grey.

Genbank Name Pathovar Strain Group Source accession

*1 PtoDC3000 tomato DC3000 1 Solanum lycopersicum AE016853

Pja301072 japonica MAFF 301072 PT*2 2 Hordeum vulgare AEAH 00000000

Ptt50252 aptata DSM50252*3 2 Beta vulgaris AEAN 00000000

Ppi1704B pisi 1704B*3 2 Pisum sativum AEAI 00000000

PsyCC1543 syringae CC1543*4 2 Fresh lake water AVEJ 00000000

Dodecantheon PsyCC1458 syringae CC1458*4 2 AVEN 00000000 pulchellum

PsyCC440 syringae CC440*5 2 Cucumis melo AVEC 00000000

PsyCC457 syringae CC457*5 2 Cucumis melo AVEB 00000000

PsyB728a syringae B728a*6 2 Phaseolus vulgaris CP000075

PssB48 syringae B48*7 2 Prunus persica LGKT 00000000

PsyB301D syringae B301D 2 Pyrus communis CP005969

Psy1212 syringae 1212 2 Pisum sativum AVCR 00000000

PsyUB303 syringae UB303*8 2 Fresh stream water AVDZ 00000000

PsyUSA011 syringae USA011*5 2 Fresh stream water AVDX 00000000

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Pac302273 aceris MAFF 302273 PT*9 2 Acer sp. AEAO 00000000

N/A Cit7*10 2 citrus leaf surface AEAJ 00000000

N/A TLP2 2 potato leaf surface Gp0012374◊

Pph1448a phaseolicola‡ 1448a*11 3 Phaseolus vulgaris CP000058

Pph1644r phaseolicola‡ 1644r*12 3 Vigna radiata AGAS 00000000

PmaES4326 maculicola¥ ES4326*13 5 Raphanus sativus AEAK 00000000

PsyUB246 syringae UB246*5 13 Fresh stream water AVEQ 00000000

128 ‡Pathovar is renamed as Pseudomonas savastanoi, not P. syringae.

129 ¥ Strain is renamed as Pseudomonas cannabina pv. asilensis.

130 ◊ JGI project id, not GenBank accession.

131 *References for strain isolation information where available:

132 1(Cuppels 1986); 2(Mukoo 1955); 3(Baltrus et al. 2011); 4(Morris et al. 2008); 5(Morris et al. 2010); 6(Loper

133 and Lindow 1987); 7(Mott et al. 2016); 8(Berge et al. 2014); 9(Sawada et al. 1999); 10(Hirano and Upper

134 1990); 11(Teverson 1991); 12(Baltrus et al. 2012); 13(Davis et al. 1991).

135

136 Pathogenicity assays utilizing artificial aphid diet (Prosser and Douglas 1991) were carried out as

137 detailed in Smee et al. (2017), with no alterations. Further details are given in Supplementary

138 Online Materials. Briefly, bacterial suspensions at an OD600 of 0.8 were added in a 1:5 ratio to

139 artificial aphid diet with 10 mM MgCl2 used for the negative control treatment. Feeding sachets

140 of diet (and bacteria) were inverted above 5-6 day old, age-controlled aphids singly placed in wells

141 of a 96-well plate, to allow monitoring of individuals. Assay plates were kept at 20°C and every

142 24 hours the feeding sachet was replaced with another sachet of sterile diet only. At 72 hours,

143 which has been previously shown to be indicative of virulence differences between strains (Smee

144 et al. 2017), mortality was recorded. Assays were replicated a minimum of three times.

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145 Assays utilizing artificial diet can be conducted in controlled environments without additional

146 biotic or abiotic factors. However, to demonstrate that the mortality rates on artificial diet aren’t

147 an artifact of keeping aphids on diet for extended periods, we infected 100 aphids on diet but

148 moved them after 24-hours to fresh two-week-old V. faba plants, and left them for another 48-

149 hours at 20°C. This was repeated twice for four strains, two that are considered highly pathogenic

150 to aphids, and two that are less so (PsyB728a; Cit7; PtoDC3000; and Pph1448a) as well as a control

151 group that had 10 mM MgCl2 buffer mixed with artificial diet instead of bacterial solution.

152 2.3 Epiphytic assays

153 To assess epiphytic ability, overnight bacterial cultures were resuspended to a final OD600 of 1.0

154 in approximately 20 mL of 10 mM MgCl2. This was then sprayed onto the upper and lower leaf

155 surfaces of eight to ten leaf pairs of V. faba (three to four individual plants in one pot) using a fine

156 mist spray bottle, until run-off. Once fully dried, plants were incubated at 22°C and 90 ± 5%

157 humidity for 48 hours. From each pot of V. faba, ten 1.27 cm diameter discs were taken using a

158 sterilized cork borer from across the leaf pairs for each of three replicates, and placed into 10 ml

159 of 10 mM MgCl2 buffer, then sonicated for 10 minutes and vortexed to dislodge epiphytic bacteria.

160 Three serial dilutions were plated from each replicate, which were averaged to give a CFU count

161 per 10 ml for each strain. The entire process was replicated a minimum of three times for each

162 strain (four replicates were achieved for two pathovars: PsyB728a and Psy1212). Only CFU counts

163 from dilutions containing 5-50 countable colonies were included, to minimize errors.

164 2.4 Impact of aphid presence on plants

165 Eight strains varying in epiphytic ability and virulence to aphids were tested to determine if the

166 presence of aphids on plants benefits epiphytic strains of P. syringae. These strains included four

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167 that were deemed to be good epiphytes and highly virulent to aphids (PsyB728a; PsyCC1458;

168 Cit7; and TLP2), and four that are poorer epiphytes and less virulent to aphids (PtoDC3000;

169 Ptt50252; PsyCC1543; and Pph1448a).

170 Overnight bacterial cultures were prepared to a final OD600 of 0.8 in approximately 40-50 ml of

171 10 mM MgCl2. Three two-week old V. faba plants, each with at least ten leaf pairs, were sprayed

172 as before. After 48 hours, long enough for the epiphytic bacterial populations to establish well,

173 200 age-controlled 5-6 day old aphids were added to one of the three plants. The aphids were

174 placed on the soil in a central location and the plant covered with a perforated 25.4 x 40.6 cm bread

175 bag. A second plant was also covered with a bread bag, but without any aphids. The final plant

176 was taken to be processed as before for a 48-hour baseline CFU count (per 10 ml). Finally, an

177 unsprayed two-week old V. faba plant had 200 aphids added, a bread bag covering it, and it was

178 placed with the two remaining plants as a control for aphid survival. Two days later, twenty aphids

179 were sampled from each of the sprayed plants and crushed individually with a pestle in 100 µl 10

180 mM MgCl2 and then the entire suspension plated with sterile glass spreading beads on King’s B

181 media with added rifampicin (50 ng/μl). Colonies were checked 2-3 days later, and an individual

182 was categorized as infected if more than ten CFUs were present. Twenty aphids from the control

183 plant were removed to ensure numbers were kept equal across all plants. The experiment ended

184 after a total of seven days of which aphids were present on plants for five. The entire process was

185 repeated three times for each strain. At the final time point we measured aphid survival on the

186 control, unsprayed, plant as well as the bacterial plants, and again tested twenty aphids for

187 infection, as before. We also took measurements of epiphytic CFU counts as before, using ten leaf

188 discs in three replicates per plant.

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189 Previous studies have shown that aphids can become infected from epiphytic bacteria on leaf

190 surfaces (Stavrinides et al. 2009; Hendry et al. 2018). Consequently, we hypothesized that the

191 mortality of aphids feeding on epiphytically infected plants should be similar to that which we see

192 when feeding bacteria suspended in artificial diet to aphids, yet likely take longer to achieve as

193 aphids would be less likely to become rapidly or heavily infected. To demonstrate this for one

194 pathovar (PsyB728a), we moved the surviving adult aphids at the end of an experiment onto fresh

195 two-week-old V. faba plants for another four days. Moving the aphids to fresh plants meant that it

196 was easier to distinguish the surviving original adults from their offspring over a longer time frame.

197 This was repeated three times.

198 2.5 Honeydew assays

199 At least one strain of P. syringae grows to high titers inside aphids and is secreted in aphids’

200 honeydew (PsyB728a: Stavrinides et al. 2009). We therefore investigated whether bacterial growth

201 inside aphids leads to higher epiphytic populations and if honeydew is beneficial to epiphytic P.

202 syringae. We used two of the strains that benefitted most from the presence of aphids: Cit7, which

203 is highly virulent to aphids, and PtoDC3000, which is much less virulent.

204 Bacterial cultures at a final OD600 of 0.8 were sprayed onto V. faba leaves, as well as added to

205 artificial diet at a ratio of 1:5 bacterial culture:diet. The experimental design, described in detail in

206 the Supplementary Online Materials, allowed us to eliminate the effect of aphid feeding inducing

207 plant-mediated effects, as the honeydew was able to fall onto leaves with epiphytic bacteria, but

208 the aphids were unable to feed on the leaves (Figure S1). This was achieved by separating aphids

209 feeding on artificial diet from sprayed leaves of V. faba with a piece of fine mesh. There were three

210 treatments replicated three times within each experimental block, which was also repeated

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211 independently three separate times. The three treatments were as follows: the entire set up with

212 just sterile diet in the feeding sachet and no aphids present; the same but with aphids present; and

213 finally, both with aphids present and also with the bacterial culture added to the feeding sachet for

214 the first 24-hours. All feeding sachets were refreshed after 24-hours, and then left for a further 48-

215 hours before the leaves were processed for epiphytic growth as before. The dishes were kept at

216 22°C and 90 ± 5% humidity for the duration of the experiment, and under UV-blocking plastic for

217 the first 24-hours whilst bacteria were present, as aphids avoid fluorescent strains of P. syringae

218 and hence may feed less (Hendry et al. 2018).To further investigate whether strains of P. syringae

219 can utilize aphid honeydew as a resource, we grew all eight strains from the previous section in

220 artificial honeydew medium. This medium included diluted artificial aphid diet, so that the sucrose

221 content was reduced to the equivalent of 2% w/v and it contained low levels of other nutrients

222 found in honeydew (Leroy et al. 2011b), and then we added fructose at 2% w/v and glucose at 4%

223 w/v as suggested by Sinka et al. (2009). We note that in nature honeydew composition will likely

224 vary depending on factors related to both the aphid and host plant (Leroy et al. 2011b; Pringle et

225 al. 2014). Our goal was not to achieve a full representation of pea aphid honeydew, but to

226 recapitulate key nutrient and sugar factors. Overnight bacterial cultures were resuspended to a final

227 OD600 of 0.8 and 20 μl added to artificial honeydew for a final volume of 180 μl and incubated at

228 22°C with constant shaking on a Bioscreen CTM (Growth Curves USA) to generate growth curves

229 over a period of 48 hours. To determine intrinsic growth rate of these strains, we repeated the

230 growth assay above using just KB media with added rifampicin (50 ng/μl).

231 Additionally, in order to eliminate possible plant-mediated effects from the benefit strains gained

232 during our original experiment on whole plants, we conducted the same experiment but instead of

233 the addition of aphids in one treatment, we sprayed plants with artificial honeydew. The honeydew

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234 was sprayed from a distance of approximately 15 cm so that individual droplets were distributed

235 across leaves and the plants were immediately placed back in high humidity. As before, these were

236 sprayed with honeydew after an initial period of 48-hours and then left another five days before

237 processing and counting CFUs to determine if there was any benefit. The two strains that benefitted

238 most in the first experiment were again used for this assay: Cit7 and PtoDC3000. This assay was

239 repeated three times.

240 2.6 Statistical Analyses

241 All statistical analyses were conducted in R version 3.5.2 (R Core Team 2018). In all analyses,

242 experimental block was initially included as a fixed effect and removed if non-significant, unless

243 otherwise stated. In addition, all models were checked for normality of residuals and

244 heteroscedasticity and were fine unless otherwise stated. All epiphytic growth data (colony-

245 forming units: CFUs) were log10-transformed for analyses. All posthoc tests were assessed using

246 the R package ‘emmeans’ (Lenth 2019), which uses a Tukey adjustment for multiple comparisons

247 and a significance level of 0.05.

248 To discern the benefit of aphid presence to epiphytic bacteria, two-tailed t-tests were conducted

249 on each pair of ‘with aphids’ and ‘no aphids’ at the 5-day timepoint. For each strain, the epiphytic

250 CFU value of the ‘no aphids’ treatment was then subtracted from the value of the ‘with aphids’

251 treatment to give a measure of benefit. Relative benefit for each strain was calculated by dividing

252 benefit by the ‘no aphids’ CFU value. Where possible, data from individual experimental blocks

253 were all used in analyses as there was much variation across blocks. However, for some Pearson’s

254 correlations with other experimental data, mean values for each strain had to be used.

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255 Differences in virulence to aphids, epiphytic ability, infection rates, survival on plants, honeydew

256 deposited on leaves and bacterial growth in artificial honeydew medium were all analyzed using

257 General Linear Models (GLMs) with Gaussian distributions. Response variables were either

258 proportion of aphids dead or infected, epiphytic growth data, or OD600 values at the 48-hour time

259 point for bacterial growth. Fixed explanatory effects were bacterial strain in most analyses, with

260 time period as well for comparing infection frequencies. When comparing survival after nine days

261 on plants, a Welch’s Two-Sample t-test was used due to unequal variances. To investigate

262 honeydew effects, the two strains were analyzed separately as we were not interested in comparing

263 across strains, only within treatments.

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274 3 Results

275 3.1 Epiphytic ability correlates with virulence to aphids

276 Some of the data presented here have been previously published by Smee et al. (2017). This

277 includes both pathogenicity to A. pisum and epiphytic assays for nine strains (Table 1, highlighted

278 in grey: PsyB728a; PtoDC3000; Pja301072; Ptt50252; Ppi1704B; Pac302273; Cit7; TLP2; and

279 Pph1448a). For the current study, this was expanded to include another 12 strains, and replicates

280 for epiphytic assays were increased from two to a minimum of three.

281 Across the 21 strains of P. syringae there was much variation in both their epiphytic ability on V.

282 faba and their pathogenicity to aphids (Figure 1: GLM: F20,171 = 22.56, p < 0.001; GLM: F20,72 =

283 5.31, p < 0.001, respectively). Some of the best epiphytes were capable of colonizing V. faba leaves

284 at over 1 x 107 CFUs per 25.8 cm2 area of leaf (the upper and lower surfaces of 10 leaf discs each

285 with 1.27 cm diameter). Three of these strains (PsyB48, PsyUSA011 and Cit7) were also amongst

286 the most pathogenic to aphids, causing over 74% death after 72 hours post-infection by artificial

287 diet. Similarly, some of the poorest epiphytes were far less virulent to aphids, indicating that the

288 two traits may be linked and there may be a common factor important for success or failure in both

289 environments (Figure 2; Pearson’s product-moment correlation: t = 4.21, d.f. = 19, p < 0.001, R =

290 0.69). Note that the correlation is not due to aphids becoming infected more by the better epiphytes,

291 since the mortality data is from controlled conditions in artificial diet, not from on plants. Also,

292 the epiphytic ability of these strains may differ in nature, as our assays started with high founding

293 populations of epiphytic bacteria and were kept at high humidity and ideal conditions for growth

294 of P. syringae. Hence, our results may reflect maximum densities achievable on fava bean plants.

295

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Figure 1 – A comparison of epiphytic ability and virulence to aphids across 21 strains of P. syringae. The

phylogenetic tree was constructed in PhyloPhlAn (Segata et al., 2013) using 382 of the most conserved protein

sequences across the 21 genomes, and rooted using the representative genome of Pseudomonas fluorescens F113

from GenBank (accession number NC_016830). All other protein sequences were also obtained from GenBank

(see Table 1). The epiphytic ability of the 21 strains is measured as log10 CFU counts per 10 leaf discs sampled, and

the y-axis starts at the minimum CFU count we might see in our dilutions, given accuracy cut-offs of 5-50 colony

counts per 5 μl droplet, taken from an initial 10 ml sample. The virulence of each strain to aphids is represented by

aphid mortality at 72 hours post-infection in artificial diet. Mean values are plotted ± standard error.

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300

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80 R = 0.69 , p = 0.00047

60

40 Aphid mortality at 72h (%) 20

5 6 7 log10 (mean CFU count per 10 leaf discs)

Figure 2 – Correlation between the epiphytic ability of each of the 21 P. syringae strains and their virulence to

aphids after 72 hours. Pearson’s correlation coefficient (R) is presented, and the grey area denotes 95% confidence

intervals. Those strains used in further experiments are shown as triangular points.

301

302 3.2 Aphid presence can benefit epiphytic bacteria

303 To determine if the correlation between epiphytic ability and virulence could be adaptive, we

304 investigated whether strains of P. syringae growing epiphytically actually benefitted from the

305 presence of aphids on plants. If using aphids as hosts is a viable ecological and evolutionary route

306 for the bacteria, we would expect that there would be some benefit gained from the two approaches.

307 Better epiphytes kill their hosts more quickly, so perhaps they would more quickly return to their

308 preferred niche of leaf surfaces. Poorer epiphytes stay in the living host for longer, which may

309 increase their epiphytic titers by dispersal via the honeydew secreted by the hosts.

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310 We selected eight strains from various points along the correlation in Figure 2, denoted by

311 triangular points on the plot. When comparing CFU counts from V. faba plants either with or

312 without aphids present for five days, several of the strains benefitted and demonstrated significant

313 increases in bacterial densities (Figure 3A). However, when comparing this benefit (change in

314 CFU counts) to the virulence of each strain, there was no correlation (Pearson’s product-moment

315 correlation: t = 0.51, d.f. = 6, p = 0.63, R = 0.21). There was also no correlation with the epiphytic

316 ability of each strain, defined using epiphytic data in Figure 1 (Pearson’s product-moment

317 correlation: t = 0.24, d.f. = 6, p = 0.82, R = 0.10). Using the relative benefit gained by each strain

318 (benefit divided by epiphytic CFU counts without aphids present) did not change these results.

A B 8 Baseline 48h *** No aphids *** R = − 0.44 , p = 0.032 ) 0.20 Strain 10 With aphids * *** Pph1448a 7 Ptt50252 0.15 PsyB728a ** PsyCC1458 PsyCC1543

0.10 Cit7 6 PtoDC3000 TLP2

0.05 e benefit of aphid presence v 5 Relati 0.00 CFU count per 10 leaf discs (log

4 −0.05 4 5 6 7 Cit7 TLP2 log10 Baseline CFU count Ptt50252 Pph1448a PsyCC1543 PtoDC3000 PsyCC1458 PsyB728a Strains Figure 3 – Benefit of aphid presence. (A) Epiphytic CFU counts for each strain sprayed on to fava beans.

Baseline values are after 48-hours, then aphids were added and five days later CFU counts were taken from

plants with and without aphids. Strains are ordered by 48-hour baseline values, smallest first. Mean values

are plotted ± standard error. Significances are given between treatments of ‘no aphids’ and ‘with aphids’ at

levels of: * p < 0.05; ** p < 0.01; *** p < 0.001. (B) The relative benefit of aphid presence after five days,

irrespective of strain, is negatively related to baseline epiphytic CFU counts at 48-hours (LM: F1,22 = 5.28,

p = 0.03, intercept = 0.35, slope = -0.04, R = -0.44).

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319 However, the baseline epiphytic growth at the 48-hour timepoint was a very strong predictor of

320 the relative benefit gained after five days, when using data from all experimental blocks (Figure

321 3B; Linear model: F1,22 = 5.28, p = 0.03). Interestingly, there was a negative relationship indicating

322 that replicates where bacteria benefitted the most from the presence of aphids had lower epiphytic

323 growth earlier in the experiment, and vice versa.

324 It is also possible that the ability to infect aphids impacts success for epiphytic bacteria such as P.

325 syringae. If some strains are less able to infect hosts from the phyllosphere, then they have less

326 chance of growing within the host at all and being dispersed via honeydew. To confirm that aphids

327 actually become infected from epiphytic populations of P. syringae we sampled aphids after being

328 on infected plants for 48-hours and also after five days (Figure 4A). All eight strains were able to

329 infect aphids, and infection rates on plants were much higher after five days than 48-hours (GLM:

330 F1,39 = 32.29, p < 0.001). There was a large range in mean infectivity levels after five days, from

331 23% for strain Ptt50252 to 100% for strain PsyCC1458, which is tightly linked to the mean

332 epiphytic density of bacteria at the same time point (Figure 4B; Pearson’s product-moment

333 correlation: t = 4.45, d.f. = 6, p = 0.004, R = 0.88), but not to the benefit (change in CFU counts)

334 of aphid presence (Pearson’s product-moment correlation: t = 0.43, d.f. = 22, p = 0.67, R = 0.09).

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335 Figure 4 – Aphid infection rates on plants. (A) After 48-hours and 5 days on plants sprayed with epiphytic

336 bacteria. Mean values are plotted ± standard error. Posthoc lettering denotes overall differences between

337 strains, irrespective of time, at p = 0.05 level. (B) Infection rates at 5 days on plants were highly correlated

338 with the epiphytic bacterial population on the leaves at that time (data from ‘with aphids’ treatment from

339 Figure 3; Pearson’s product-moment correlation: t = 4.45, d.f. = 6, p = 0.004, R = 0.88).

340

341 In previous experiments, epiphytic ability was connected with virulence on artificial diet, not

342 virulence directly from epiphytic populations of bacteria. Hence, we investigated whether aphid

343 mortality on plants was similar to that on artificial diet, and therefore if our expectations would be

344 the same. After five days on infected plants, there was no difference in aphid mortality across the

345 eight strains being tested, or when compared to a control treatment that had only buffer sprayed

346 onto the leaves (Figure S2A; GLM: F8,28 = 0.52, p = 0.84). However, if aphids were infected on

347 artificial diet initially, then placed onto V. faba plants for 72 hours, the mortality rates varied

348 among strains and were significantly different to the control treatment, and closer to what we

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349 would expect from our previous data (Figure S2B; GLM: F4,5 = 33.18, p < 0.001). This confirms

350 that it is not the feeding on artificial diet per se that promotes quicker death, but perhaps the uptake

351 of more bacterial cells. Consequently, as the dosage of epiphytic bacteria ingested by aphids is

352 likely much lower on plants than when fed artificial diet, we expected the effect to take longer

353 when on plants. For one strain, PsyB728a, we extended the experiment on plants only and found

354 that during an extra four days, to make nine days total on plants, a significant difference did emerge

355 between aphid mortality on control plants and infected plants (Figure S2C; Welch Two Sample t-

356 test: t = -4.83, df = 2.26, p = 0.03). We also observed transfer of bacteria onto the fresh V. faba

357 plants from the aphids initially fed bacterial solutions via artificial diet.

358 3.3 Honeydew may provide a valuable resource

359 We hypothesized that bacteria could benefit both from growth inside aphids and also from

360 honeydew, so we sought to determine the relative benefit of growth inside aphids versus increased

361 epiphytic growth from the addition of honeydew to leaf surfaces by experimentally separating

362 these components. We would expect that with the addition of bacteria growing inside the aphid

363 host and being excreted in honeydew would amplify the populations of bacteria on the leaf surface.

364 Alternatively, bacteria already on a leaf might benefit from additional nutrients deposited on the

365 leaf in honeydew and grow to higher titers.

366 Two strains were chosen for this assay, PtoDC3000 and Cit7, as both had shown that they

367 benefitted from the presence of aphids on plants (Figure 3) but differed in their virulence to aphids

368 and their epiphytic ability (Figure 1). CFU values of both strains varied over experimental blocks

369 (PtoDC3000: GLM: F2,49 = 7.59, p = 0.001; Cit7: GLM: F2,49 = 3.65, p = 0.03), but there was no

370 significant interaction, so the pattern reported did not change. For both strains, the treatment that

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371 had no aphids present repeatedly gave the lowest CFU counts, compared to treatments with aphids,

372 whether or not they were also feeding on bacteria (Figure 5A: PtoDC3000: GLM: F2,49 = 8.92, p

373 < 0.001; Cit7: GLM: F2,49 = 4.95, p = 0.01). Therefore, at least for these two strains, the presence

374 of honeydew appeared to increase population densities, but additional growth of bacteria inside

375 the aphids did not contribute to the increased titers of bacteria on leaves.

376

A B 48−hour baseline 7.0 Five days without honeydew No aphids Five days with honeydew b b With aphids b a Aphids & bacteria 8 6.5 z ) ) 10 a a 10 z z y y 6.0 7

y 5.5 6

5.0

5 CFU count per 10 leaf discs (log 4.5 CFU count per 10 leaf discs (log

4.0 4 Cit7 PtoDC3000 Cit7 PtoDC3000

Strain Strain 377 Figure 5 – The effect of honeydew on leaves. (A) Epiphytic CFU counts from leaves sprayed with bacteria

378 and then with either no aphids added, or aphids feeding on artificial diet suspended above the leaves, with

379 or without added bacterial suspensions. The presence of aphid honeydew on leaves increases bacterial CFU

380 load, regardless of whether or not the aphids had ingested bacteria. (B) Epiphytic CFU counts from whole

381 plants 48-hours after being sprayed with bacteria, and then after another five days with or without artificial

382 honeydew sprayed onto leaves. Even without the presence of aphids feeding, honeydew is beneficial to

383 these epiphytic strains of P. syringae. Mean values are plotted ± standard error, and lettering indicates

384 results of post-hoc tests, with strains analyzed separately.

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385 We further demonstrate that it is honeydew that benefits the epiphytic bacteria, and not plant-

386 mediated effects of aphids feeding on plants, by excluding aphids completely from our assays on

387 whole plants. Both Cit7 and PtoDC3000 showed a significant increase in population densities

388 when plants were sprayed with artificial honeydew and left for five days, compared to unsprayed

389 control plants which did not differ from baseline values after 48-hours (Figure 5B: PtoDC3000:

390 GLM: F2,22 = 10.48, p < 0.001; Cit7: GLM: F2,22 = 20.55, p < 0.001). Across experimental blocks

391 there was some variation in PtoDC3000 densities, but not Cit7 (PtoDC3000: GLM: F2,22 = 8.98, p

392 = 0.001; Cit7: GLM: F2,22 = 1.09, p = 0.36).

393 We then grew all eight strains in artificial honeydew, to confirm whether the ability to utilize

394 honeydew as a resource leads to epiphytic benefit for particular strains. All eight strains grew well

395 in the artificial honeydew medium. From a starting OD600 of 0.1 ± 0.01, strain PsyCC1458 grew

396 the most, up to an OD600 of 1.73 in 48 hours, and strain PsyCC1543 grew the least, to an OD600 of

397 1.17 (Figure S3A). The eight strains showed a significant amount of variation in their ability to

398 grow in the honeydew medium at the final time point (GLM: F7,72 = 219.77, p < 0.001), but also

399 showed no correlation with the benefit gained by each strain in Figure 3 (Pearson’s product-

400 moment correlation: t = -0.18, d.f. = 6, p = 0.87, R = -0.07). These growth patterns do not reflect

401 the intrinsic ability of each strain to grow, as there is no correlation with their growth patterns in

402 KB medium (Figure S3B).

403

404

405

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406 4 Discussion

407 As perhaps the best-studied plant pathogen to date, and an influential model system for studying

408 plant-microbe interactions and the evolution of virulence, P. syringae provides a powerful tool for

409 exploring more complex, tripartite interactions in the phyllosphere. More recent genomic tools

410 have revealed the impressive diversity of this bacterium in nature (Baltrus et al. 2011; Berge et al.

411 2014; Thakur et al. 2016). However, much of the literature has focused on revealing variation in

412 pathogenicity traits associated with plants (Hwang et al. 2005; Lindeberg et al. 2012). We show

413 that in addition to this, across 21 strains of P. syringae that vary in plant virulence, epiphytic

414 growth on fava beans is also highly variable and strongly correlated with virulence to aphids,

415 building on a previous study by Smee et al (2017). This would suggest that the two traits are

416 somehow linked and that those strains most often encountered on plants are likely to be

417 entomopathogenic.

418 Out of the eight strains chosen to further investigate this relationship between epiphytic ability and

419 virulence to aphids, five were shown to significantly benefit from the presence of aphids on plants.

420 If gaining a benefit from the presence of aphids is adaptive, we hypothesized that there may be a

421 trade-off between virulence and dispersal, in that less virulent strains remain in the aphid host for

422 longer, allowing more dispersal, and in which case those strains might benefit more from the

423 presence of aphids. We found that this was indeed the case for these P. syringae strains as a whole,

424 although irrespective of specific strain. Epiphytic growth at 48-hours was found to significantly

425 predict the relative benefit aphids provided to bacteria after being on plants for five days, with

426 poorer epiphytes benefitting more. As there was much variation across experimental blocks within

427 single strains this effect was lost if only using the mean of each strain, suggesting that the extent

428 to which strains benefit is likely context dependent. There is often a lot of variability in how cells

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429 react to different environmental stimuli, so drawing overarching conclusions about each strain at

430 this point is difficult.

431 Low virulence to aphids may have evolved in poorer epiphytes that benefit more from keeping

432 aphids alive for longer. Better epiphytes that don’t benefit from the presence of aphids can

433 consequently evolve high virulence. When correlating the mean benefit gained by each strain

434 against the mean virulence to aphids, or our initial measure of epiphytic ability (sprayed at an

435 OD600 of 1.0), there were no significant relationships, although this could again be due to losing

436 variation through the use of mean data points for each strain. Short-term mortality of aphids on

437 plants with epiphytic bacteria was found to be less extreme than when aphids were infected on

438 artificial diet. This was somewhat unsurprising, assuming aphids ingest far more bacterial cells

439 when feeding on artificial diet than when feeding on infected plants. However, over longer time

440 periods, the relative differences in virulence between the strains is likely to be the same as on

441 artificial diet as infection rates were shown to be highly correlated with epiphytic growth at five

442 days, and once infected aphids cannot recover. We also show, for one pathovar PsyB728a, that

443 over another few days mortality is closer to the level seen on artificial diet, and significantly

444 different to controls. It is possible that we would see differences in how strains benefit over longer

445 time scales when aphid death would vary more between bacterial strain treatments. Alternatively,

446 it is possible that the relationship between epiphytic ability and virulence observed here occurs

447 because whatever trait causes a strain to grow well on a leaf surface is also the same trait that

448 causes them to grow well inside an aphid and causes death to the aphid host. Given the lack of

449 correlation between epiphytic growth or virulence and benefit from aphids found here, we

450 conclude that there is no adaptive reason for at least some P. syringae strains to use aphids as

451 alternative hosts.

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452 In further support of the idea that P. syringae does not benefit from using aphids as an alternative

453 host, we found that there was no additional impact on epiphytic cell titers of bacterial growth inside

454 the aphids when honeydew was deposited on leaves, for the two strains tested. If the growth inside

455 an aphid is not the source of benefit to epiphytic P. syringae, then ultimately it is only the bacterial

456 cells already on the leaf surface that benefit from aphids. It is possible that strains other than Cit7

457 and PtoDC3000 might show different patterns, or that with different experimental conditions we

458 would see different effects. However, we did find support to conclude that some strains benefit

459 from aphid presence due to honeydew excretion. It could be that particular strains of P. syringae

460 have evolved mainly to utilize aphids not as hosts, but instead the sugar-rich honeydew waste

461 secreted by aphids onto leaves, in order to boost bacterial densities on leaves. Our data indicate

462 that strains of P. syringae differ in their ability to use honeydew as a growth medium, and that

463 across the eight strains tested this was uncorrelated and therefore unpredictable from their growth

464 in KB media. Hence, strains are differentially adapted to utilize this potentially abundant resource.

465 However, their growth in the honeydew medium was unrelated to the benefit of aphid presence. It

466 could be that our artificial honeydew was not realistic enough, or that as honeydew on leaf surfaces

467 dries and becomes more concentrated, then different bacteria are better or worse equipped to utilize

468 it. Honeydew is carbon-rich, and hence a potential energy source for many organisms in the

469 phyllosphere, but the composition differs depending on both the species of aphid and also the host

470 plant they feed on, which has further knock-on effects to other organisms such as ants that feed on

471 honeydew (Leroy et al. 2011b; Pringle et al. 2014). Cinara species of aphid also spread honeydew

472 which has been shown to encourage growth of bacteria, yeasts and filamentous fungi (Stadler and

473 Müller 1996). If honeydew is important in structuring communities on both the leaf surface, but

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474 also the surrounding habitat (Stadler and Müller 1996; Milcu et al. 2015), then aphids are surely

475 engineers of both the phyllosphere microbiome, and also the wider ecosystem.

476 Most studies to date that illustrate an effect of honeydew, or herbivores in general, on bacterial

477 populations in the phyllosphere have not managed to remove the potential plant-mediated effects

478 of herbivore presence (Stadler and Müller 1996, 2000). By restricting aphids from feeding on

479 leaves, but allowing honeydew still to be deposited or by spraying artificial honeydew on to plants,

480 we show that the significant increase in growth of epiphytic P. syringae is not due to plant-

481 mediated effects caused by herbivore feeding (Humphrey and Whiteman 2019). Despite this, there

482 may have been differential plant-mediated effects acting in response to different strains of P.

483 syringae or even the presence of honeydew on the leaves in experiments using aphids. Plant

484 responses to bacteria are variable, and in nature may make plants more susceptible to herbivores

485 due to crosstalk between JA and SA (Humphrey et al. 2014), which in turn may also positively

486 affect the abundance and diversity of bacteria on some plants or trees (Stadler and Müller 2000;

487 Müller et al. 2003). However, here we show that aphids can interact directly with epiphytic bacteria

488 to influence their population densities. We further speculate that in natural, diverse communities

489 differences in the bacterial ability to utilize honeydew would also impact competition between

490 bacterial strains and species to influence community composition.

491 In conclusion, our results support the hypotheses (i) that there is a link between P. syringae

492 virulence to the pea aphid, A. pisum, and epiphytic growth ability across a diverse set of strains,

493 (ii) there is a benefit of aphid presence to some epiphytic bacterial populations, (iii) this benefit is

494 not due to plant-mediated effects, nor bacterial growth within aphid hosts, but likely an interaction

495 between poorer epiphytes and the excretion of honeydew onto leaf surfaces. Success in the

496 phyllosphere is crucial for many P. syringae pathovars and environmental strains. The ability to

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497 successfully utilize ephemeral resources such as carbon-rich honeydew secretions from aphids

498 could provide a competitive advantage to epiphytic bacteria, leading to higher densities on leaf

499 surfaces, greater opportunity to infect other hosts, and even further reaching and less explored

500 consequences, such as attracting a plethora of other vectors or hosts (Farré-Armengol and Junker

501 2019).

502

503 5 References

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