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3-13-2019 Manipulating Wild and Tamed Phytobiomes: Challenges and Opportunities Terrence H. Bell The Pennsylvania State University

Gwyn A. Beattie Iowa State University, [email protected]

Kurt P. Kowalski U.S. Geological Survey

Amy T. Welty Iowa State University, [email protected] et al.

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Abstract This white paper presents a series of perspectives on current and future phytobiome management, discussed at the Wild and Tamed Phytobiomes Symposium in University Park, PA, USA, in June 2018. To enhance plant productivity and health, and to translate lab- and greenhouse-based phytobiome research to field applications, the academic community and end-users need to address a variety of scientific, practical, and social challenges. Prior discussion of phytobiomes has focused heavily on plant-associated bacterial and fungal assemblages, but the phytobiomes concept covers all factors that influence plant function. Here we discuss various management considerations, including abiotic conditions (e.g. soil, nutrient applications), microorganisms (e.g. bacterial and fungal assemblages, bacterial and fungal inoculants, viruses), macroorganisms (e.g. arthropods, plant genetics), and societal factors (e.g. communication approaches, technology diffusion). An important near-term goal for this field should be to estimate the potential relative contribution of different components of the phytobiome to plant health, as well as the potential and risk of modifying each in the near-future.

Disciplines Agriculture | Ecology and Evolutionary Biology | Plant Sciences

Comments This is a manuscript of a proceeding published as Bell, Terrence H., Kevin Hockett, Ricardo Ivan Alcalá- Briseño, Mary Barbercheck, Gwyn A. Beattie, Mary Ann Bruns, John Carlson et al. "Manipulating Wild and Tamed Phytobiomes: Challenges and Opportunities." Phytobiomes Journal (2019). doi: 10.1094/ PBIOMES-01-19-0006-W.

Rights Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The onc tent of this document is not copyrighted.

This conference proceeding is available at Iowa State University Digital Repository: https://lib.dr.iastate.edu/plantpath_conf/7 Page 1 of 112

1 Manipulating Wild and Tamed Phytobiomes: Challenges and

2 Opportunities

3 Terrence H. Bell1,2,3 †, Kevin L. Hockett1,2,3 †, Ricardo I. Alcalá-Briseño4,5,6, Mary

4 Barbercheck7,8, Gwyn A. Beattie9, Mary Ann Bruns2,3,10, John E. Carlson3,10, Taejung Chung3,11,

5 Alyssa Collins1, Bryan Emmett12, Paul Esker1,8, Karen A. Garrett4,5,6, Leland Glenna3,8,13, Beth

6 K. Gugino1, María del Mar Jiménez-Gasco1,8, Linda Kinkel14, Jasna Kovac3,11, Kurt P.

7 Kowalski15, Gretchen Kuldau1,8, Johan H.J. Leveau16, Matthew J. Michalska-Smith14,17, Jessica

8 Myrick18, Kari Peter1, Maria Fernanda Vivanco Salazar8,13, Ashley Shade19,20,21,22, Nejc

9 Stopnisek19,20,21, Xiaoqing Tan3,11, Amy T. Welty9, Kyle Wickings23, Etienne Yergeau24

10 † authors co-chaired the symposium and contributed equally to this manuscript

11

12 Affiliations:

13 1- Department of Plant Pathology and Environmental Microbiology, The Pennsylvania State

14 University, University Park, PA, USA

15 2- Intercollege Graduate Degree Program in Ecology, The Pennsylvania State University,

16 University Park, USA

17 3- Microbiome Center, The Huck Institutes of the Life Sciences, The Pennsylvania State

18 University, University Park, PA, USA

19 4- Plant Pathology Department, University of Florida, Gainesville, FL, USA

20 5- Institute for Sustainable Food Systems, University of Florida, Gainesville, FL, USA

21 6- Emerging Pathogens Institute, University of Florida, Gainesville, FL, USA

22 7- Department of Entomology, The Pennsylvania State University, University Park, PA,

23 USA Page 2 of 112

24 8- International Agriculture and Development Graduate Program, College of Agricultural

25 Sciences, The Pennsylvania State University, University Park, PA, USA

26 9- Department of Plant Pathology and Microbiology, Iowa State University, Ames, IA, USA

27 10- Department of Ecosystem Science and Management, The Pennsylvania State University,

28 University Park, USA

29 11- Department of Food Science, The Pennsylvania State University, University Park, PA,

30 USA

31 12- Boyce Thompson Institute, Ithaca, NY, USA

32 13- Agricultural Economics, Sociology, and Education Department, The Pennsylvania State

33 University, University Park, PA, USA

34 14- Department of Plant Pathology, University of Minnesota, St. Paul, MN, USA

35 15- U.S. Geological Survey, Great Lakes Science Center, Ann Arbor, MI, USA

36 16- Department of Plant Pathology, University of California, Davis, CA, USA

37 17- Department of Veterinary Population Medicine, University of Minnesota, Saint Paul,

38 MN, USA

39 18- Bellisario College of Communications, The Pennsylvania State University, University

40 Park, PA, USA

41 19- Department of Microbiology and Molecular Genetics, Michigan State University, East

42 Lansing, MI, USA

43 20- The Plant Resilience Institute, Michigan State University, East Lansing, MI, USA

44 21- The DOE Great Lakes Bioenergy Research Center, Michigan State University, East

45 Lansing, MI, USA Page 3 of 112

46 22- Department of Plant, Soil and Microbial Sciences; and Program in Ecology, Evolutionary

47 Biology and Behavior, Michigan State University, East Lansing MI, USA

48 23- Department of Entomology, Cornell University, Cornell AgriTech at the New York State

49 Agricultural Experiment Station, Geneva, NY, USA

50 24- Centre INRS-Institut Armand-Frappier, Institut National de la Recherche Scientifique,

51 Laval, QC, Canada

52

53

54

55 ABSTRACT

56 This white paper presents a series of perspectives on current and future phytobiome

57 management, discussed at the Wild and Tamed Phytobiomes Symposium in University Park, PA,

58 USA, in June 2018. To enhance plant productivity and health, and to translate lab- and

59 greenhouse-based phytobiome research to field applications, the academic community and end-

60 users need to address a variety of scientific, practical, and social challenges. Prior discussion of

61 phytobiomes has focused heavily on plant-associated bacterial and fungal assemblages, but the

62 phytobiomes concept covers all factors that influence plant function. Here we discuss various

63 management considerations, including abiotic conditions (e.g. soil, nutrient applications),

64 microorganisms (e.g. bacterial and fungal assemblages, bacterial and fungal inoculants, viruses), Page 4 of 112

65 macroorganisms (e.g. arthropods, plant genetics), and societal factors (e.g. communication

66 approaches, technology diffusion). An important near-term goal for this field should be to

67 estimate the potential relative contribution of different components of the phytobiome to plant

68 health, as well as the potential and risk of modifying each in the near-future.

69

70 ABBREVIATIONS AND GLOSSARY

71 2,4-DAPG: 2,4-diacetylphloroglucinol.

72 Biocontrol: The use of living organisms to suppress pests.

73 C: Carbon.

74 CBC: Conservation biological control. Biocontrol that exploits natural enemies through

75 modification of the environment or management practices.

76 CMD: Cassava mosaic disease.

77 Dormancy: Period in which an organism’s function is slowed, which is reversible under certain

78 conditions.

79 Endophyte: Organisms (generally fungi, bacteria, and viruses) that colonize internal plant tissue

80 without causing obvious visible symptoms.

81 HIPV: Herbivore-induced plant volatile.

82 Holobiont: Host and all of its associated partners.

83 ITS: Internal transcribed spacer.

84 Microbiome: All of the microorganisms found in a particular environment. The boundaries on

85 microbiomes are often challenging to define and depend on individual study parameters.

86 N: Nitrogen.

87 P: Phosphorous. Page 5 of 112

88 PapMV: Papaya mosaic virus.

89 Phytobiome: All of the biotic and abiotic factors that jointly determine a plant’s health and

90 growth.

91 PRSV: Papaya ringspot virus.

92 PVX: Potato virus X.

93 PVY: Potato virus Y.

94 SCMV: Sugarcane mosaic virus.

95 SynComs: Synthetic microbial communities. Groups of microorganisms assembled for

96 experimentation by combining multiple isolates.

97

98 INTRODUCTION

99 Phytobiomes are not simply the collection of microorganisms associated with a plant,

100 but all of the biotic and abiotic factors that influence the health and productivity of plants in a

101 defined biome. Many living components of the environment shape plant productivity, including

102 other plants, large animals, and various microorganisms, such as viruses, bacteria, fungi,

103 oomycetes, amoebae, and algae. Abiotic factors such as soil and climate also impact plants and

104 regulate the presence and function of the many organisms that interact with them.

105 Historically, agricultural and natural systems have been managed by focusing on

106 individual components of the phytobiome (e.g. nutrient applications, pesticides, removal of

107 invasive organisms). However, managing the phytobiome as an integrated system of components

108 has greater potential to achieve optimal and sustainable ecosystem health and productivity. As a

109 step toward advancing phytobiome science and translating lab- and greenhouse-based

110 phytobiome research to the field, approximately 150 people came together for a workshop and Page 6 of 112

111 symposium entitled Wild and Tamed Phytobiomes on 19-22 June 2018. This symposium was the

112 21st installment of the Penn State Plant Biology Symposium series in University Park, PA, USA.

113 This white paper contains perspectives and findings that were shared among the attendees.

114 Previous perspective papers and reviews have identified key barriers, needs, and

115 opportunities related to plant-associated microbiome research (Busby et al. 2017; Farrar et al.

116 2014; Hawkes and Connor 2017; Lebeis et al. 2012; Mueller and Sachs 2015), and another has

117 presented an overarching vision for integrated phytobiome research

118 (American_Phytopathological_Society 2015;

119 www.phytobiomes.org/Roadmap/Documents/PhytobiomesRoadmap.pdf). Here, we present

120 insights from discussions on the opportunities and challenges related to understanding and

121 manipulating the wide array of factors that influence the function of both wild and tamed plant

122 systems, as well as considerations on communicating this science to stakeholders and the broader

123 public.

124

125 PERSPECTIVES ON MANAGING AND MANIPULATING PLANT-ASSOCIATED

126 MICROBIOMES

127 How might soil properties constrain phytobiome manipulation?

128 Contributed by Mary Ann Bruns

129 Wild and tamed phytobiomes correspond to plants growing in undisturbed and

130 domesticated soils, respectively (Amundson et al. 2015). Tamed systems offer the greatest

131 potential for effective manipulation. Generally speaking, inherent soil properties (e.g., climate,

132 depth to bedrock, slope, texture) are geospatially determined, making them difficult or

133 impossible to change, and imposing a priori constraints on which phytobiomes can be Page 7 of 112

134 established. Alterable soil properties (e.g., organic matter content, pH, porosity, biological

135 activity, macronutrients) are strongly shaped by biotic factors, being modified by vegetation,

136 microbial activity, and human management. For phytobiomes, resource availability is the most

137 important category of properties influencing the strength and direction of plant-soil feedbacks.

138 Under low nutrient availability, plants allocate less photosynthate to roots than they do under

139 high nutrient availability, leading to lower overall soil microbial biomass (Treseder 2008) and

140 weaker interactions between roots and microbial biotrophs (Johnson et al. 2010). Soil microbial

141 succession, adaptation, and C cycling are also affected by resource availability, thereby resulting

142 in persisting legacy effects (Kaminsky et al. 2018; Martiny et al. 2017; Treseder et al. 2011).

143 Revillini et al. (2016) showed that plant biomass was higher in sterile soils inoculated with

144 microbial communities from a nonfertilized soil than with communities from a fertilized soil.

145 Thus, the success or failure of phytobiome manipulation may depend on compatible adjustments

146 of resource availability through soil management.

147 Soil properties are dynamic and will affect functional (saprotophs, biotrophs, specialists)

148 and morphological microbial groups in different ways (Delgado-Baquerizo et al. 2016; Tedersoo

149 et al. 2014). Microbial activity itself results in changes in soil structure through aggregate

150 formation and stabilization (Six et al. 2004), macropore creation, and altered hydraulic

151 conductivity, all of which have contrasting effects on growth and dispersal of filamentous and

152 non-filamentous microbes (Wolf et al. 2013). Interdependencies among alterable properties, such

153 as pH, redox potential, and nutrient chemical speciation, also result in direct and indirect impacts

154 on microbial habitats. Soil pH, for example, is a highly influential but spatially variable factor

155 affecting bacterial diversity (Lauber et al. 2009). In the rhizosphere, pH can be determined by the

+ - 156 predominant N species taken up by plant roots (NH4 or NO3 ), so that source and type of Page 8 of 112

157 fertilizer N could influence diversity of rhizosphere microbes. Because root sensing and

158 signaling are also affected by rhizosphere pH (Xuan et al. 2017), fertilizer type would influence

159 the ability of roots to attract compatible microbial partners. While inherent soil properties

160 determine land use suitability and vegetation type, alterable properties can be fine-tuned to shape

161 soil-phytobiome feedbacks, particularly in agricultural soils and engineered growth media.

162

163 Managing agricultural soils to enhance the phytobiome

164 Contributed by Bryan Emmett

165 Phytobiome-based management presents an opportunity to support plant productivity,

166 while limiting saturating external nutrient inputs. The last two decades of research have

167 increasingly highlighted mechanisms of microbial facilitation of plant nutrient use. Nutrients that

168 are not readily mineralized in the absence of the plant can be available to plant-microbe

169 interactions, including widespread priming effects on C and N mineralization and solubilization

170 of phosphate in the rhizosphere (Cheng et al. 2014; Zhang et al. 2016; Zhu et al. 2014a).

171 Certain nutrient fluxes are driven largely by well-known mutualisms (e.g. N2 fixation). However,

172 others are mediated by the broader microbial community and optimizing this phytobiome-

173 mediated nutrient supply necessitates a systems-based approach that accounts for the soil habitat

174 and nutrient pools available throughout the phytobiome, rather than focusing solely on known

175 beneficial taxa.

176 Recent research highlights the interdependency of phytobiome components that are

177 responsive to management. For example, over-wintering cover crops and diversified rotations are

178 effective tools to increase soil C, N, and microbial biomass in soils (McDaniel et al. 2014). In

179 turn, these parameters are key drivers of soil- and plant-associated microbiome composition Page 9 of 112

180 (Berthrong et al. 2013; Hamel et al. 2018). Moreover, Berthrong et al. (2013) found that

181 management-induced increases in soil C and N stocks and associated shifts in soil microbial

182 community composition led to increased N mineralization following labile C addition,

183 potentially linking rhizosphere effects and improved plant N supply under diversified rotations.

184 The challenge of the next decades will be to move from experimental examples to a

185 robust understanding of the management levers influencing agricultural phytobiomes and a

186 prescriptive knowledge of the rotations, crop selection, residue management, and tillage practices

187 that optimize phytobiome function. The inherent variability of diverse soils and sites presents a

188 challenge to this effort. A network of researchers collecting data and assessing management

189 interventions across sites will be essential to address this challenge. Working farms have always

190 been an incubator for agricultural innovation and experimentation. Data generation and

191 processing capabilities are now scaling to a point where it is possible to envision harnessing this

192 experimentation at a scale that can unravel the complexity present across sites and soils, and

193 identify microbial taxa, networks, and functions that are responsive to management.

194

195 Delving into the plant-microbiome-environment nexus with a focus on drought

196 Contributed by Gwyn Beattie and Amy Welty

197 Due to their sessile state, plants are inherently vulnerable to the vagaries of weather and

198 soil conditions. The mobility of microbes coupled with their potential to form intimate

199 associations with plants enables microbial-induced phenotypic plasticity that modulates plant

200 responses to stress. Knowledge of the contributions of individual microbes to plant stress

201 tolerance has been increasing, particularly with respect to drought (Dimkpa et al. 2009; Kasim et

202 al. 2013; Latef et al. 2016; Ngumbi and Kloepper 2016; Rho et al. 2018; Rolli et al. 2015). Page 10 of 112

203 Given the huge global diversity of plant species and of dynamic plant habitats, the beneficial

204 plant-microbe interactions that are currently characterized represent only a fraction of the

205 interactions that bolster plant fitness. In-depth knowledge of the plant-microbiome-environment

206 nexus is key to managing crop phytobiomes, and to harnessing beneficial associations in the face

207 of challenges such as climate change, water scarcity, and degraded soils.

208 Many approaches can lead to the discovery of microbes and microbial community traits

209 that enhance crop performance under non-ideal conditions. Historically, cultivated isolates have

210 been screened for beneficial impacts, and some have been developed into agricultural products,

211 such as those that enhance drought tolerance. Recent studies characterizing the soil microbiome

212 as a whole have demonstrated that, during the growth of plants under drought stress, changes

213 occur in soil microbial assemblages that favor plant fitness in subsequent generations if those

214 plants are also grown under drought conditions (Lau and Lennon 2012). Beattie and colleagues

215 have investigated the nature of these microbial assemblage changes by profiling communities

216 during multi-generational growth of soybeans in low and high soil moisture conditions. They

217 discovered a notable enrichment in members of the phylum Actinobacteria in roots under

218 drought stress (unpublished data). These findings echo those of two recent studies (Fitzpatrick et

219 al. 2018; Xu et al. 2018), which also documented drought-associated increases in root-associated

220 Actinobacteria in diverse plant species.

221 The complexity of a phytobiome can be daunting when searching for the functional

222 interactions among the many involved players. Although the functional relevance of interactions

223 at the crop-Actinobacteria-drought nexus have not yet been demonstrated, the identification of

224 consistent associations among these players is a critical step toward both understanding

225 phytobiomes and using this information to improve crop health and productivity under drought Page 11 of 112

226 conditions. Given the increasing challenge that drought poses to global crop productivity

227 (Daryanto et al. 2017; Lesk et al. 2016), strategies that exploit all components of phytobiomes

228 are needed to maximize the resilience of our agroecosystems.

229

230 Assembly and activation of the rhizosphere microbiome during plant stress

231 Contributed by Ashley Shade and Nejc Stopnisek

232 Interactions between bacteria and plants in the rhizosphere are important for plant

233 wellness and productivity (Berendsen et al. 2012; Coleman-Derr and Tringe 2014; Pieterse et

234 al. 2014). Assembly and maintenance of rhizosphere bacterial assemblages are primarily driven

235 by root exudates that add C and nutrients to the soil system (Hu et al. 2018; Zhalnina et al.

236 2018). However, changes in root exudates can occur when plants experience biotic or abiotic

237 stress, which can in turn alter the composition or activities of the rhizosphere microbiota (Lebeis

238 et al. 2015; Sasse et al. 2018). Rhizosphere soils are a resource-rich hotspot of microbial activity

239 (Ma et al. 2018). However, soils harbor a sizeable diversity reservoir, including numerous rare

240 and inactive (dormant) taxa (Lennon and Jones 2011). Dormant microbial taxa can resuscitate,

241 and rare taxa can bloom to provide pulses of activity under specific environmental conditions

242 (Shade and Gilbert 2015). The roles of diversity reservoirs in the assembly and dynamics of the

243 rhizosphere community microbiomes are unknown, as are the changes in microbial activities in

244 response to changes in root exudates during stress.

245 To improve understanding of the phytobiome, there is a need to consider the dynamics of

246 microbial dormancy, resuscitation, and changes in relative activities in the rhizosphere during

247 plant stress. An integrated approach that considers both the microbiology and chemistry of the

248 phytobiome is likely to be particularly fruitful. First, a baseline of precise chemistry of root Page 12 of 112

249 exudates in “good” times of plant wellness is required. This information should include the

250 resolved spatial dynamics of root exudates, given root architecture and its micron-scale

251 heterogeneity, as well as the resolved temporal dynamics over development of the growing

252 season for both perennial and annual plant life history strategies. This information can then be

253 compared to changes in exudates during stresses like drought, increased temperature, resource

254 excess and limitation, herbivory, and pathogen infection. Finally, quantitative measurements of

255 microbial activity and dormancy are required to develop an understanding of the dynamics of

256 microbial diversity reservoirs, including dormant and rare taxa (Bowsher et al. 2018). The

257 conditions of dormancy and rarity are temporary, and reactivation of these microbiome

258 constituents can fundamentally change the functional profile of the rhizosphere. Development of

259 high-throughput, quantitative methods for measuring microbial activity in situ will allow for

260 precise tracking of key microbiome functions over time and in response to stress. Likewise,

261 expanded infrastructure and standard analysis workflows for high-throughput metabolite analysis

262 will be key for supporting comparisons across studies and plants.

263

264 Manipulating the crop holobiont for increased stress resistance

265 Contributed by Etienne Yergeau

266 It is estimated that by 2050, drought episodes will impact >50% of the world’s arable

267 land, causing serious yield losses in major crops (Ngumbi and Kloepper 2016). An interesting

268 framework for studying the adaptation of host-associated microorganisms to stressful events is

269 the hologenome theory of evolution. This theory postulates that the complex interacting network,

270 involving a eukaryotic host and all of its associated microorganisms and other partners (the

271 holobiont), acts as a unit of evolution (Zilber-Rosenberg and Rosenberg 2008). As such, rapid Page 13 of 112

272 adaptations of holobionts are thought to be more likely related to changes in plant-partnered

273 organisms, through mechanisms such as recruitment, amplification/reduction, and horizontal

274 gene transfer, suggesting a clear path to the improvement of plant resistance and resilience to

275 stress (Berendsen et al. 2012; Duhamel and Vandenkoornhuyse 2013; Nogales et al. 2016;

276 Rosenberg and Zilber-Rosenberg 2016). Most efforts to date have focused on trying to direct

277 microbial recruitment by the plant by providing selected microbial strains or assemblages. For

278 instance, the priming of seeds with single microorganisms has often been used to supplement the

279 rhizosphere microbiome (Parnell et al. 2016). However, this technique can sometimes be of

280 limited success, mainly because it underestimates the importance of the biotic context (microbe-

281 microbe and plant-microbe interactions) for optimized colonization (Rivett et al. 2018). Complex

282 communities that target multiple niche spaces and exhibit functional redundancy might be better

283 at entering and surviving a new environment than single species (Yergeau et al. 2015).

284 Knowledge of how microbiomes coalesce is still minimal (Rillig et al. 2015), but we

285 know that established soil inhabitants can prevent the success of newcomers, a phenomenon

286 referred to as priority effects (Vannette and Fukami 2014). Priority effects might be overcome

287 when established microbial assemblages are weakened by environmental stressors (Calderón et

288 al. 2016), such as drought. Such processes are potentially more complex under the influence of a

289 plant, which can partly determine which microorganisms stay, and which do not (Mueller and

290 Sachs 2015). Translating this theoretical framework into applied solutions for rapidly adapting

291 crops to abiotic stresses remains an unmet challenge.

292

293 Storage and reproduction of microbiomes

294 Contributed by Terrence Bell Page 14 of 112

295 Controlled systems, such as incubators, growth chambers, and greenhouses, are key tools

296 for parsing the factors that shape soil and plant-associated microbiome structure and function.

297 Although conditions such as temperature, moisture, and plant variety are easy to control, it is

298 challenging to standardize the microorganisms that we work with. This complicates the

299 comparison of studies run by different research groups and at different points in time. When

300 searching the methods section of such studies, it is common to find that the soil and associated

301 microorganisms came from a nearby experimental station, or an investigator’s backyard. This

302 contrasts with descriptions of biological material used in cell culture, which are more in line with

303 the following: “MDA-MB-231 cells were obtained from the American Type Culture Collection”

304 (Li et al. 2017).

305 Synthetic microbial communities (SynComs), i.e. mixtures of cultivated bacterial

306 isolates, have been applied to address this issue in some studies (e.g. Castrillo et al. 2017; Lebeis

307 et al. 2015). SynComs allow for the removal or replacement of specific isolates, to investigate

308 the impacts of each isolate individually. Some disadvantages of this approach, as currently

309 applied, are that 1) yet-to-be-cultivated microbial lineages are excluded, and 2) a group of

310 unconditioned isolates will lack many of the interactions that would be selected for over time in a

311 true assemblage. Several studies show that microbial phenotypes can differ dramatically after

312 multiple generations of selection in isolation, as opposed to the same amount of selection within

313 a multi-strain assemblage (Fiegna et al. 2015; Ketola et al. 2016; Lawrence et al. 2012). This

314 latter issue could be resolved through differential conditioning of SynComs, followed by

315 preservation at specified generations.

316 However, the former issue suggests that there is value in cultivation-independent

317 approaches to microbiome preservation and transfer. In addition to retaining yet-to-be-cultivated Page 15 of 112

318 microbes, preserving microbiomes directly from soils without cultivation would facilitate

319 screening for desirable group phenotypes (e.g. low carbon dioxide output, high N retention)

320 across many soil types and locations. Although it may be challenging or impossible to directly

321 reproduce microbiomes found in the field, the generation of replicable storage and experimental

322 conditions should allow for reasonable reproduction of lab-adapted microbiomes across

323 generations (Auchtung et al. 2015). Williams and Marco (2014) showed that cryopreservation

324 only minimally impacted the microbiome transferred from field-grown lettuce to lettuce grown

325 in the lab, indicating the potential to maintain stored microbiome collections. Beyond simply

326 preserving microbiomes, it is important to consider the impacts of different microbiome transfer

327 approaches, which can also have large impacts on microbiome composition in study systems

328 (Howard et al. 2017).

329 Moving forward, we should consider how best to preserve and distribute both intact

330 microbiomes and diverse SynComs for replicated experimental design through space and time.

331 Given that barcode identifications are not sufficient for predicting all microbial functions and

332 traits, these resources should be preserved with metadata that provides insight into the observed

333 function of the assemblages at the time of preservation.

334

335 Shaping microbiomes with microbial warfare

336 Contributed by Kevin Hockett

337 Plant-associated microbes have long been known to produce chemicals and proteins that

338 are antagonistic toward potentially co-colonizing microbes. These include secondary metabolites

339 (e.g. antibiotics, Haas and Defago 2005), contact-dependent mechanisms (type V and type VI

340 secretion systems, Hayes et al. 2014), protein toxins (bacteriocins, Ghequire and De Mot 2014), Page 16 of 112

341 and other protein exoenzymes. Although substantial research has demonstrated the activity of

342 antagonistic systems in culture environments and has allowed prediction of the genetics

343 underlying such systems in genomic and metagenomic studies of plant-associated microbes, our

344 understanding of how most antagonistic traits influence plant-associated microbial assemblages

345 under natural conditions remains limited.

346 One of the best understood examples of antagonism under field settings is the

347 suppression of take-all (a soil-borne root disease caused by Gaeumannomyces graminis var.

348 tritici) of wheat by fluorescent Pseudomonas spp. in disease-suppressive soils. This antagonism

349 is linked to a suite of secondary metabolites produced by these organisms, chief among them

350 being 2,4-diacetylphloroglucinol (2,4-DAPG, Haas and Defago 2005). Although the role and

351 activity of 2,4-DAPG producing Pseudomonas spp. in suppressing soilborne fungal diseases has

352 been elucidated through decades of research conducted by several laboratories, such detailed

353 understanding of antagonism in most other systems is lacking. One reason the causal link

354 between 2,4-DAPG production and inhibition of take-all has been so strong is that the discovery

355 of 2,4-DAPG producers stemmed from an initial observation of take-all decline, where the

356 severity of take-all decreases over successive seasons of continuous wheat monocropping (Kwak

357 and Weller 2013). This is in contrast to the majority of instances, in which plant-associated

358 isolates are screened for antagonistic activity in culture first, then assayed for pathogen inhibition

359 within a plant environment.

360 While most research on antagonism has focused on taking advantage of these systems for

361 the purposeful exclusion of a pathogen, more research should be directed toward understanding

362 the role of microbial warfare under natural conditions. Chief among the questions that should be

363 asked is: under what conditions does a given form of antagonism contribute to ecological fitness Page 17 of 112

364 for the producing organism? Antagonistic traits are costly, and thus are almost certainly

365 regulated in a manner that provides the greatest net benefit to the producer. Future research

366 should be aimed at understanding the specific environmental and ecological context(s) in which

367 a given antibiotic or bacteriocin provides such a benefit and when such contexts(s) arise in

368 agricultural or other managed settings. Recent work by Dorosky et al. (2018) demonstrated that

369 loss of production of either of two distinct bacteriocins resulted in reduced persistence within a

370 wheat rhizosphere environment. Conversely, neither bacteriocin affected persistence in bulk soil

371 or in a rhizosphere environment that lacked microbial competitors. Similar work investigating

372 competition between two Xanthomonas tomato pathogens indicated that bacteriocin-mediated

373 inhibition was more prominent on the leaf surface than leaf apoplast (Hert et al. 2009). This may

374 be a contrived example, however, as the bacteriocin-producing strain was engineered to be

375 compromised in pathogenicity.

376 Regardless, a better understanding of the natural role of microbial warfare will result in a

377 better sense of how best to take advantage of these systems for phytobiome benefit. For instance,

378 rather than attempting to use these systems for excluding a given pathogen, they may provide

379 more reliable benefit in ensuring competitive colonization by beneficial organisms, or potentially

380 in promoting greater diversity within the microbial fraction of the phytobiome, which itself may

381 result in plant benefit in a number of different ways. Broadening our perspective on the utility of

382 microbial antagonism beyond pathogen suppression will likely provide useful strategies for

383 phytobiome management.

384

385 Wild, tamed, then back to where the wild things are: discovery and use of biocontrol agents

386 Contributed by Johan Leveau Page 18 of 112

387 Plant pathologists tend to classify individual plant-associated microorganisms as either

388 capable of causing disease or not capable of causing disease. The traditional path to pathogen

389 discovery involves culture-dependent isolation of candidate microorganisms, based on consistent

390 association with disease symptoms, characterization in the laboratory, and inoculation of plants

391 in the greenhouse or field, with the intent of reproducing symptoms and confirming

392 pathogenicity. Complications may arise when a pathogen is not culturable (e.g. plant viruses or

393 bacteria with Candidatus status) or when a disease is ‘complex’, i.e. caused by a combination of

394 two or more pathogenic microorganisms. In such cases, a culture-independent, DNA-based

395 approach may offer further insight. Typically, this involves high-throughput sequencing of whole

396 plant-associated microbiomes, also known as metagenomics (Leveau 2007), or with a specific

397 focus on taxonomically informative gene amplicons, such as 16S rRNA genes for bacteria, or the

398 ITS region for fungi (Rastogi et al. 2013). The success of this approach depends on many

399 factors, but it is critical to compare and contrast microbial profiles from diseased and healthy

400 plant material, to identify candidate pathogenic taxa as those that are only, or more abundantly,

401 associated with diseased plants.

402 Biocontrol of plant diseases is a cultural practice that aims to lower the impact of plant

403 pathogens through the use of microbial agents. The typical pipeline for discovery of biocontrol

404 agents is very similar to that described above for pathogens. It typically involves the culture-

405 dependent isolation of ‘wild’ microbes from plants, soils, or other environments, screening them

406 for antagonistic activity in the lab, and taking these ‘tamed’ candidates to the greenhouse or field

407 (‘where the wild things are’), in the hopes that they reduce symptom formation on pathogen-

408 challenged plants. As with pathogens, there are likely to be microorganisms with biocontrol

409 potential that are not readily culturable. In addition, there are probably plant-protective effects Page 19 of 112

410 that are a function of the interaction between two or more microbial taxa (i.e. complex

411 biocontrol; Xu et al. 2011). Such microorganisms may be of limited practical interest, as their

412 resistance to culture, or dependence on other microorganisms, may pose considerable challenges

413 for commercial exploitation. As with pathogens, the choice of source material for discovering

414 biocontrol agents may be guided by the principle of consistent association. A classic example is

415 the phenomenon of disease-suppressive soils (Schlatter et al. 2017), from which biocontrol

416 agents have been successfully identified through both culture-dependent and -independent

417 means.

418 DNA-based interrogation of plant-associated microbiomes may one day become the basis

419 for decision-making tools that help farmers/growers manage their fields and maintain healthy

420 and productive crops. To be most effective, such tools should detect not only pathogens, but also

421 microorganisms with pathogen-suppressive potential. Based on profiling of complete plant-

422 associated microbiomes, a farmer may calculate a pathogen:antagonist ratio for their field

423 (analogous to a pest:defender or prey:predator ratio as used in integrated pest management, see

424 e.g. Naranjo and Ellsworth 2009) and choose to rely on the native microbiota to keep a pathogen

425 in check, or opt for intervention by specific stimulation of pathogen-suppressive taxa. If such

426 taxa are absent, this could occur through the introduction of biocontrol agents that are

427 specifically tailored to target the pathogen, and specifically fit for ‘where the wild things are’.

428 Progress towards this form of microbiota-informed disease management requires many more

429 field observations of natural and experimentally-imposed conditions of consistent association, to

430 establish cause and effect of pathogen and non-pathogen co-occurrence. In other words, to tame

431 your plant-associated microorganisms is to know your plant-associated microorganisms.

432 Page 20 of 112

433 Can we manage endophyte functionalities?

434 Contributed by María del Mar Jiménez-Gasco and Gretchen Kuldau

435 Endophytes are organisms (generally fungi, bacteria, and viruses) that colonize internal

436 plant tissue without causing obvious visible symptoms (Petrini 1991; Wilson 1995). Although

437 we focus here on fungal endophytes, these concepts apply to other microorganisms. There is

438 little evidence that we can easily manage endophyte functionalities. The best known, studied, and

439 commercialized example of endophyte management is that of temperate grass endophytes in the

440 genus Epichloë (Schardl et al. 2004; Tanaka et al. 2012). These naturally-occurring endophytic

441 fungi are demonstrated to confer benefits to their host, such as drought tolerance and anti-

442 herbivory, mediated by the production of an array of secondary metabolites (Kuldau and Bacon

443 2008). The protective characters of species in this genus resulted in commercialization of grass

444 seed containing the fungus, which is vertically transmitted through seed for forage and turf

445 applications (Johnson et al. 2013a). Adoption of endophyte-infected tall fescue in the

446 southeastern United States is high, since this forage crop is able to withstand high summer

447 temperatures (Bacon 1995). However, cattle grazing on this crop often have compromised health

448 and productivity, due to the presence of ergot alkaloids produced by the endophyte (Beck et al.

449 2008). Identification of a naturally-occurring isolate that does not produce the ergot alkaloids led

450 to development of seed colonized by a “friendly endophyte”, which still confers drought

451 tolerance, without negatively impacting cattle health (Beck et al. 2008). This example of

452 manipulating endophyte function is based on decades of research by numerous researchers.

453 However, such manipulation is typically limited by substantial knowledge gaps.

454 A major gap is our lack of understanding of the ecological roles of fungal endophytes,

455 despite the ubiquity and abundance of this functional guild. Epichloë endophytes, described Page 21 of 112

456 above, are specialized, obligate, highly co-evolved beneficial mutualists (Schardl et al. 2004).

457 This sort of relationship likely represents a very small fraction of all endophyte-host

458 relationships. A growing body of work suggests that the leaf endophytes of deciduous woody

459 perennials might act as saprotrophs in decaying leaf litter, and that the endophytic habit is a

460 transient life stage (Guerreiro et al. 2018). Similarly, root-inhabiting dark septate endophytes

461 may be acting as saprophytes, given their demonstrated capabilities in the degradation of detritus

462 (Caldwell et al. 2000).

463 One of the main questions that needs to be addressed is the extent to which we can assign

464 functionality to identified endophytes. Most fungal endophytes are generalists, not obligate

465 symbionts (Rodriguez et al. 2009). Rodriguez et al. (2009) put forth a classification of endophyte

466 types, based largely on location of growth within host plants. This work also highlighted the

467 ubiquitous nature of fungal endophytes: class 1, Clavicipitaceous (including Epichloë); class 2,

468 non-Clavicipitaceous fungi colonizing the entire plant; class 3, non-Clavicipitaceous hyper-

469 diverse fungi colonizing aerial plant tissues; and class 4, dark septate endophytes. One example

470 is Fusarium oxysporum, a species generally recognized as a plant pathogen. However,

471 saprophytic and endophytic lifestyles are more common for F. oxysporum than plant

472 pathogenicity (Demers et al. 2015; Stergiopoulos and Gordon 2014), and may be the result of a

473 continuum of interactions with the plant. Another example of well-known plant pathogens with

474 endophytic biology is Verticillium dahliae. The same genotype of V. dahliae may be pathogenic

475 to one host, but act as a non-harmful endophyte in another. In addition, changes between

476 endophytic and pathogenic interactions may depend on factors such as host plant genotype or

477 environmental conditions (Malcolm et al. 2013). Page 22 of 112

478 Interest in microbial endophytes stems in part from their potential for beneficial

479 interactions with their hosts. Generally fungal endophytes are known for production of bioactive

480 secondary metabolites and have been studied extensively in search of compounds with

481 pharmaceutical value. But fungal endophytes have also been attributed numerous other beneficial

482 traits, mostly through unknown mechanisms. Fungal endophytes can confer plant growth

483 promotion mediated by phytohormones (e.g. Ali et al. 2017). Pest and pathogen

484 resistance/deterrence and biocontrol activity have been associated with plant colonization by

485 fungal endophytes, through induction of resistance mechanisms in the plant such as systemic

486 acquired resistance, among others (Quesada Moraga et al. 2014). Resistance/tolerance to

487 environmental stress has been well documented for the endophyte Fusarium culmorum,

488 conferring salt tolerance to salt-sensitive plants (Rodriguez et al. 2008; Sikora et al. 2008).

489 Finally, non-mycorrhizal root endophytes (dark septate endophytes) have been shown to improve

490 P solubilization and plant uptake (Newsham 2011). Plant growth promotion through P

491 mineralization and solubilization, as well as uptake, may be increased by the synergistic

492 relationship between arbuscular mycorrhizal and dark septate endophytes (Della Monica et al.

493 2015).

494 The ecological role of fungal endophytes and the secondary metabolites they produce is

495 mostly unknown. However, the role of several Epichloë endophyte metabolites such as peramine

496 and loline in insect deterrence is elucidated (Johnson et al. 2013a). Understanding metabolite

497 roles in the ecology and biology of their producers, and their interactions with host plants, is

498 needed to advance a more complete picture of these fungi in the larger phytobiome context. The

499 importance of understanding this complexity is illustrated by microbial co-culturing studies,

500 which indicate that the presence of other microbes can both elevate levels of secondary Page 23 of 112

501 metabolite production, and in some cases, induce the production of secondary metabolites not

502 previously known to be produced by the organism under study (Abdalla et al. 2017). Similarly,

503 the host plant, and specific plant environment, can influence expression of secondary metabolites

504 by endophytes (Schmid et al. 2017).

505

506 Virome management

507 Contributed by Ricardo I. Alcalá-Briseño and Karen Garrett

508 Virome management is an important frontier for phytobiome research. Viruses are

509 particularly abundant and diverse organisms (Rodrigues et al. 2017; Suttle 2005). In the plant

510 virome, spatial structuring of viruses is generally based on the compartmentalization of the cell:

511 typically RNA viruses replicate in the cytoplasm, associated with membranes or replication

512 complexes, and DNA viruses replicate in the nuclei. This spatial structuring may prove to have

513 an important role in virus-virus interactions, analogous to larger-scale spatial structuring of

514 bacteria or fungi growing in soil, on plant surfaces, or as endophytes.

515 Viruses and their interactions result in a range of plant symptoms, from necrosis, to foliar

516 deformation, to common mosaic symptoms, and even to a persistent asymptomatic state (King et

517 al. 2011; Roossinck et al. 2011). Strains of potato virus Y (PVY) cause a range of symptoms on

518 potato, from mild to necrotic strains (Cuevas et al. 2012). However, interactions with other

519 viruses, such as potato virus X (PVX) can cause an increase or decrease in the severity of the

520 disease. This type of interaction has also recently been described in papaya, with the interaction

521 of papaya ringspot virus (PRSV), a potyvirus, and papaya mosaic virus (PapMV), a potexvirus

522 (Chavez-Calvillo et al. 2016; Vance 1991). Severe diseases can be caused by interacting viruses,

523 such as maize lethal necrosis caused by the interaction of maize chlorotic mottle virus, a Page 24 of 112

524 tombusvirus, and at least one of four species of potyvirus within the sugarcane mosaic virus

525 (SCMV) group (Adams et al. 2013; Adams et al. 2005). Another example of multiple

526 interactions causing a devastating agricultural problem is cassava mosaic disease (CMD), where

527 up to seven begomovirus species, and recombinants, are the cause of a pandemic in East and

528 Central Africa (Legg et al. 2015). A sharp increase in the abundance of the whitefly vector was

529 associated with the spread of CMD (Legg et al. 2011; Legg et al. 2015). In the case of PVY,

530 high aphid species richness may slow spread of the pathogen (Claflin et al. 2017). Warmer

531 temperatures often result in larger populations of aphids, one of the main vectors of plant viruses,

532 while temperatures around 30°C have been reported to increase virus fitness in plants. Climate

533 change impacts host-pathogen dynamics and host-pathogen-vector interactions, and these

534 changes could impact crop yields (Boyko et al. 2000; Canto et al. 2009; Durak et al. 2016;

535 Kido et al. 2008; Roos et al. 2011).

536 As plant viromes are more commonly studied, many new types of virus interactions will

537 likely be revealed, and understanding these interactions may be useful for anticipating disease

538 emergence. Complex viromes, including both RNA and DNA viruses, as well as phytobiomes

539 more generally, can be analyzed using bipartite networks. In this context, the direct links in

540 bipartite networks may represent associations between virus species nodes and “sample type”

541 nodes – where sample types might represent host species, vector species, or environmental types.

542 These sample types may be linked indirectly to each other through shared viruses (as discussed

543 in Garrett et al. 2018). With the use of high-throughput sequencing technologies, asymptomatic

544 infections have increasingly been reported in crops and wild plants (Roossinck 2015). Co-

545 infections of plant viruses are now being studied that would likely have been overlooked by

546 more specific serological or molecular techniques (Massart et al. 2014). As plant viromes are Page 25 of 112

547 more commonly studied, many new types of virus interactions will likely be revealed, and

548 understanding these interactions may be useful for anticipating disease emergence and for

549 identifying antagonistic interactions that can be used to manage viruses.

550 More complete analyses of viromes will include persistent plant viruses whose role

551 remains unknown, and environmental viruses such as genomoviruses identified in many types of

552 environments, including plants. Understanding the interactions within the phytovirome will

553 contribute to a fuller understanding of the phytobiome, with the potential to inform novel

554 strategies for predicting disease emergence, and for improving integrated plant health

555 management more generally.

556

557 Dissecting the phytobiome: network analyses as a tool for linking biological organization to

558 organismal and holobiont phenotypes

559 Contributed by Linda Kinkel and Matthew J. Michalska-Smith

560 Despite the widespread recognition of the phytobiome as an important ecological unit of

561 organization, we remain limited in our understanding of the ways in which phytobiome

562 phenotype (hereafter, “phenotype”; e.g. yield, health, productivity, stress resilience) is mediated

563 by the complex array of coexisting organisms and their interactions. There is increasing

564 evidence that the overwhelming majority of species in the phytobiome are involved in myriad

565 interactions, both between individual microbes and with the plant host. Importantly, these

566 pairwise interactions can be further mediated by the presence of other species and/or interactions.

567 However, we have limited insight into the ways in which the biological organization of the

568 holobiont, and its associated array of species interactions, impact phenotype. Moreover, we have Page 26 of 112

569 little in the way of analytical, experimental, or conceptual frameworks for building this

570 understanding.

571 Network analyses offer a useful starting point for exploring the biological organization of

572 phytobiomes, by characterizing the relationships among species or operational taxonomic units

573 within a community. Co-association networks have been widely used to describe plant

574 microbiomes (Bakker et al. 2014; Poudel et al. 2016; Shi et al. 2016), partly because they can

575 be readily created using amplicon sequencing data. Specifically, co-association networks identify

576 pairs of species whose abundances are significantly correlated across space or time. Connections

577 between species can represent positive or negative co-associations. Such networks provide a

578 holistic perspective on the biological organization of the phytobiome, for example in quantifying

579 community connectivity (the total number of associations within the community). Communities

580 with low connectivity have few consistently co-associated species, suggesting either weak or

581 inconsistent organization across the landscape, while high connectivity might suggest strong

582 environmental determinants of community structure. In one study, community connectivity was

583 reported to increase in rhizosphere microbiomes over the course of a growing season (Shi et al.

584 2016), implying an increasing role for microbiome community organization and consistent

585 assembly dynamics as the season progresses.

586 It is critical to note also a significant limitation of co-association networks to

587 understanding species interactions within the phytobiome. Species may coexist because of shared

588 habitat preferences (passive co-association) or explicit species interactions (e.g. syntrophy,

589 nutrient complementarity, or mutualisms). Analogously, species may be negatively correlated

590 across phytobiomes because of distinct habitat preferences or due to direct antagonistic or

591 competitive species interactions. Thus, positive or negative co-associations do not in themselves, Page 27 of 112

592 per se, prove a role for species interactions in community assembly or functional characteristics

593 (Freilich et al. 2018). Despite this limitation, co-association analyses have also been commonly

594 used to define `modules’, or collections of species that tend to have similar abundance profiles

595 across samples.

596 Species interaction networks offer a more definitive means for evaluating the biological

597 organization of individual communities. In recent research, we have mapped species resource

598 competitive, antagonistic, and signaling interactions among microbial populations within

599 individual holobionts. In contrast to co-association networks, interaction networks detail explicit

600 pairwise competitive, inhibitory, or signaling interactions among populations (Figure 1). Though

601 the extent of such detailed pairwise interaction networks is limited by our ability and capacity for

602 culturing isolates, these networks highlight the complexity of, and variation in, species

603 interactions among plant microbiomes. For example, connectivity differs substantially among

604 these three communities, but so does the distribution of interactions among species (Figures

605 2,3).

606 By identifying specific microbial isolates that are either especially impacted by species

607 interactions (high in-degree) or are dominant in impacting other species (high out-degree), we

608 can also begin to characterize the roles occupied by specific microbes within the network (e.g.

609 Figure 2 and 3). Understanding the extent to which particular taxa are likely to be significantly

610 engaged in species interactions, and which taxa are largely independent (non-interactive) is

611 important to understanding the ways in which various taxa contribute to the aggregate

612 phenotype. For example, highly interactive species within the microbiome may have little direct

613 impact on the plant, but still play critical roles in determining phenotype due to their impacts on

614 other organisms. For these species, microbiome composition may be especially critical to Page 28 of 112

615 determining their impact on the phytobiome. In contrast, interaction-independent taxa may have

616 consistent impacts on phenotype, regardless of the composition or relative abundances of other

617 microbes in the phytobiome. Similarly, highly interactive pathogens may be more amenable to

618 biological control than non-interactive pathogen populations.

619 Our goal in effective management of phytobiomes is to optimize holobiont phenotypes.

620 The complexity of phytobiomes can be daunting, but network analyses offer an important tool

621 for building our understanding of biological organization within the phytobiome, and a means for

622 quantifying variation in community structure. Systematic evaluation of co-association and

623 species interaction networks among phytobiomes are needed to shed light on the ways in which

624 the biological organization of the phytobiome and its array of species interactions impact

625 phenotype, and provide a path towards effective management.

626

627 Epidemiological perspectives on scaling phytobiome evaluation / network analysis

628 Contributed by Karen Garrett and Ricardo I. Alcalá-Briseño

629 Many concepts from epidemiology can inform phytobiome analyses, particularly when

630 the focus is on pathogens, their vectors, and other microbes that interact with them. One key

631 emphasis of epidemiology is dispersal, a challenge for understanding complete phytobiomes,

632 where a wide range of mechanisms and scales of dispersal may be important. A second key

633 component of epidemiology is integration of human decision-making and its influence on both

634 small- and large-scale phytobiomes. Network analysis is a powerful tool for evaluating microbe

635 dispersal and the spread of information among microbes, plants, and humans, with effects on

636 decision-making across taxa and scales (Garrett 2012; Garrett et al. 2018). Networks of

637 microbial taxon association can be used to identify candidate assemblages for biocontrol (Poudel Page 29 of 112

638 et al. 2016). Global crop breeding networks describe the potential for human influence on the

639 global dispersal of crop genotypes and associated phytobiomes (Garrett et al. 2017). In

640 individual farmers’ fields, particularly for resource-poor farmers who may have limited options

641 for purchasing seed, an “integrated seed health strategy” can support decision-making about

642 balanced use of quality-declared seed, crop resistance to disease, and on-farm management

643 (Thomas-Sharma et al. 2017). Local networks of crop seed exchange, in combination with

644 farmers’ strategies, determine the final distribution of crop genotypes and pathogens, including

645 the distribution of crop genes that select for particular microbiomes (Andersen et al. 2019;

646 Buddenhagen et al. 2017).

647

648 INCREASING FOCUS ON MACROORGANISMS IN THE PHYTOBIOME

649 Contribution of soil animals to soil microbiome function

650 Contributed by Kyle Wickings

651 While microbes are acknowledged as the dominant driver of ecosystem processes in the

652 rhizosphere, invertebrates can have both direct and indirect effects on rhizosphere processes

653 through their interactions with microorganisms. Soil animals contribute directly to soil

654 microbiome composition and function through the consumption of bacterial cells and fungal

655 hyphae, and, through their movement, also aid in microbial dispersal throughout the soil profile

656 and the colonization of new resources (Crowther et al. 2012; Lilleskov and Bruns 2005).

657 Invertebrates also make a number of indirect contributions to soil microbial communities and

658 processes including modification of the distribution and chemical composition along with the

659 rate of introduction of new microbial resources in the soil profile and within soil aggregates Page 30 of 112

660 (Chamberlain et al. 2006; Shuster et al. 2001; Wickings and Grandy 2011). These changes often

661 stimulate microbial activity, increase community size, and alter community structure.

662 Interest in manipulating microbial communities for enhancing ecosystem services such as

663 plant productivity, pest suppression, nutrient cycling and soil organic matter formation warrants

664 a more complete understanding of the biotic factors that govern microbial functioning in soils.

665 Given the growing awareness of the importance of invertebrates as moderators of microbial

666 function in the rhizosphere, it is critical that they be better integrated into the soil microbiome

667 framework. For decades, soil ecologists have acknowledged that the effects of soil fauna on

668 belowground ecosystem services stem both from their direct interaction with plant residues and

669 their alteration of soil microbial communities (Seastedt 1984); however, direct and indirect

670 effects of soil animals are difficult to distinguish experimentally. Consequently, the relative

671 importance of animal-microbe interactions in governing microbial control of soil processes

672 remains poorly understood, and such interactions require more attention in modern soil

673 microbiome research. This knowledge gap will become increasingly relevant when attempting to

674 manipulate microbiome traits in managed ecosystems receiving diverse plant, soil, and pest

675 management practices, many of which are known to alter both microbial and animal

676 communities belowground (Gan and Wickings 2017; Wickings and Grandy 2013). Such

677 diversity in ecosystem management will be important to account for when predicting the

678 functional output of soil microbiome manipulations in agricultural production systems.

679

680 Modifying the composition and function of plant-associated arthropods

681 Contributed by Mary Barbercheck Page 31 of 112

682 Arthropods comprise the largest and most varied group of invertebrates on Earth and are

683 essential to ecosystem health. They contribute to many critical ecological processes, including

684 decomposition, pollination, and predation. In agricultural and other managed ecosystems, some

685 plant-feeding arthropods can become pests, which are estimated to cause a loss of about 18-26%

686 of world crop production (Culliney 2014). The most widespread approach to controlling

687 arthropod plant pests and associated losses in commercial agriculture is through the application

688 of insecticides. Although the use of insecticides has contributed to agricultural productivity, the

689 risks for negative non-target impacts to the environment, humans, and other animals, including

690 natural enemies of arthropods, and loss of efficacy through development of resistance, illustrate

691 the need for integration of novel approaches to arthropod pest management (Damalas and

692 Eleftherohorinos 2011; Squillace et al. 2002).

693 Alternative approaches to pesticide-intensive modification of arthropod community

694 composition and function need to address ecological and organismal processes at multiple spatial

695 and temporal scales, and their mediation by biotic and abiotic components of the environment

696 and management practices (Begg et al. 2017). Two general factors in trophic interactions that

697 drive populations of herbivorous arthropods that vary in relative strength among communities

698 and ecosystems can be targeted for manipulation (Walker and Jones 2001). “Top-down” factors

699 are those related to the action of natural enemies (e.g., predators, parasitoids, pathogens) on

700 plant-feeding arthropods, whereas “bottom-up” factors include those related to food quality (e.g.,

701 nutrient content or secondary metabolites) or supply. Approaches that combined top-down and

702 bottom-up factors could improve efficacy of arthropod management strategies (Jaber and

703 Ownley 2018; Peterson et al. 2016).

704 Top-down factors Page 32 of 112

705 At the field and landscape scale, manipulating the biotic and abiotic components of the

706 environment to support populations and promote the efficacy of natural enemies of plant-feeding

707 arthropods is the basis of conservation biological control (CBC) (Chaplin-Kramer et al. 2011;

708 Tscharntke et al. 2008). To implement CBC to achieve predictable reductions of plant-feeding

709 arthropods is complex, and focuses on habitat management to add diversity to resources that are

710 limiting to natural enemies, e.g., shelter, nectar, alternate prey, or pollen, resulting in an increase

711 in natural enemies for pest suppression (Begg et al. 2017; Letourneau et al. 2011). Common

712 practices in CBC include reducing the use of broad-spectrum insecticides and increasing the

713 diversity of floral and other plant-associated resources through cover crops and establishing edge

714 or in-field plantings of floral resource plants (insectaries), beetle banks, and hedgerows. For

715 example, the floral resource plant, sweet alyssum, Lobularia maritima (L.) Desv. (Brassicaceae),

716 supported the suppression of aphids (Hemiptera: Aphididae) by the hoverfly, Eupeodes

717 fumipennis (Thomson) (Diptera: Syrphidae) in California lettuce fields. The presence of alyssum

718 enhanced hoverfly egg production, resulting in more hoverfly larvae and fewer aphids. Hoverfly

719 survival was unaffected by alyssum, indicating that the effect of alyssum on aphids was mediated

720 primarily through enhanced reproduction by the hoverfly (Hogg et al. 2011).

721 Bottom-up factors

722 Plants have evolved multiple defense strategies against herbivorous organisms, including

723 arthropods (Erb et al. 2012; Johnson et al. 2016). A major defense against herbivory by

724 arthropods involves chemical defenses, represented by constitutive or induced secondary

725 metabolites (reviewed in Mithofer and Boland 2012). The phytohormones jasmonic acid (JA)

726 and salicylic acid (SA) coordinate the complex signaling pathways involved in these multitrophic

727 interactions (Pieterse et al. 2014; Robert-Seilaniantz et al. 2011). Chemical defenses can be Page 33 of 112

728 toxic, repellent, or anti-nutritive for herbivores directly, or they may affect herbivores indirectly

729 via the attraction of natural enemies. Direct defenses include changes in plant nutrient profiles,

730 production of toxins, digestion inhibitors, and herbivore-induced plant volatiles (HIPVs)

731 (Johnson et al. 2013b; Kessler and Baldwin 2001; Turlings et al. 1990), and include compounds

732 such as alkaloids, cyanogenic glycosides, glucosinolates, and terpenoids. HIPVs can be toxic to

733 some members of the phytobiome, e.g., microbes and herbivorous arthropods (van der Meijden

734 and Klinkhamer 2000), and therefore, can have complex effects on the phytobiome, with both

735 positive and negative consequences for the plant (Poelman 2015).

736 In indirect defenses, plants under attack increase production of HIPVs that can be used by

737 predators as cues for locating their herbivore prey (Khan et al. 2008; Poveda et al. 2010). For

738 example, above-ground, egg deposition by stemborer moths, Chilo partellus, on corn was

739 associated with emission of HIPVs that attracted the egg parasitoid, Trichogramma bournieri,

740 and the larval parasitoid, Cotesia sesamiae (Tamiru et al. 2012). The authors suggested that these

741 results implied a sophisticated defense strategy whereby plants recruit parasitoids in anticipation

742 of egg hatching. In another example, the volatile sesquiterpene (E)‐caryophyllene serves as a

743 signal involved in plant defense against below-ground herbivorous arthropods (Rasmann et al.

744 2005; Tamiru et al. 2012). Roots of some varieties of corn release caryophyllene in response to

745 feeding by larvae of the Western corn rootworm, Diabrotica virgifera virgifera, a beetle pest of

746 corn. This compound attracts a natural enemy of rootworms, the entomopathogenic nematode,

747 Heterorhabditis megidis. Field experiments showed a fivefold higher nematode infection rate of

748 D. v. virgifera larvae on a corn variety that produced the signal compared with a variety that did

749 not (Rasmann et al. 2005). Page 34 of 112

750 Root-associated microbes, including symbiotic fungi such as mycorrhizae and

751 endophytes, may trigger physiological changes in the host plant that induce systemic host plant

752 defenses and influence interactions between plants and aboveground insects at several trophic

753 levels, including herbivores, predators and parasitoids (Pangesti et al. 2013; Pineda et al. 2017;

754 Pineda et al. 2013). An interesting example is the interaction between fungi historically

755 described as entomopathogens that can also grow as endophytes in a variety of host plants, where

756 they function as antagonists of herbivorous arthropods and plant pathogens (Jaber and Ownley

757 2018), plant growth promoters (Bamisile et al. 2018; Sasan and Bidochka 2012), and

758 rhizosphere colonizers (Hu and St Leger 2002; Leger 2008; Pava-Ripoll et al. 2011). The

759 negative effects of endophytic fungal entomopathogens on herbivorous arthropods have been

760 attributed to induced systemic plant resistance (Akello and Sikora 2012; Lopez and Sword 2015;

761 Lopez et al. 2014; Martinuz et al. 2012). The suggested mode of action for antagonism of

762 herbivorous arthropods is feeding deterrence or antibiosis due to fungal metabolites secreted in

763 planta (Akello et al. 2008; Akutse et al. 2013; Golo et al. 2014; Gurulingappa et al. 2010;

764 Mantzoukas et al. 2015; Mutune et al. 2016; Muvea et al. 2014; Rios-Moreno et al. 2016);

765 however, studies have not examined specific mechanisms for induced resistance associated with

766 endophytic entomopathogens.

767 Several questions remain to be answered to improve our ability to manipulate plants, the

768 phytobiome, and their environment to enhance beneficial processes (e.g., biological control) and

769 reduce herbivory and losses of yield and quality. Can we breed for plant volatiles to improve

770 plant resistance against key pests directly (bottom-up) and indirectly via improved biological

771 control (top-down)? What are the roles of rhizosphere organisms and endophytes in mediating

772 arthropod interactions with other arthropods and plants? What is the role of rhizosphere Page 35 of 112

773 competency and in planta competitiveness in the establishment of plant-protective endophytic

774 microbes by seed or soil inoculation? How do crop cultivars, soil types, microclimates,

775 agronomic practices, and the presence of other organisms, including other herbivores,

776 endophytes, plant pathogens, omnivores, and predators affect the establishment of beneficial

777 organism(s) (Rosenheim et al. 2011; Wani et al. 2015)? Can we breed plants to modify the

778 phytobiome to alter the quality of hosts for multiple herbivorous arthropods and produce

779 herbivore-induced HIPVs that are cues for natural enemies (Douglas 2015; Stam et al. 2014;

780 Zhu et al. 2014b)? Should the development of plant-protective microbes focus on individual

781 species or compatible consortia of microorganisms? Can we alter the phytobiome to

782 simultaneously manage pests and pathogens (Pineda et al. 2017)?

783

784 The role of host plant genetics in shaping phytobiomes

785 Contributed by John Carlson

786 Improving plant climatic resilience, food-fiber-fuel production and ecosystem

787 sustainability in concert, requires a phytobiomes-based systems biology approach that considers

788 all factors that influence plant growth and productivity (Busby et al. 2017). A plant’s genotype

789 plays a major role in shaping plant growth and productivity. However, whether in highly

790 managed agricultural cropping (eco)systems or in unmanaged natural ecosystems, plant growth

791 and productivity are the result of highly complex gene networks and genotype-by-environment

792 interactions (Stuber 1994).

793 Plant growth and productivity are complex multigenic traits, with each of the many genes

794 involved across the genome exerting only minor control on each trait. Complex trait variation is

795 typically then “quantitative”, in which natural populations display a normal distribution of Page 36 of 112

796 phenotypic values resulting from the contributions of varying accumulations of alleles for the

797 many genes involved (Stuber 1994). Complex traits tend to have lower heritability than simple

798 gene-for-gene traits, i.e. performance differs among generations and different conditions. Low

799 heritability in complex trait phenotypes is in large part because the many underlying genes

800 respond differently to different environmental conditions (Eversole 2016). Thus complex traits

801 reflect both the underlying genotypic variation and the environment in which the genes were

802 expressed.

803 We now have the means to efficiently and comprehensively catalogue genotypes of

804 individual plants by whole genome sequencing. However, pinning down all factors in the

805 environmental variables in the phenotype = genotype x environment equation for complex plant

806 phenotypes like yield has remained elusive (Busby et al. 2017; Eversole 2016).

807 In practice, natural selection and breeding (artificial selection) succeed in improving

808 mean growth and productivity of crops by shaping (matching) factors, such as composition and

809 function of the microbial community with each plant’s genotypes. To shape phytobiomes, we

810 will need to decipher the many interactions of genotype and environment at a more

811 comprehensive, systems level. Experimentally, this will require studies that compare replicates

812 of multiple genotypes at multiple sites differing environmentally, over multiple years and

813 generations. Fortunately, this is what quantitative geneticists (breeders) do. They have provided a

814 rich literature, data collections, and even long-term field trials for perennial crops, documenting

815 the genetic contributions to phenotypes in many crops and tree species from past and ongoing

816 breeding activities.

817 Furthermore, the field of ecological genomics is contributing important insights into the

818 profound effects from a community genetics perspective of the genotype of host plants on Page 37 of 112

819 shaping multi-trophic interactions of perennial plants, such as trees, with other organisms such as

820 herbivores and symbionts (Keith et al. 2010; Ungerer et al. 2008). In exchange, a holistic

821 understanding of phytobiomes should strengthen the predictive value of breeding and

822 quantitative ecosystem models (Singh et al. 2018; Stewart et al. 2018). To achieve a

823 comprehensive understanding of phytobiomes for improving crop productivity, it will be

824 necessary to include the effect of the host plant’s genotype on shaping, and elucidating, the

825 important factors influencing genetic-by-environment interactions (Busby et al. 2017).

826

827 PHYTOBIOME MANIPULATION BEYOND AGRICULTURAL PRODUCTION

828 Post-harvest management of crop microbiomes

829 Contributed by Jasna Kovac, Taejung Chung, and Xiaoqing Tan

830 Understanding the taxonomic composition and functional role of post-harvest crop

831 microbiomes is essential for enhancement of the quality and safety of plant foods (Bokulich et al.

832 2016). Early challenges with the sensitivity of direct metagenomic sequencing for detecting low-

833 abundant microbial taxa of interest relevant to plant food quality and safety have led to a

834 paradigm shift in approaches to microbial hazard detection. As opposed to direct detection of

835 low abundance pathogens of interest, changes to microbiomes in which pathogens and spoilage

836 microorganisms commonly occur are beginning to be explored. Detection of such microbiome

837 perturbations requires a baseline database of microbiomes and their transcriptomes in samples

838 collected throughout the food supply chain. These are being catalogued by the Sequencing the

839 Food Supply Chain consortium led by the International Business Machines (IBM) Corporation,

840 in collaboration with other industry and academic partners, among others (Fontanazza 2016). Page 38 of 112

841 Development of new methods, and optimization of existing methods, for characterization

842 of plant food supply microbiomes is leading to a better understanding of microbial ecology at

843 different stages in the food supply chain (Bokulich et al. 2016; Busby et al. 2017; Jarvis et al.

844 2015; Lebeis 2014; Leff and Fierer 2013; Rossouw and Korsten 2017). Relating microbiome

845 composition to function, and identifying factors influencing function, is the first step towards

846 shaping agricultural pre- and post-harvest microbiomes for increased food quality and safety.

847 However, several challenges remain to be overcome. As in any system, the key influential

848 environmental and anthropogenic factors shaping microbiomes need to be identified (Figure 4).

849 Second, identified factors need to be categorized based on the ability to control or manage them

850 by changing pre-harvest agricultural and post-harvest food processing practices. A number of

851 environmental factors are impacted by anthropogenic activities (e.g., air, weather, soil, plant

852 surface), but not all can be practically managed. For example, the air microbiome has been

853 shown to overlap with the plant phyllosphere microbiome, but cannot be readily controlled in the

854 field (Ottesen et al. 2016). Nevertheless, there is great potential in controlling soil and plant

855 microbiomes that serve as inocula for built post-harvest crop processing environments.

856 Post-harvest environment microbiomes are typically managed through physical and

857 chemical cleaning and sanitizing procedures. These procedures, even though controlled, often

858 fail to control pathogens such as Listeria monocytogenes that are commonly associated with

859 fresh produce outbreaks (Angelo et al. 2017; Garner and Kathariou 2016). The difficulties in

860 effective cleaning and sanitizing of poorly accessible parts of built environments are recognized

861 as the main cause of foodborne pathogen persistence in these environments; often in biofilms

862 (Colagiorgi et al. 2017). Food safety challenges associated with produce supply chain are driving

863 a new wave of research focused on investigating the protective potential of microbial biocontrol Page 39 of 112

864 strains. Manipulations of post-harvest food processing environments, with the goal of pathogen

865 exclusion, have been successfully demonstrated in the animal food supply chain (Zhao et al.

866 2013), but are yet to be assessed in plant processing environments. Going forward, these types of

867 control agents will likely need to be carefully tailored to allow for their successful growth and

868 competition with the baseline microbiomes under specific environmental conditions (e.g.

869 different temperatures, humidity levels, surface materials).

870

871 Can microbial intervention be used as a strategy to manage invasive plants?

872 Contributed by Kurt Kowalski

873 Invasive plants are a global problem, including in highly valued coastal and wetland

874 systems. As non-native plants (e.g., common reed in the Great Lakes system; Phragmites

875 australis (Cav.) Trin. ex Steud.) invade, they often displace native flora, reduce biodiversity,

876 degrade fish and wildlife habitat, reduce recreational opportunities, increase fire hazards, and

877 decrease property values (Great_Lakes_Phragmites_Collaborative 2018; Isely et al. 2017;

878 Weinstein and Balletto 1999). Therefore, resource managers invest substantial time and

879 resources in management and control (Braun et al. 2016; Hazelton et al. 2014), often with little

880 long-term success (Martin and Blossey 2013). Similarly, the agricultural industry is seeking new

881 ways to control weeds (often non-native) sustainably and maximize crop growth. However, the

882 underlying biological mechanisms that help invasive plants outcompete native plants, or improve

883 the yield of agricultural crops, are not clear.

884 We know that microbes (e.g., bacteria, fungi, oomycetes) exist in soil, water, on plants,

885 and in plants during all growth stages. We also know that P. australis and other invasive plants

886 host a tremendous diversity of endophytic microbes (Bickford et al. 2018; Bowen et al. 2017; Page 40 of 112

887 Clay et al. 2016; Reinhart and Callaway 2006; Shearin et al. 2018; Soares et al. 2016), both in

888 their native range and introduced range. Microbes are known to form symbiotic relationships

889 with plants that generate many plant benefits, including increased tolerance to heat, drought, and

890 other stresses, accelerated development of seedlings, and increased growth and yield (de Vrieze

891 2015). Further, plants can manipulate the microbiome to influence the local environment (Bowen

892 et al. 2017; Funck-Jensen and Hockenhull 1984; White et al. 2018) and create conditions that

893 support enhanced growth. Increased stress tolerance and growth characteristics are, in part, why

894 non-native plants can be so invasive on the landscape.

895 The symbiotic relationships among microbes and plants, therefore, can be targeted for

896 disruption or enhancement, depending on the purpose of the management actions (Kowalski et

897 al. 2015). Once microbial composition is characterized and the roles of individual microbes are

898 known, new techniques can be developed to disrupt symbiotic relationships and decrease the

899 benefits that they generate for invasive plants (Kennedy 2018; Kowalski et al. 2015), or

900 alternately, promote microorganisms that negatively impact invasive plant health and abundance.

901 Similarly, symbiotic relationships that promote growth in native species, agricultural crops, or

902 desired species can be targeted for enhancement. Combining these two approaches may generate

903 innovative treatments that combat invasive plant growth while promoting growth of desirable

904 species. Advancements in research to identify the microbes in invasive plants of aquatic and

905 terrestrial systems, and the roles that they play in growth and production, are leading to exciting

906 new approaches for invasive species management, habitat restoration, and agricultural

907 production.

908 Page 41 of 112

909 PRACTICAL AND SOCIAL CONSIDERATIONS FOR PHYTOBIOME

910 MANIPULATION

911 Communicating phytobiome management to stakeholders and consumers

912 Contributed by Jessica Myrick

913 Phytobiome scientists are eager to avoid public misconceptions and potential

914 politicization of their work, similar to what has occurred with genetically modified organisms. It

915 may be tempting to suppose that simply communicating more frequently about phytobiome

916 science will prevent stakeholders and the public from misunderstanding the science.

917 Nonetheless, facts alone are often not enough. It used to be a common assumption that if

918 scientists could educate the public and remove any knowledge deficits that existed, then people

919 would accept the science and act in line with it. However, research science communication

920 shows this “deficit model” does not hold up to empirical scrutiny

921 (National_Academies_of_Sciences_Engineering_and_Medicine 2017). This does not mean that

922 facts cannot help shift attitudes; however, it does mean that when communicating information

923 about the phytobiome, scientists need to keep in mind the goals and motivations of different

924 audiences.

925 The empirical study of communication and media effects offers phytobiome scientists

926 some strategies for more effectively sharing their research with stakeholders and consumers. For

927 example, using stories or narrative structures is more engaging than listing facts alone

928 (Dahlstrom 2014). The story could be the researcher’s path to the topic, the narrative of how the

929 study came to be and then progressed, the tale of the study of the phytobiome more generally, or

930 even a single plant’s “story.” Page 42 of 112

931 Consider using emotionally evocative content strategically, but not just fear. When

932 people become really scared, many tune out and avoid the topic at hand. Think about ways to

933 talk about solutions to pressing phytobiome issues in order to leave audiences with something

934 hopeful to ponder that can then motivate them to further engage with the topic (Nabi and Myrick

935 2018). Curiosity is another key driver for why people seek science news (Shearer 2018), one that

936 could also help increase interest in the phytobiome. Awe and wonder about the natural world are

937 also powerful drivers of attention and action (Keltner and Haidt 2003), ones which phytobiome

938 scientists could harness for conveying the importance of their research to stakeholders and the

939 public.

940

941 Hurdles to adopting new production practices

942 Contributed by Alyssa Collins, Paul Esker, Beth Gugino, and Kari Peter

943 Although research can identify promising new strategies for phytobiome management,

944 this is not enough for widespread implementation. Farmers/growers consider a number of factors

945 when deciding whether to adopt a new technology, including: 1) Does the technology address a

946 farmer-based need or perceived need?; 2) What is the cost?; 3) How easily can it be integrated

947 with existing farming practices and equipment?; and 4) How will it improve production in the

948 short- and long-term? Farmers must weigh both immediate and long-term benefits, and the

949 importance of each will vary based on the financial security and cropping system(s) of each

950 farmer. Additionally, new approaches are unlikely to work equally across different cropping

951 systems and farm scales. Since profit margins also vary by crop type, an approach may be

952 technically feasible across systems, but only economically feasible in some. Page 43 of 112

953 Adoption of new technologies is largely impacted by attitudes of farmers towards new

954 technology (Mase et al. 2017), i.e. where they lie on the technology adoption curve, as well as

955 whether or not their consumer market will accept the technology. Late adopters are unlikely to

956 make a change without substantial precedent in the farming community, even if shown the

957 benefits of a new technology. Those working to bridge research and practice, such as extension

958 educators, aim to identify early innovators, who can then help to diffuse technology to other

959 farmers. Concepts should be easily communicated through this route, as individual farmers will

960 aim to innovate and adapt new approaches within their personal operation scheme and

961 production constraints.

962 Farmers are cognizant of the fact that there are no silver bullets, and this should be

963 considered in communication. Agricultural practices are meant to be integrated, with additive

964 and/or synergistic impacts on crop health. Farmers are also seeking consistency in production

965 benefits, which leads to predictability in year-to-year yields. For example, it would generally be

966 preferable to see an incremental productivity or profit increase in nine out of ten growing years,

967 as opposed to a large increase in one or two. In addition to the predictability of management

968 impacts, cost-benefit tradeoffs, ease of adoption, and consumer acceptance of the technology are

969 primary concerns of farmers when considering new approaches or technologies.

970

971 Thinking about social innovation as agricultural scientists

972 Contributed by Leland Glenna and Maria Fernanda Vivanco Salazar

973 The likelihood that phytobiome research will contribute to public wellbeing is contingent

974 on the social context in which the research is conducted and diffused. In the United States,

975 agricultural research and development policies and research funding since the late 1970s and Page 44 of 112

976 early 1980s has focused on generating private goods and attracting private research investments

977 (Glenna et al. 2015). Research indicates that this privatization approach to agricultural research

978 and development has failed to generate the promised public benefits (Glenna et al. 2015; Heisey

979 and Fuglie 2018; Lopez and Galinato 2007).

980 The social innovation concept emerged after analysts came to realize that privatization

981 failed to deliver widespread social benefits from science and technology research (Moulaert et al.

982 2013). Rejecting the idea that society is best coordinated through self-regulating markets, social

983 innovation proponents claim that direct policy interventions, institutional change, and other

984 collective actions are necessary to achieve socially equitable outcomes (Jessop et al. 2013). This

985 leads to the question of whether university research is likely to contribute to social innovation.

986 We conducted a survey of 1126 agricultural scientists at 71 United States land-grant

987 universities and analyzed how scientists perceive their research activities contributing to social

988 innovation. The survey measured scientist norms, beliefs, and values regarding how they

989 envision science and technology contributing to social outcomes and how their perspectives

990 relate to their research activities and outputs. Principle components analysis divided scientists

991 into two factors: private-science norms and public-science norms. Since just over one-third of

992 respondents agreed or strongly agreed with the statement that scientists should focus on research

993 with market potential and about two-thirds agreed or strongly agreed that scientists should focus

994 on research with public benefits, it is reasonable to conclude that approximately one-third of

995 scientists adhere to private-science norms and two-thirds adhere to public-science norms.

996 Additional statistical analysis found that those conforming to the private-science factor were

997 significantly more likely than those conforming to the public-science factor to be correlated with

998 the belief that markets are best able to evaluate the social value of science and technology and Page 45 of 112

999 that markets are best at solving social problems like hunger. The private-science factor was also

1000 significantly correlated with less basic science research, with industry funding and industry

1001 collaborations, and with more excludable research outputs.

1002 The findings have implications for national, state, and university policies directed at

1003 promoting social innovations from university-based agricultural research. Traditionally,

1004 university scientists have been expected to conduct basic research, to generate public goods, and

1005 to be dedicated to solving social problems that are not easily addressed with a commercial

1006 product. If one-third of public agricultural scientists are committed to private-science norms and

1007 practices, the capacity for land-grant universities to contribute the public goods needed to solve

1008 social problems will likely be compromised.

1009

1010 CONCLUSIONS

1011 Both wild and tamed phytobiomes have been modified by humans for centuries, but

1012 without an understanding of how most components of the system were being changed or

1013 impacting plant growth. The goal of phytobiome modification, as discussed here and in our

1014 symposium, is to understand how the many elements of the phytobiome interact, and how

1015 specific management of previously ignored factors can help to meet societal needs.

1016 A discussion within a larger group in our workshop identified many potential challenges

1017 to phytobiome manipulation, encompassing scientific, practical, and social barriers. Within each

1018 of these three categories, numerous factors could feasibly be modified (e.g. crop varieties,

1019 microorganisms, public and grower engagement), whereas many others could not, at least in the

1020 short term (e.g. soil type, culturally-driven norms). Phytobiome science encourages a systems

1021 view of plant production and management, and yet not all components of the system present Page 46 of 112

1022 effective management targets. An important near-term goal for this field should be to estimate

1023 the potential relative contribution and mutability of distinct phytobiome components. Such

1024 estimations should consider both the physical limitations and the practical/social constraints on

1025 management change.

1026 Effective phytobiome management will depend on integrating scientific advances with

1027 farming, management, and/or societal realities; this presents opportunities for innovative and

1028 creative thinking. An important step in this nascent field of science is to identify research

1029 directions that have a high potential for impacting the health and productivity of our natural and

1030 managed landscapes in the face of our currently limited understanding of the complex

1031 interactions within phytobiomes and the level of specificity of these interactions to distinct

1032 biomes and plant species. Given this complexity, inter- and cross-disciplinary communication

1033 across phytobiome researchers, and broadening of the disciplines working in this field, will help

1034 maximize innovation. Ultimately, the health of our environment and the productivity of our

1035 agricultural enterprise require a new approach to managing both wild and natural phytobiomes

1036 into the future.

1037

1038 ACKNOWLEDGEMENTS

1039 We thank Joy Drohan for recording notes during the workshop, which contributed to the

1040 structuring of this manuscript. We also thank all workshop and symposium participants for

1041 insightful discussions on these and other topics that came up during the event. Funding support

1042 for the Wild and Tamed Phytobiomes Symposium was provided by Penn State University,

1043 through the College of Agriculture, Eberly College of Science, and the Huck Institutes of the

1044 Life Sciences, with additional funding support from WrightLabs, Pall Laboratory, Pacific Page 47 of 112

1045 Biosciences, and NewLeaf Symbiotics. Any use of trade, firm, or product names is for

1046 descriptive purposes only and does not imply endorsement by the U.S. Government.

1047

1048 FIGURE CAPTIONS

1049 Figure 1: Species interactions networks for three microbial communities (A-C). Each red

1050 circle represents an individual microbial isolate, and interactions between isolates are

1051 characterized here with directional arrows. Thus, for example, when considering inhibitory

1052 interactions (antibiosis), an arrow indicates the capacity of one isolate (the arrow source) to

1053 inhibit another isolate (the arrow head). Similarly, resource competition can be captured by

1054 arrows that identify capacities of isolates to consume some threshold proportion of another’s

1055 resource niche space. Additional species interactions, including both directional and non-

1056 directional interactions (e.g. signaling, syntrophy, parasitic or mutualistic relationships) may also

1057 be summarized using networks.

1058

1059 Figure 2: Degree distribution for each of these three microbial communities (A-C). To

1060 generate the degree distribution, the number of connections each isolate has to other isolates

1061 within the community (the degree for an isolate) is first determined for every isolate. For directed

1062 interactions (e.g. inhibitory interactions illustrated here), every isolate has both outgoing

1063 interactions (out-degree, or the number of antibiotic targets) and incoming interactions (in-

1064 degree, or the number of organisms to which an isolate is susceptible). The distribution of in-

1065 degrees and out-degrees among isolates within a community sheds light on the different roles

1066 that individual isolates play within the community. For example, communities having greater

1067 abundance of isolates that have high out-degrees may be dominated by these `superkillers’ that Page 48 of 112

1068 inhibit most other community members. Similarly, variation in the frequency of low vs. high in-

1069 degrees among communities provides insight into the extent to which interactions are centered

1070 on a small number of highly sensitive isolates vs. more broadly distributed among many

1071 moderately resistant isolates, facilitating inferences about functional diversity.

1072

1073 Figure 3: Out-degrees vs. in-degrees for every isolate in the same three communities (A-C).

1074 In these plots, each isolate is represented by a point whose position is dictated by the

1075 combination of incoming and outgoing connections. This allows for enhanced inference about

1076 specific isolates and their roles within the network. For instance, despite the visual similarity in

1077 their networks (Figure 1), communities A and C have quite distinct relationships between in-

1078 and out-degrees among isolates, while those for networks A and B are highly similar.

1079 Communities A and B are composed of a range of highly inhibitory/highly resistant isolates

1080 (high out-degree/low in-degree) and weak inhibitory/low resistance (small out-degree/high in-

1081 degree), while community C is composed of isolates having more broadly similar resistance and

1082 inhibitory capacities. Variation in the strength and direction of out-degree vs. in-degree

1083 relationships among communities provides a basis for quantifying differences in interaction

1084 structure among communities.

1085

1086 Figure 4. Potential factors in natural and built environments that may shape food crop

1087 microbiomes. This illustration is based on an apple supply chain, but represents the type of

1088 interactive processes that govern microbial flow between environments and/or management

1089 stages, as well as the factors that constrain microbiome composition. Here, environmental and

1090 anthropogenic factors are influencing microbiome composition on food crops throughout the Page 49 of 112

1091 food supply continuum. This includes natural environments in the pre-harvest chain and built

1092 environments in the post-harvest chain.

1093

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1693 the sorghum root microbiome and enriches for monoderm bacteria. P. Natl. Acad. Sci. 1694 USA 115:E4952-E4952. 1695 Xu, X. M., Jeffries, P., Pautasso, M., and Jeger, M. J. 2011. Combined use of biocontrol agents to 1696 manage plant diseases in theory and practice. Phytopathology 101:1024-1031. 1697 Xuan, W., Beeckman, T., and Xu, G. H. 2017. Plant nitrogen nutrition: sensing and signaling. 1698 Curr. Opin. Plant Biol. 39:57-65. 1699 Yergeau, E., Bell, T. H., Champagne, J., Maynard, C., Tardif, S., Tremblay, J., and Greer, C. W. 1700 2015. Transplanting soil microbiomes leads to lasting effects on willow growth, but not 1701 on the rhizosphere microbiome. Front. Microbiol. doi: 10.3389/fmicb.2015.01436. 1702 Zhalnina, K., Louie, K. B., Hao, Z., Mansoori, N., da Rocha, U. N., Shi, S. J., Cho, H. J., Karaoz, U., 1703 Loque, D., Bowen, B. P., Firestone, M. K., Northen, T. R., and Brodie, E. L. 2018. Dynamic 1704 root exudate chemistry and microbial substrate preferences drive patterns in 1705 rhizosphere microbial community assembly. Nat. Microbiol. 3:470-480. 1706 Zhang, L., Xu, M. G., Liu, Y., Zhang, F. S., Hodge, A., and Feng, G. 2016. Carbon and phosphorus 1707 exchange may enable cooperation between an arbuscular mycorrhizal fungus and a 1708 phosphate-solubilizing bacterium. New Phytol. 210:1022-1032. 1709 Zhao, T., Podtburg, T. C., Zhao, P., Chen, D., Baker, D. A., Cords, B., and Doyle, M. P. 2013. 1710 Reduction by competitive bacteria of Listeria monocytogenes in biofilms and Listeria 1711 bacteria in floor drains in a ready-to-eat poultry processing plant. J. Food Protect. 1712 76:601-607. 1713 Zhu, B., Gutknecht, J. L. M., Herman, D. J., Keck, D. C., Firestone, M. K., and Cheng, W. X. 2014a. 1714 Rhizosphere priming effects on soil carbon and nitrogen mineralization. Soil Biol.

1715 Biochem. 76:183-192. 1716 Zhu, F., Poelman, E. H., and Dicke, M. 2014b. Insect herbivore-associated organisms affect plant 1717 responses to herbivory. New Phytol. 204:315-321. 1718 Zilber-Rosenberg, I., and Rosenberg, E. 2008. Role of microorganisms in the evolution of 1719 animals and plants: the hologenome theory of evolution. FEMS Microbiol. Rev. 32:723- 1720 735. 1721 A B C Page 64 of 112 ● ● ● 10 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

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Natural Environment Built Environment Employee personal hygiene Organic vs. conventional Control of built growing practices environment microbiome Human (harvester) by introduction of Storage microbiome biocontrol microbial strains environment and conditions

Environmental microbiome: Handling of Introduction of the Cleaning and (air, rain, irrigation water, wildlife, soil) finished microbiome from sanitizing product natural to built practices environment

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The Wild and Tamed Phytobiomes Symposium was held from 19-22 June 2018 in University Park, PA, USA, and was co-chaired by Kevin Hockett and Terrence Bell. Other members of the organizing committee were Carolee Bull, Cristina Rosa, Teh-Hui Kao, Siela Maximova, Mary Barbercheck, Jasna Kovac, and Kara Krall. This symposium was the 21st installment of the Penn State Plant Biology Symposium Series, which is hosted by the Intercollege Graduate Degree Program in Plant Biology.

The goal of this symposium was to highlight cutting edge research in plant-microbe relationships, but to also highlight research focused on the many other organisms and factors that impact phytobiome health and productivity in wild and tamed systems. The symposium brought together presenters with extremely diverse research backgrounds, with some talks also focusing on societal, economic, and practical hurdles to phytobiome manipulation and management.

The Keynote Speakers were Mary Firestone (UC Berkeley), Gwyn Beattie (Iowa State University), and George Kowalchuk (Utrecht University). The oral sessions were organized as follows:

SECTION 1: NATURAL PROCESSES AND PHYTOBIOMES

Evolutionary vs. Ecological Processes Speakers: Linda Kinkel (University of Minnesota), Joel Sachs (UC Riverside), Joanne Emerson (UC Davis), Jesse Lasky (Penn State University)

Multipartite Interactions Speakers: Gary Felton (Penn State University), Kyle Wickings (Cornell University), Tory Hendry (Cornell University), Corlett Wood (University of Toronto), Kevin Rice (University of Missouri)

Lessons from Epidemiology Speakers: Andrea Otteson (FDA CFSAN), Karen Garrett (University of Florida), Alejandra Huerta (Colorado State University), Brian Tague (Wake Forest University)

SECTION 2: MANAGED PHYTOBIOMES Scientific Opening Act (student speakers) Speakers: Mia Howard (Cornell University), Norma Morella (UC Berkeley), Sarah Gibert (Rutgers University), Jedaidah Chilufya (University of Massachusetts Amherst)

Who Needs Whom: Forces in Recruitment and Assembly Speakers: Sarah Lebeis (University of Tennessee), Joana Falcao Salles (University of Groningen), Ashley Shade (Michigan State University)

Manipulating Phytobiomes Page 69 of 112

Speakers: Etienne Yergeau (Institut Armand-Frappier), Mary Barbercheck (Penn State University), Johan Leveau (UC Davis), Richard Lankau (University of Wisconsin- Madison), Ellen Yerger (Indiana University of Pennsylvania), Rodrigo Valverde (Louisiana State University)

Socioeconomic Issues Related to Phytobiomes Speakers: Trudi Baker (Indigo Ag), Leland Glenna (Penn State University)

WORKSHOP Manipulating Phytobiomes: Challenges and Opportunities Speakers: Bryan Emmett (Cornell University), Mary Ann Bruns (Penn State University), Terrence Bell (Penn State University), Kevin Hockett (Penn State University), John Carlson (Penn State University), Maria Jimenez-Gasco (Penn State University), Gretchen Kuldau (Penn State University), Beth Gugino (Penn State University), Paul Esker (Penn State University), Jessica Myrick (Penn State University), Jasna Kovac (Penn State University), Kurt Kowalski (USGS), Thomas Butzler (Penn State University)

The included abstracts are only from authors that agreed to publication. The full presenter list for the symposium can be found at the end of this document.

Using A Small Aquatic Plant To Study Plant Microbiome Assembly, Structure, And Function

Kenneth Acosta1, Sarah Gilbert1, Jenny Xu1, and Eric Lam1 1Rutgers University, New Brunswick, New Jersey, USA

The application of next-generation sequencing technologies to study the plant microbiome has revealed insights into its composition and structure. However, a greater mechanistic understanding of plant microbiota assembly and function is needed to harness the potential to improve plant health. To this end our group has taken both a culture-independent and culture-dependent approach to study the plant microbiome using the aquatic monocot Duckweed as a model. Duckweed is a family of small aquatic plants that are found in many different regions around the world. It’s small size, fast growth rate, and natural aquatic habitat combine to make Duckweed a facile system to investigate interactions with microbes. In our culture-independent approach we harvested Duckweed from different natural habitats to reveal conserved, environment-specific, and species-specific microbiota. For our culture-dependent approach we have established a sequenced culture collection of 50 putative Duckweed endophytes. We are currently working on characterizing this culture collection for phytohormone production, growth promotion, and attachment in both mono-associations and a community context. We hope through using these novel approaches to unravel various factors, both environmental and biological, which can shape plant microbiota assembly as well as affect functional output. Page 70 of 112

Harnessing nature: The multi-functional role of Metarhizium as a plant protectant

Imtiaz Ahmad1, Brianna Flonc1, Christina Mullen1, Mary E. Barbercheck1, Dawn S. Luthe2, Maria Jimenez-Gasco3 1 Department of Entomology, The Pennsylvania State University, University Park, PA, USA 2 Department of Plant Sciences, The Pennsylvania State University, University Park, PA, USA 3 Department of Plant Pathology and Environmental Microbiology, The Pennsylvania State University, University Park, PA, USA

Recent studies have revealed that many insect-pathogenic fungi, including Metarhizium (Hypocreales: Clavicipitaceae), are also endophytes that can benefit their host plant through plant-disease antagonism and plant growth promotion. Metarhizium is a commonly occurring insect-pathogenic fungus that can establish endophytic relationship with a variety of plants. My research focuses on the molecular and physiological aspects of plant-Metarhizium-insect interactions. Translation elongation factor (TEF) sequencing of 130 Metarhizium isolates from soil indicates that the dominant species at our research sites is M. robertsii. Moreover, in field experiments, cover crop diversity was not related to Metarhizium species diversity. In greenhouse experiments, endophytic colonization of M. robertsii was greater in maize (90%), than in Austrian winter pea (62.5 %), canola (52 %) and cereal rye (50%) plants grown from seeds exposed to a spore suspension of M. robertsii. Re-isolation of M. robertsii from exposed plants was more frequent from roots than from leaves in all crop plants. Endophytic colonization resulted in greater plant height of maize, Austrian winter pea and cereal rye compared to control plants. Similarly, the above-ground biomass of M. robertsii-colonized maize, Austrian winter pea, and canola plants was greater than for plants grown from unexposed seed. In detached-leaf feeding bioassays, the relative growth rate of 2nd instar black cutworm (Agrotis ipsilon) was lower than when feeding on corn leaves from M. robertsii-colonized plants compared to non- treated control plants. My future research goals focus on determining the role of endophytic Metarhizium in plants on Cochliobolus heterostrophus, the causative agent of southern corn leaf blight, and the effects of endophytic M. robertsii on the regulation of gene expression in plants. These newly emerging, but not yet fully understood, ecological roles hint at the potential for the further development of Metarhizium as an inundative biopesticide and protective seed coating.

Redefining the bounds of the bargain – trends in patent eligible subject matter for phytobiome innovations Trudi A. Baker1 1Indigo Ag, Inc., 500 Rutherford Avenue, Charlestown, MA 02129 The US Constitution grants Congress the power “to promote the progress of science and useful arts, by securing for limited times to authors and inventors the exclusive right to their respective writings and discoveries” – this is the bargain of the patent, a limited Page 71 of 112

period of exclusivity is granted to the inventor in return for public disclosure of how to practice the invention. Several key court decisions of the last decade have significantly redefined the bounds of patent eligible subject matter, particularly as related to innovations in the natural and computational sciences. I will discuss these trends in the context of their relative impacts on agricultural innovations and regional and global economic development.

Exploring the impact of plant enrichment for beneficial rhizosphere communities under drought stress

Gwyn A. Beattie1 and Chiliang Chen1 1Department of Plant Pathology & Microbiology, Iowa State University, Ames IA 50014

Breeding plants that foster beneficial interactions with their associated organismal communities requires understanding how plants select for beneficial communities. We predict that the selection pressure for beneficial organisms is strongest under non-ideal plant growth conditions. Although many community members in the phytobiome likely influence plant resiliency to non-ideal growth conditions, the microbial component of phytobiomes is currently the least explored. In this study, we investigated the benefits of rhizosphere microbial communities to plant drought tolerance. We selected drought as a stressor due to the ease of experimentally manipulating soil water content, the numerous mechanisms by which microbes are known and proposed to influence water stress

tolerance in plants, and the importance of drought tolerance given the current threat of water insecurity to crop production in many regions. The studies were performed with legumes due to the extreme sensitivity of biological nitrogen fixation to drought stress, and thus the potential for the results to impact the water and nitrogen status of plants. We characterized the impact of soil water deficits on soil, ectorhizosphere and endosphere communities during successive growth cycles of soybean and bean plants under water- limited and water-rich conditions in field soils in the laboratory. We observed an increase in the plant water use efficiency during the late growth cycles, supporting the possibility that the plants enriched for communities that enhance their tolerance. Our results also suggested that bacterial exposure to drought-induced changes in plants (e.g., ROS, hormone or metabolite changes) impact community composition more strongly than direct bacterial exposure to water deficits; this was evidenced by the greater impact of water limitation on bacterial communities within roots than in the ectorhizosphere or bulk soil. A similar, but less pronounced, trend occurred with fungal communities. Select fungal and bacterial phyla differed greatly in the root endosphere under low versus high soil water content conditions. In particular, members of the Actinobacteria phylum were strongly enriched in the root endosphere under drought conditions. Network analyses based on co-occurrence showed that most of these Actinobacteria were in modules composed primarily of Actinobacteria, suggesting that Actinobacteria interact primarily with other Actinobacteria. Among these, however, one genus, the Glycomyces, was highly linked with diverse members of the community, and was notable for being strongly enriched during each of four successive cycles of serial plant growth under drought conditions. These results support our prediction that plants foster specific organismal associations under stressful growth conditions, and provide a foundation for Page 72 of 112

future work harnessing knowledge of these associations for manipulating both plant genetics and microbial communities to enhance plant drought tolerance.

Evolutionary genomics of sorghum-symbiont interactions

Emily Bellis1, Lua Lopez1, Eric Wafula1, Loren Honaas2, Sarah Hearne3, Claude dePamphilis1, Geoff Morris4, Jesse Lasky1 1The Pennsylvania State University 2United States Department of Agriculture 3International Maize and Wheat Improvement Center 4Kansas State University

Researchers have been rapidly developing of genomic approaches to identify the basis of genotype-environment interactions (GxE). At the same time, researchers studying host- symbiont systems have increasingly emphasized the importance of abiotic environmental variation in modulating symbiotic interactions. We are developing a framework for extending association studies to interactions between host and symbiont genotypes (GxG) that depend on environment. To identify primary environmental factors limiting the distribution of host-specific parasite populations, we developed ecological niche models for Striga hermonthica, a weed that parasitizes cereal crop hosts and is a major biotic constraint to food production in sub-Saharan Africa. Striga germinates upon detection of strigolactones, hormones exuded by host roots that are used to signal mycorrhizal fungi. Sorghum hosts exhibit genetic variation in strigolactones and genes in their biosynthesis pathways that confer resistance. However, our modeling shows likely costs to host alleles resistant to parasitism, suggesting that tradeoffs associated with strigolactone production maintain diversity. As global change impacts host-symbiont interactions, we hope our approach will be broadly useful for understanding genomics in other host-symbiont systems, particularly in non-model systems.

Source partitioning of soil N2O emissions in response to manure and cover crop management in organic systems

Arnab Bhowmik1*, Debasish Saha2*, Armen R. Kemanian2, Jason P. Kaye1, and Mary Ann Bruns1 *Authors with equal contribution 1 Dpt. of Ecosystem Science and Management, Pennsylvania State University, University Park, PA 2 Dpt. of Plant Science, Pennsylvania State University, University Park, PA

Manure and cover crop management determine the synchrony between crop nitrogen (N) demand and supply in organic systems. In a typical corn-soybean-winter grain rotation in the Mid-Atlantic region, manure is incorporated in spring after cover crop termination before corn planting. This practice creates a short temporal window when carbon (C) and N availability from manure and decomposing cover crops exceeds corn N demand and Page 73 of 112

may favor nitrous oxide (N2O) emissions from microbial nitrification and denitrification. The objective of this study was to assess the effects of manure and cover crop exclusion on N2O emissions in a corn-soybean-winter grain rotation in the USDA certified Reduced Tillage Organic Systems Experiment at the Penn State Agronomy Farm, Rock Springs, PA. The treatments included 1) both cover crop and manure addition (+M+C), 2) cover crop exclusion (+M-C), 3) manure exclusion (-M+C) and 4) both manure and cover crop exclusion (-M-C). In this rotation, corn receives 274 kg of manure N and a hairy vetch- triticale cover crop mixture biomass of 5 Mg/ha contributes 190 kg N to the total plant available N pool, calculated to be 268 kg N/ha. Results showed that peak N2O emission occurred 10 days after manure and cover crop residue incorporation and followed the trend: +M+C = -M+C (0.5 kg N/ha/d) > -M-C (0.4 kg N/ha/d) > +M-C (0.3 kg N/ha/d). We found that exclusion of cover crop incorporation reduced N2O emissions by 60% as compared to control. Shifts in denitrifier nosZ and bacterial amoA nitrifier gene copy numbers at field scale indicated their potential use as a microbiological soil metric that corresponds to N cycling processes responsible for N2O emission. Isotopomer analysis suggested that denitrification was the main source of N2O.

Can rhizosphere microbial communities inform management strategies for apple replant success?

Ari Fina Bintarti1, Julianna Wilson2, Marisol Quintanilla2, and Ashley Shade1,3 1Dept. of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing MI 48824 2Dept. of Entomology, Michigan State University, East Lansing MI 48824 3Dept. of Microbiology and Molecular Genetics, and The Plant Resilience Institute, Michigan State University, East Lansing MI 48824

Soil microbial communities are important for soil health because they can suppress soil pathogens, enhance plant resistance to biotic and abiotic stresses, and support crop growth and productivity. One major challenge faced by apple growers when replanting old orchards with new trees is apple replant disease. The disease is defined by poor performance of young apple trees, and is characterized by symptoms including stunted growth, shortened internodes, discolored roots, and reduction of root biomass; these symptoms collectively lead to yield reduction and economic losses. It is thought that replant disease is caused by a suite of contributing factors, including disturbance of the soil microbial community by long-term replanting and monoculture agroecosystems. As part of a soil health assessment in old orchards before replanting with new trees, we investigated the rhizosphere microbial community structure of apple trees prior to anticipated replant. We collected 45 soil samples taken from 20 geographic “blocks” of apple orchards that represent a gradient of environmental conditions for apple orchards in Michigan. In addition, we collected 30 soil samples from Michigan State University’s Clarksville Research Center as a baseline to compare with a suite of pre-replant management practices. We sequenced the V4-V5 region of the bacterial and archaeal 16S rRNA gene and the fungal ITS1-2 region using high-throughput Illumina sequencing. Combined with analysis of the nematode communities, rhizosphere soil chemistry, and Page 74 of 112

the influences of rootstock and cultivars, we hoped to identify the important biotic and abiotic drivers of microbial communities and later relate these to apple replant outcomes. We found that apple rhizosphere microbial communities were diverse, including ~50 bacterial and archaeal phyla and ~12 fungal phyla. The dominant phyla consist of Proteobacteria, Acidobacteria, Bacteroidetes, Verrucomicrobia, Planctomycetes, and Actinobacteria. The bacterial and fungal community composition differed among blocks (P < 0.001) and rootstocks (P < 0.001). Year of planting, soil pH, lime index, K, Ca, Mg, NO3N, NH4N, sand and silt percentage, the local nematode populations of Bacterial Feeders, and Oligochaetes had significant explanatory value for the bacterial and archaeal communities. The major fungal phyla include Ascomycota, Basidiomycota, and Mortierellomycota. Year of planting, K, NO3N, NH4N, sand and silt percentage, the local nematode populations of Ring, Pin, Stunt, Tylenchs, and Bacterial Feeders had significant explanatory value for the fungal communities. Our results provide insights into the key belowground players in apple orchard soil and we will next determine their value in deciding management actions before planting new trees.

Provision of agroecosystem services by soil-surface microbial consortia

Mary Ann Bruns1,3, Xin Peng1, Ryan Trexler2, and Terrence Bell2,3 1Department of Ecosystem Science and Management 2Department of Plant Pathology and Environmental Microbiology 3Dual Title PhD Program in Biogeochemistry, The Pennsylvania State University, University Park, PA 16802

Cyanobacteria have been recognized as important N2-fixing members in biological soil crusts of semiarid regions, but they have received little attention for their potential contributions to carbon and nitrogen inputs in agricultural soils of more humid regions. Photosynthetic microbial consortia on soil surfaces of agricultural fields have been observed every year for the past 18 years in Central Pennsylvania, USA. These soil surface consortia (SSCs) could provide renewable sources of biologically fixed N and a mechanism to add carbon and make soils more cohesive and erosion-resistant. Here we describe a consortium (DG1) enriched from Penn State Agronomy Farm soils using serial transfers in N-free culture media and robust growth in soil microcosms. Attempts to purify the cyanobacterial strain(s) from other bacteria in the DG1 enrichment were unsuccessful. Based on metagenomics analysis, the DG1 enrichment was a consortium made up of one or more closely related Cylindrospermum spp., which are heterocystous cyanobacteria in the Nostocacae family, and six other bacterial genotypes including Ensifer adherens. The presence of bacterial associates did not interfere with rapid growth and high biomass density in soil microcosms, as well as high SSC stability in water-flush tests. The growth of DG1 in N-free medium compared favorably with pure cultures of commercial strains of heterocystous cyanobacteria as tracked using increases in chlorophyll a and resistance to water-flush tests. Flocculated growth in liquid culture appeared to be associated with greater soil adherence. While commercially available Nostoc spp. formed stable SSCs in soil microcosms, they exhibited lower growth rates Page 75 of 112

and biomass densities than DG1. The SSCs formed by DG1 showed good potential for use as SSC amendment.

Structure and functions of rhizosphere microbiomes on shrub willow cultivars

John E. Carlson1, Wanyan Wang1, Eric S. Fabio2, Lawrence B. Smart2 1Ecosystem Science and Management, The Pennsylvania State University, University Park, PA, USA 2 Department of Horticulture, Cornell University, Geneva, NY USA

Plant roots are colonized by tens of thousands of microorganisms and these microbes and their metabolites have profound effects on host physiology and development. However, the factors which determine the rhizosphere microbial profile are ambiguously understood. This study compared the effects of geographic location and host genotype on rhizopheric microbial communities of shrub willow grown in plantations at 3 sites in Northeast US. In total, 130 rhizosphere soil samples were collected from 12 willow genotypes at the Rock Springs in Pennsylvania, Fredonia in New York and Mylan Park in West Virginia. Total soil DNA compositions were determined spanning a 3-year period from planting time to harvest time. via Metagenomics analyses revealed that geography and host genotype were both found to have significant effects on the structure and function of willow rhizosphere microbiomes. The effect of geography predominated. The bacterial genera Mycobacterium, Methylobacterium, Fankia, Rhodopseudomonas and Nitrobacter, which include well-studied plant growth-promoting microorganisms, showed significant correlations with willow biomass yields in the states of NY and PA, indicating shared microbiome structural features and vital roles in willow growth and yield.

Orchestration of plant holobiont composition and activities

Sarah Stuart Chewning, Katherine Moccia, Bridget O'Banion, Andrew Willems, David Grant, and Sarah L. Lebeis1 1Department of Microbiology, University of Tennessee, 1414 Cumberland Ave., Knoxville, TN 37996

Plant-associated microbial communities promote plant health by: improving nutrient acquisition, producing plant growth hormones, and protecting plants from abiotic and biotic stress. While agricultural biotechnology companies work to translate microbial mechanisms of plant growth promotion into biological products for crop yield improvement, there remain opportunities to understand plant-microbe and microbe- microbe relationships to facilitate increased consistency and widespread success of these biological products. Among key traits of successful biological products are robust colonization and reliable activity despite significant microbial competition and a sophisticated plant immune system. These studies will investigate if microbial traits that venefit plant health are altered in a complex microbial community. Further, we explore Page 76 of 112

potential host-mediated and microbe-mediated mechanisms gating the transition from bulk soil to rhizosphere and finally into the internal root microbiome. Although the mechanisms of plant microbiome assembly are complicated and interweaving, massive parallel sequencing of plant grown in natural soils and synthetic communities composed of a well- defined mixture of bacterial root isolates has uncovered common activities associated with robust colonizers of the root microbiome. Further, these approaches have identified critical chemicals produced by plants and microbes that shape their relationships. Together, the studies described here begin to define the orchestration of multiple selective mechanisms to control root microbiome membership.

Transmembrane Domain in a Syntaxin Protein involved in Legume-Rhizobia Symbiotic Interaction May Determine its Differential Subcellular Localization

Jedaidah Chilufya1, Xiaoyi Wu2 and Dong Wang1, 2 1Plant Biology Graduate Program 2Biochemistry and Molecular Biology Department, Plant Biology, University of Massachusetts Amherst MA 01003

Syntaxins are a conserved family of proteins involved in the fusion of vesicles with target membranes to deliver proteins essential for different physiological functions including growth, development and plant-microbe interactions. In rhizobia and arbuscular mycorrhizal (AM) symbioses, syntaxins contribute to the secretion of proteins needed for the development of the plant cells that interact with the beneficial microbe. This interaction subsequently contributes to the acquisition of growth-promoting nutrients from the soil. In particular, nitrogen fixation symbiosis (NFS) allows legume plants to thrive in nitrogen-limited soils because they form a symbiotic association with nitrogen- fixing bacteria called rhizobia, which convert atmospheric inert nitrogen gas into bio- available ammonia. This NFS is achieved in specialized root organs called nodules where hundreds of rhizobia divide and differentiate into mature nitrogen-fixing forms called bacteroids. The bacteroids are completely and individually enclosed in a plant-derived organelle-like compartment called symbiosome and are surrounded by the symbiosome membrane. Formation of symbiosomes require abundant proteins and lipid material. We are interested in the question of how legume plants influence the successful formation and development of symbiosomes. Previous studies have shown that a symbiosome protein in Medicago truncatula legume species called syntaxin132 (MtSYP132), is part of the nodule-specific secretory pathway, necessary for supplying the symbiosome with plant-host proteins essential for its formation and development. Our lab recently showed that the MtSYP132 gene undergoes alternative cleavage and polyadenylation during transcription, giving rise to two isoforms; MtSYP132A and MtSYP132C (Pan, Oztas et al, 2016). In AM symbiosis, MtSYP132A was required for the penetration of fungi into the plant roots and the development of the peri-arbuscular membrane. In rhizobia symbiosis, knock-down of the symbiosome-localized MtSYP132A resulted in nodules that did not fix nitrogen and differentiation of symbiosomes inside these nodules was blocked. Additionally, these isoforms exhibited differential subcellular localizations; MtSYP132C localizes on the plant plasma membrane, while MtSYP132A localizes on Page 77 of 112

the symbiosome membrane. Our goal is to understand how these two SYP132 protein isoforms localize to distinct membrane compartments: the plasma membrane and the symbiosome membrane. Sequence alignment showed that the two SYP132 isoforms are conserved in many species forming symbioses with rhizobia or AM fungi but there are striking differences in some amino acids in the transmembrane (TM) domain between SYP132A and SYP132C. We hypothesize that these amino acids are important for the difference in subcellular localization patterns. To test this hypothesis, plant transformation with constructs containing GFP-tagged-SYP132A/C ‘swaps’ in the TM domain is underway, and the localization patterns of these mutants will be examined using confocal microscopy. We expect that domain swapping between the TM domains will result in novel localization patterns for the corresponding proteins. Because SYP132 protein is a component of peri-microbial compartments in both NFS and AM symbioses, detailed knowledge of this protein can be potentially used to genetically manipulate crop plants in order to enhance their nitrogen and phosphorus acquisition, respectively, for crop productivity.

Assessing effects of tillage on soil microbial N dynamics and crop N uptake in a 40- year experiment

Mara Cloutier1,2, Sjoerd Duiker3, Mary Ann Bruns1,2

1Department of Ecosystem Science and Management 2Biogeochemistry Dual Title PhD Program 3Department of Plant Science, The Pennsylvania State University, University Park PA 16802

Natural ecosystems are hypothesized to have more effective plant-microbe interactions resulting in increased nutrient cycling efficiency. Disturbances to ecosystems such as tillage after land conversion to agriculture result in changes to carbon and nitrogen cycling in soil. The overall goal of this project is to assess how functional bacterial biomarkers are affected by the type of tillage disturbance maintained for the past 40 years (moldboard plow, chisel disking, or no-till). The biomarkers correspond to bacterial genes responsible for two N cycling processes which could increase N retention and lower nitrous oxide (N2O) emissions, namely nitrate reduction to ammonium (NA) and N2Oreduction to di-nitrogen (N2) gas. To address this, bulk soil and rhizosphere samples will be taken from main crops (corn and soybean) and cover crops (wheat and rye). Measurements of N2O emissions will be made through the growing seasons in 2018 and 2019. Crop N uptake will be estimated from biomass N of main crops and cover crops. . Both quantitative PCR and Illumina Miseq sequencing will be performed on genes encoding enzymes forNA and N2O consumption, namely nrfA, nosZI, and nosZII. It is hypothesized that tillage will have a greater impact on rhizosphere communities than crop type. Additionally, loss of reactive N is hypothesized to be lower in no-till plots compared to reduced/conventional tillage. Results from this research will provide much needed information regarding the effects of long-term treatments on soil N cycling. Page 78 of 112

Arbuscular mycorrhizal associations impact plant resistance to a belowground feeding herbivore

Elizabeth Davidson-Lowe1, Swayamjit Ray1 and Jared Ali1 1Department of Entomology, Penn State University, University Park PA 16802

Plants interact with multiple organisms throughout their lifetime and respond dynamically to changes in their environment and within their community. Arbuscular mycorrhizal fungi (AMF) form mutualistic associations with the roots of most land plants. Plants provide AMF with carbohydrates in exchange for mineral nutrients, such as phosphorous. However, AMF colonization can also facilitate changes in phytochemistry and defenses. The majority of studies that focus on AMF-related changes in plant defenses focus on responses to aboveground damage. In this study, we investigated how AMF colonization in Zea mays affects protease inhibitor activity to belowground damage by the root-feeding larvae of western corn rootworm (Diabrotica virgifera virgifera). Larvae were added to plants with and without AMF colonization. Above and belowground plant tissue was collected for plant defense analyses and larval survival was used as a measure of herbivore performance. This study demonstrates that mycorrhizal associations can impact herbivore performance by altering plant defenses, which has significant implications in agricultural practices for improved pest management.

Wheat Variety-Specific Recruitment of Bacterial Communities Towards Soil-borne Disease Suppression

Christine Jade Dilla-Ermita1, Ricky Lewis2, Tarah Sullivan2, and Scot Hulbert1,2 1Department of Plant Pathology, Washington State University, Pullman, WA 99164 2Department of Crop and Soil Sciences, Washington State University, Pullman, WA 99164

Soil microbial communities are involved in diverse ecological services in agriculture such as improved soil quality and nutrient uptake, abiotic stress tolerance and soil-borne disease suppression. With this in consideration, attention have been drawn to the recruitment and manipulation of these microbiomes as advancements in DNA sequencing technologies have enabled the efficient examination of soil microbiomes. The identification of disease-suppressive soil microbiomes has become the “holy grail” of soil-borne disease management, especially in direct-seeded wheat. The root rot disease in wheat caused by Rhizoctonia solani AG8 has remained challenging to control in direct- seeded wheat due to lack of sources of resistance in the wheat germplasm. Despite discovery of fluorescent pseudomonads as biological control agent of soil-borne diseases, inherent microbial community dynamics and edaphic factors that affect survival of introduced bacteria constrained the success of disease control. Planting of specific wheat varieties that strongly recruit disease “suppressive” microbiomes is critical in the manipulation of useful microbiomes. Our group have previously found differences in rhizosphere bacterial communities among several wheat varieties after being grown in the field for two successive seasons. In my research, I am testing growth chamber assays to Page 79 of 112

develop more rapid assays for characterizing microbiomes of wheat varieties planted in different soil types and evaluate disease suppression. To identify soil microbiomes that are wheat variety-specific, six winter wheat varieties (Madsen, Lewjain, Eltan, Hill81, PI561725, and PI561727) were planted in pots for three 35-day cycles in the growth chamber to mimic three seasons of wheat cropping. Pullman (high rainfall) and Lind (low rainfall) soils were used, and watering was regulated to mimic the amount of water available to plants in these two rainfall zones. Rhizosphere soil was collected after the third cycle and the DNA was sequenced to identify bacterial taxa composing communities in each variety. To determine potential “suppressiveness”, remaining soils planted with the six wheat varieties were inoculated with Rhizoctonia solani AG8-C1 and Alpowa seeds were sown in all of the soils. Shoot length and root disease were assessed after 14 days. The 35-day cycles in both Pullman and Lind generated significantly different microbiomes among wheat genotypes in the two soil types. Based on Wald test, 85 and 53 bacterial taxa in Pullman and Lind soils, respectively, were differentially abundant across the six wheat varieties. There are some differences found in the root disease scores and shoot length of Alpowa planted in soils cultivated with the six wheat varieties but were not statistically different. Further experiments are needed to determine optimum cycle lengths to recruit significant disease “suppressive” microbiomes in wheat.

The effect of the host environment on bacteriocin-mediated competition between bacteria

Hanareia Ehau-Taumaunu1, Kevin Hockett1,2 1Department of Plant Pathology and Environmental Microbiology, Penn State University, University Park PA 16802 2The Huck Institutes of the Life Sciences, Penn State University, University Park PA 16802

Bacteriocins are narrow-spectrum toxins encoded by bacteria that antagonize closely related competitors. Bacteriocin-mediated interactions have been extensively investigated both in animal and laboratory systems to discover the potential benefits and costs of bacteriocin production. Conversely, there is a discernable absence of research of bacteriocin-mediated dynamics within a plant host context. In this study, we compared the antagonistic interactions in planta and in vitro between two strains of Pseudomonas syringae, a globally distributed and economically important plant pathogen. Our model systems consisted of a bacteriocin producer strain P. syringae pv. syringae (Psy), which targets the sensitive strain P. syringae pv. phaseolicola (Pph). A series of co-infiltrations (leaf apoplast inoculation) experiments were performed in Phaseolus vulgaris (common bean) and in culture over a six-day period with either Psy or a bacteriocin-deficient mutant of Psy (Psy Rrbp) against Pph. The populations of each strain were measured at day 0, 4, and 6 for all treatments. Additionally, the presence or absence of bacteriocins within the plant host was evaluated. In 1:1 competition, both Psy and Psy Rrbp strains reduced the population growth of Pph in the host plant. At day 4, the growth of Pph was reduced by 100-fold and 10-fold when co-inoculated with Psy or Psy Rrbp, respectively, compared to individual infiltration. Page 80 of 112

By day 6, the Pph population was reduced by 100-fold by co-infiltration with either Psy and Psy Rrbp. The Psy and Psy Rrbp populations were unaffected by co-infiltrations, compared to individual infiltration. In culture, the Pph population was reduced 100-fold by co-inoculation with either Psy or Psy Rrbp by day 4, compared to individual inoculations. Bacteriocin production by Psy appears to reduce Pph to a larger extent in planta than in culture. This may be related to differential bacteriocin production by Psy or differential sensitivity by Pph in the plant environment. Although our data demonstrates that the Psy bacteriocin negatively affects the Pph population in planta, we were unable to detect a fitness benefit conferred by Psy by bacteriocin production. Future experiments will assess whether bacteriocin production confers a detectable benefit to Psy in epiphytic co-inoculations, or when Psy is rare relative to Pph (i.e. does bacteriocin production benefit invasion of the host, rather than suppression of a competitor).

Host-linked soil viral ecology along a permafrost thaw gradient

Joanne B. Emerson1*, Simon Roux2, Jennifer R. Brum3, Benjamin Bolduc4, Ben J. Woodcroft5, Ho Bin Jang4, Caitlin M. Singleton5, Lindsey M. Solden4, Adrian E. Naas6, Joel A. Boyd5, Suzanne B. Hodgkins7, Rachel M. Wilson7, Gareth Trubl4, Changsheng Li8§, Steve Frolking8, Phillip B. Pope6, Kelly C. Wrighton4, Patrick M. Crill9, Jeffrey P. Chanton7, Scott R. Saleska10, Gene W. Tyson5, Virginia I. Rich4, Matthew B. Sullivan4,11

1Department of Plant Pathology, University of California, Davis, One Shields Ave., Davis, CA 95616 (most work conducted at The Ohio State University4) 2United States Department of Energy Joint Genome Institute, Lawrence Berkeley National Laboratory, 2800 Mitchell Dr., Walnut Creek, CA 95418 (most work conducted at The Ohio State University4) 3Louisiana State University, Baton Rouge, LA 70803 (most work conducted at The Ohio State University4) 4Department of Microbiology, The Ohio State University, Columbus, OH 43210 5Australian Centre for Ecogenomics, University of Queensland, Brisbane 4072, Australia 6Faculty of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences, Ås, Norway 7Department of Earth, Ocean, and Atmospheric Science, Florida State University, Tallahassee, FL 32306 8Earth Systems Research Center, Institute for Study of Earth, Oceans, and Space, University of New Hampshire, 8 College Rd., Durham, NH 03824 9Department of Geological Sciences, Stockholm University, Stockholm 106 91, Sweden 10Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, AZ 85721 11Department of Civil, Environmental and Geodetic Engineering, The Ohio State University, Columbus, OH 43320 §Deceased

The role of viruses, known to affect microbial dynamics, metabolism, and biogeochemistry in the oceans, remains largely unexplored in soil. Here, we investigated Page 81 of 112

how soil viruses influence microbial ecology and carbon cycling in peatlands along a permafrost thaw gradient marked by differences in plant community composition in arctic Sweden. We recovered 1,907 viral populations (genomes and large genome fragments) from 197 bulk soil and size-fractionated metagenomes, approximately doubling known prokaryotic viral genera. In silico predictions linked 35% of these viral populations to microbial host populations, including methanogens and methanotrophs. Lineage-specific virus:host abundance ratios varied with thaw, suggesting that viral infection dynamics may impact microbial responses and contributions to a changing climate. The recovery of virus-encoded glycoside hydrolases suggests that viruses may have direct impacts on complex carbon degradation. Overall, results suggest that soil viruses have multiple contributions to carbon cycling, and the recovery of hundreds of previously unknown soil viruses in a single ecosystem highlights the potential for incredible soil viral biodiversity and ecological impacts yet to be discovered. Ongoing efforts to study viral communities in agricultural systems using similar approaches may also be presented.

Fungal-bacterial interactions in the hyphosphere: Profiling microbial communities associated with arbuscular mycorrhiza fungi Bryan D Emmett1, Veronique Levesque-Tremblay1, Maria J Harrison1 1Boyce Thompson Institute, Ithaca, NY 14853

Arbuscular mycorrhizal fungi (AMF) are ancient plant symbionts, which evolved a symbiotic relationship with plant hosts over 400 million years ago and associate with approximately 80% of all land plants. This symbiosis does not take place in isolation but in the context of close association with soil microbial communities. Extraradical hyphae (ERH) extend the influence of the root and plant derived carbon into the soil environment, modulating soil microbial communities and nutrient cycling in the hyphosphere. The potential importance of ERH associated microbial communities is high owing to their role in mediating nutrient availability to the fungus and plant. Previous work has highlighted compositional shifts in the soil microbial community in the presence of ERH and attachment of particular bacterial strains to ERH in vitro. However, the composition, function and interactions that give rise to the microbial community closely associated with ERH in soil remains largely unexplored. To address this knowledge gap an experiment was undertaken to detail the ERH- associated microbial community in three agricultural soils. Mesocosms were planted with Brachypodium distachyon and inoculated with Glomus versiforme. After establishment, a 50 m mesh core (1.5 cm x 13.5 cm) containing a sand and soil mixture (1:1 v/v) was inserted to allow hyphal colonization of the substrate while excluding plant roots. Hyphae were harvested by removing the core, submerging the soil/sand matrix and collecting hyphae under a dissecting scope for downstream DNA extraction. Tagged 16S rRNA gene amplicon libraries were prepared at the University of Minnesota Genomics Center (Minneapolis, MN) and sequenced on the Illumina MiSeq system. Preliminary data reveal an ERH associated community distinct from both rhizosphere and bulk soil and that this community forms in a soil-dependent manner. Hyphal communities were dominated by representatives of the Gamma-, Delta- and Page 82 of 112

Betaproteobacteria, as well as the Cytophagia (Bacteroidetes). Most OTUs were enriched in the hyphal samples from one or two of the field soils, but several were enriched across the three field soils assayed. These results suggest the experimental system and sampling approach can isolate and characterize hyphal associated communities. Future work will focus on the ecology of the ERH associated communities and their modulation of the plant-fungus symbiosis. Knowledge of the composition and function of AMF associated microbial communities may ultimately contribute to the management of the symbiosis to support plant productivity.

Microbial Mediation of Plant-Herbivore Interactions

Gary W. Felton1, Ching-Wen Tan1, and Michelle Peiffer1 1Department of Entomology, Penn State University, University Park, PA 16823

Plants are subject to attack by an onslaught of microbes and herbivores, yet are able to specifically perceive the threat and mount appropriate defenses. Plants have evolved two primary defense pathways: one regulated by jasmonic acid (JA), which defends against herbivorous insects, the other by salicylic acid (SA), which responds to microbial pathogens and is frequently antagonistic with JA. Chewing herbivores cause massive damage when crushing plant tissues with their mandibles, thus releasing an array of specific cues that may be perceived by the plant, which mobilizes plant defenses.

While specific cues in the oral secretions of herbivores such as caterpillars and beetles trigger plant defenses, we have found that bacteria associated with these secretions can trigger the SA pathway, which benefits the herbivore by suppressing JA regulated defenses. These results reveal a new strategy for how herbivores evade plant defenses by using symbiotic bacteria that deceive the plant into perceiving a herbivore threat as microbial, thus resulting in suppression of plant defenses against herbivores.

In another recent study, we have found that insect parasitoids that parasitize caterpillars may indirectly have a strong impact on plant defenses. Along with injecting an egg inside the caterpillar, the parasitoid injects symbiotic polydnaviruses, which disable the caterpillar’s immune system. As part of this immunosuppression, one component in the caterpillar’s saliva known to trigger plant defenses is dtrongly suppressed. These striking findings indicate that a symbiotic virus found in parasitoids not only causes a massive suppression of the caterpillar’s immune system, but also compromises the plant’s immunity or defense against herbivores.

These findings indicate that microbes are the “hidden” players in mediating plant- herbivore interactions. Our evidence from several plant-herbivore systems indicates that insect-associated microbes can have a profound effect on the ability of a plant to perceive herbivores and thus trigger plant defenses. Page 83 of 112

A web of interactions shapes microbial communities and processes in an annual grass rhizosphere

Mary Firestone*1,2, Javier A. Ceja-Navarro2, Evan Starr1, Henrik Krehenwinkle1, Shengjing Shi3, Angela Hodge4, Rosemary Gillespie1, Eoin Brodie1,2, Jizhong Zhou2,5, Jillian Banfield1,2, Jennifer Pett-Ridge6 1University of California, Berkeley 2Lawrence Berkeley National Laboratory 3AgResearch Ltd, Christchurch, New Zealand 4University of York, York UK 5University of Oklahoma, Norman 6Lawrence Livermore National Laboratory

Rhizosphere organisms use root input of exudates and debris to develop a complex web of cross-kingdom interactions that shape carbon transformation and nutrient supply to plants. Three classes of interactions will be discussed: arbuscular mycorrhizal fungi, bacteria, and roots; phage and their microbial hosts; and rhizosphere fauna (proto- and metazoan) and their prey.

13 CO2-grown plants introduce carbon into soil to delineate the complex web of biotic interactions. Isotope-enabled genomics and genome-resolved metagenomics reveal phage, bacterial, fungal, and faunal participants; food-web and network modeling identify and quantify guilds contributing to soil C persistence. Avena fatua, (common in

13 Mediterranean grasslands) was grown under CO2 and rhizosphere and bulk soil collected over time. Extracted DNA underwent density-gradient centrifugation, yielding discreet ranges of 13C label; DNA was then sequenced, data were assembled, binned for host genomes, and phage contigs.

Populations of phages that incorporated label into their genomes differed from non- labelled phages, indicating that the development of rhizosphere-competent bacterial consortia enabled production of different phage populations. Several phages were linked with hosts; phage-host abundances differed between samples. Rhizosphere phages infected root-carbon-dependent bacteria.

The physical separation of nematodes and protists from soil involved gradient centrifugation and size selection by differential filtration. Subsamples of isolated nematodes and protists underwent DNA extraction, metabarcoding, fixing, and imaging. Sequenced libraries yielded 50-70% faunal sequences. Nematode and protist densities were thus assessed in bulk and rhizosphere. Predicted functional roles of these guilds suggest direct root feeding and bacterial and fungal predation. These functional predictions are being evaluated using isotopic methods. Page 84 of 112

Understanding the photophysiology and vulnerability of scleractinian corals

Claudia Tatiana Galindo-Martínez1, Tomás López-Londoño, Darren J. Brown, Kelly J. Gómez- Campo, Luis Alejandro Gonzalez-Guerrero, Roberto Iglesias-Prieto. 1Department of Biology, Penn State University, University Park PA 16802

Corals in symbiosis with photosynthetic microalgae have dominated the shallow oligotrophic waters of tropical and subtropical oceans for the last 200 million years. In these tropical areas, the coral reefs are built when the rates of calcium carbonate production, mainly by corals, are larger than the physical, chemical and biological erosion rates. The success of the corals as reef-builders is the result of the metabolic advantages derived from the symbiosis. Algal photosynthesis is responsible for the high rates of coral calcification (light enhanced calcification); therefore light is one of the principal factors within the reef system and it has a fundamental role in the understanding of different process that affect it. We will discuss how the high irradiance absorption efficiency of corals improves coral performance and explains their success in oligotrophic environments, although, this efficiency exposes them to an extreme fragility under climate change. We will discuss how the algal capacity to repair the light-induced damage of the photosynthetic machinery plays an important role during coral bleaching. Finally, based on the understanding of the metabolic balance in symbiotic corals, we propose different bio-optical models that: 1) explain the differential vertical distribution of coral species along depth mediated light gradients, 2) predict coral responses to future ocean acidification scenarios, and 3) increase our knowledge about the mechanisms of calcification in symbiotic corals.

Perspectives on Social Innovation Among Agricultural Scientists

Leland L. Glenna1 and Maria Vivanco1 1Agricultural Economics, Sociology, and Education Department, The Pennsylvania State University, University Park, PA, USA

The social innovation concept emerged after analysists came to realize that market fundamentalism failed to deliver widespread social benefits from science and technology research. Rejecting the idea that society is best coordinated through self-regulating markets, social innovation proponents claim that direct policy interventions, institutional change, and other collective actions are necessary to achieve socially equitable outcomes. We use a survey of 1126 agricultural scientists at 71 United States land-grant universities (LGUs) to analyze how scientists perceive their research activities contribute to social innovation. We measured scientist norms, beliefs, and values regarding how they envision science and technology contributing to social outcomes and how their perspectives relate to their research activities and outputs. Principle components analysis divided scientists into two factors: private-science norms (market fundamentalism) and public-science norms. Since just over one-third of respondents agreed or strongly agreed with the statement that scientists should focus on research with market potential and about two-thirds agreed or strongly agreed that scientists should focus on research with Page 85 of 112

public benefits, it is reasonable to conclude that approximately one-third of scientists adhere to private-science norms and two-thirds adhere to public-science norms. Additional statistical analysis found that those conforming to the private-science factor were significantly more likely than those conforming to the public-science factor to be correlated with the belief that markets are best able to evaluate the social value of science and technology and that markets are best at solving social problems like hunger. The private-science factor was also significantly correlated with less basic science research, with industry funding and industry collaborations, and with more excludable research outputs. The findings have implications for national, state, and university policies directed at promoting social innovations from university-based agricultural research. Traditionally, university scientists have been expected to conduct basic research, to generate public goods, and to be dedicated to solving social problems that are not easily addressed with a commercial product. If one-third of public agricultural scientists are committed to private-science norms and practices, the capacity for LGUs to contribute the public goods needed to solve social problems will likely be compromised.

Managing the plant phytomicrobiome for increased disease resistance

Andie Gonzales and David C. Haak

Fusarium head blight (FHB), or scab, is a devastating wheat disease. USWBSI-supported efforts to combat FHB have focused primarily on breeding resistant cultivars and degrading mycotoxin. However, one untapped resource for combatting FHB lies in microbiome-mediated resistance. Increasingly, we recognize that host genotype- microbiome interactions mediate phenotypic responses. For example, the chickpea microbiome can reduce growth of pathogenic Fusarium graminearum, but the outcome varies with host genotype. Therefore, to successfully manipulate this interaction between host genotype, microbiome, and pathogens to fight disease, will require a systems-level approach. This is particularly true for managing FHB, as the plant health impacts and agronomic properties are strongly integrated, genetic resistance requires multiple resistance loci, and microbes can reduce FHB impacts on grain quality. Thus, it should be possible to dissect the phytomicrobiome-geneotype-phenotype interaction in this system. We are going to characterize differences in phytomicrobiome structure and plant performance among 3 wheat lines differing in susceptibility to FHB with replication in 3 locations and 5 developmental stages.

The beech leaf microbiome: Examining effects of beech leaf disease

Adam J. Hoke1 and David J. Burke1,2 1The Holden Arboretum, Kirtland, OH 44094 2Department of Biology, Case Western Reserve University, Cleveland, OH 44106

We are currently using molecular methods to examine fungal, bacterial, and nematode community structure on leaves and dormant buds of American beech (Fagus grandifolia) Page 86 of 112

affected with “beech leaf disease” (BLD). Although the condition was first noticed in the Lake County Metroparks in 2012, the condition was not observed at the Holden Arboretum until 2014. The condition involves characteristic interveinal thickening and darkening of tissue followed by blackening of tissue and leaf curling. The cause of BLD is unknown but several microbial agents, including fungi, have been suggested. We have found that bacterial and fungal communities differ significantly between leaf and bud tissue, but BLD symptomology does not alter bacterial community composition. However, fungal community composition on buds showed significant effect of treatment (P<0.001) between symptomatic and asymptomatic tissue using non-metric multidimensional (NMS) scaling ordination techniques. We also detected a significant effect of treatment driven by differences in tissue type: Leaf vs. buds (P<0.001). Subsequent sequence libraries found and increase in some fungal groups between symptomatic and asymptomatic tissue (e.g. sequences with affinity to the genus Taphrina increase from 24% to 57% of clone libraries). Although Taphrina species can cause leaf disease in other plants (e.g. peaches), it is uncertain whether this is the cause of BLD or simply an organism that grows opportunistically on leaves damaged by another microbe or insect. Additionally, in agreement with other research groups, we have detected nematodes on both leaves and buds of beech using molecular methods (PCR of LSU rDNA). Species identity is unknown at this point, but we will continue to investigate these data further.

Ralstonia solanacearum bacteriocin diversity and specificity within the species complex

Alejandra I. Huerta1, Florent Ailloud2 and Caitilyn Allen1

1Department of Plant Pathology, University of Wisconsin-Madison, Madison 53706 2CIRAD, UMR Peuplements Végétaux et Bioagresseurs en Milieu Tropical, CIRAD- Université de la Réunion, Pôle de Protection des Plantes, Saint Pierre, La Réunion, France

Inter-species competition has been observed across all organismal levels, including the genetically and ecologically diverse group of strains that comprise the Ralstonia solanacearum species complex. Many of these strains cause bacterial wilt diseases of plants and are globally distributed. However, naturally infected plants have not been reported to contain more than one strain. K60, a warm-temperate R. solanacearum strain from North America, was found to outcompete a R3bv2 strain in the stems and rhizospheres of tomato plants. This competitive fitness correlated with production of secreted strain-specific growth inhibitors. We hypothesized that bacteriocins were responsible for the inhibition. We screened a K60 mutant library for loss of inhibitory ability and identified two genes encoding Rhs Polymorphic Toxins (Rhs PT). K60 Rhs PT mutants lost their competitive advantage over R3bv2 strain UW551 in tomato plants and in culture. Gene complementation restored inhibition in culture and competitive fitness in plants. Furthermore, a high diversity of Rhs PT was found across 27 strains in the R. solanacearum species complex. Pairwise inhibition assays showed complex inhibition and susceptibility profiles (Figure 1). This knowledge may be useful in Page 87 of 112

developing fine targeted management tactics for bacterial wilt diseases.

Host plant and population source drive diversity of microbial gut communities in two polyphagous insects

Asher G. Jones1, Charles J. Mason1, Gary W. Felton1, Kelli Hoover1 1Department of Entomology, Penn State University, 501 ASI Building, University Park 16802

Symbiotic associations between insects and microbes are ubiquitous, but vary greatly in their degree of association, mode of transmission, and location in which they are harbored in the insect. The Lepidoptera represent one of the largest, most economically important insect orders; yet, in many cases the ecology and functions of their gut microbiomes are largely unresolved. We used high-throughput Illumina sequencing of 16S-rRNA to characterize and compare bacterial midgut communities of wild-collected fall armyworm (Spodoptera frugiperda) and corn earworm (Helicoverpa zea) from two locations in Pennsylvania. Although we found variation in the composition and structure of bacterial communities between conspecific individuals, midgut microbial communities from fall armyworm were distinct from those of corn earworm collected from the same host plant at the same site. This suggests that gut physiology of different insect species may play a role in shaping bacterial communities. We also found that corn earworm

bacterial communities from locations in Pennsylvania were distinct, indicating that variables associated with location can also affect composition of midgut microbiota. To further investigate factors shaping bacterial communities, fall armyworm larvae were collected from different egg population sources (field or laboratory) and were fed corn or soybean foliage. We then analyzed the microbiota of the regurgitant (foregut) and the midgut. Host plant had a much greater impact on fall armyworm bacterial community composition and structure than egg population source, for both regurgitant and midgut communities. When we compared regurgitant and midgut bacterial communities they were significantly different, but this was driven by a single Enterococcus OTU. The gut microbiome was primarily composed of members of the Enterobacteriaceae, Pseudomonadaceae and Enterococcaceae, which were also detected via Sanger sequencing of cultures isolated from the regurgitant of wild fall armyworm larvae collected from Pennsylvania and Puerto Rico. Our results demonstrate that ecological factors can affect microbial interactions at different levels of scale. Host plant appears to be a major driver shaping gut microbiota in the species we studied, although differences in insect physiology and gut region likely also contribute to variation in gut microbiota. Additional studies are needed to understand the mechanisms by which these factors affect insect microbiomes and the ecological implications of variation in bacterial gut communities. Page 88 of 112

Medicago sativa has reduced biomass and nodulation when grown with soil microbiomes adapted to high phosphorous inputs

Laura M. Kaminsky1,2, Grant L. Thompson1, Ryan V. Trexler2, Terrence H. Bell1,2, Jenny Kao-Kniffin1 1School of Integrative Plant Science, Cornell University, Ithaca, NY, USA 2Department of Plant Pathology and Environmental Microbiology, The Pennsylvania State University, University Park, PA, USA

Over-fertilization is common in agricultural systems and may impact crop growth-promoting plant-microbe relationships. It is unclear if soil microbial assemblages adapt quickly to changes in fertilizer inputs once conditioned to specific nutrient regimes. We conducted a growth chamber study assessing the response of root-associated microbiomes of Medicago sativa to nutrient regime changes, and consequences for plant growth. Plants were grown with a common starting soil microbiome under four nutrient input conditions: control (no fertilizer), organic fertilizer (compost), low inorganic P (low triple superphosphate, TSP) and high inorganic P (high TSP). After four planting generations, where root-associated microbiomes were transferred forward based on highest plant biomass, microbiome composition and hydrolytic enzyme activity were distinct across nutrient conditions. The resulting microbiomes were then transplanted into soils receiving one of each nutrient treatment, leading, generally, to taxonomic convergence with others transplanted into the same nutrient regime. However, high inorganic P-conditioned microbiomes were more resistant to compositional change. Correspondingly, M. sativa grown with high inorganic P-conditioned microbiomes had lower biomass, fewer nodules, and lower %N than plants grown under the same nutrient regime with other microbiomes. These findings suggest prolonged impacts of excessive fertilization on soil microbiomes, potentially altering plant growth-promoting qualities.

Pseudomonas syringae target cells exhibit multiple survival responses upon exposure to lethal doses of a phage-tail like bacteriocin

Prem P. Kandel1, Kevin L. Hockett1 1Department of Plant Pathology and Environmental Microbiology, Penn State University, University Park, PA, 16802

Pseudomonas syringae is an opportunistic plant-pathogenic bacterium causing serious diseases in crops of economic importance. Pathovars of P. syringae can produce a variety of antimicrobials, including bacteriocins, a group of antibacterial peptides or proteins synthesized and released from bacterial cells to suppress competitors that, in most cases, belong to the same species or genus. Bacteriocins have been used as food preservatives in the past and are proposed as antibiotic alternatives by numerous studies. Although promising, the likelihood of breakdown in bacteriocin-mediated pathogen control is currently unknown. Such breakdown could occur through a number of mechanisms, including evolution of resistance, or the development of non-heritable physiological tolerance (such as a ‘persister cell’ phenotype). To better understand the dynamics Page 89 of 112

associated with bacteriocin resistance and tolerance, we used a model system, where a bacteriocin produced by P. syringae pv. syringae B728a (Psy) is lethal to cells of P. syringae pv. phaseolicola 1448a (Pph). Stationary and logarithmically growing Pph cells were treated with a saturating concentration of partially purified bacteriocin for 1-24 hours under an in vitro broth condition, and surviving cells were enumerated after removing the bacteriocin. Upon 1 hour of bacteriocin treatment, a significantly higher survival was observed in stationary cells [(23.8 ± 5.94)%, mean ± SEM, n=21] than in the log cells (0.068 ± 0.032)%. The majority (85-100%) of the surviving cells from both cultures were sensitive upon bacteriocin re-treatment, suggesting that bacteriocin tolerance, rather than resistance, was the underlying cause of survival. No additional reduction in the surviving population beyond that which occurred in the first hour was observed upon treatment for up to 8 hours. However, when subjected to constant bacteriocin exposure, the bacterial population increased by >10-fold by 24 hours of incubation. Upon tailocin re-treatment, the surviving cells (from 24-hour treatment) showed three phenotypes; tolerance (a non-heritable fully sensitive phenotype), incomplete resistance (a heritable phenotype in which cells show increased frequency of survival compared to the wild type strain), and resistance (a heritable bacteriocin insensitive phenotype). Although the tolerant phenotype dominated the stationary survivors (96.9%), incomplete resistance was the dominant phenotype among the 24-hour log phase survivors (81.2%). The resistant phenotype was rarely detected among stationary survivors, but was detected in 10, 65, 38, and 0% of the log survivors after 1, 4, 8, and 24 hours of bacteriocin exposure, respectively. The underlying mechanism of incomplete and full resistance is being investigated by genome sequencing and preliminary results suggest mutation of various genes involved in lipopolysaccharide synthesis. Future research will seek to understand the dynamics of tolerance and resistance during bacteriocin exposure within a plant host environment. A better understanding of these survival mechanisms will have implications in designing bacteriocins as pathogen control agents.

Establishing the core microbial community of Vaccinium species as a prerequisite for studying replant disease

1 2 1 Joseph Kawash , Peter V. Oudemans , and James J. Polashock 1USDA-ARS, 125A Lake Oswego Rd., Chatsworth, NJ 08019 2PE Marucci Center, Rutgers University, 125a Lake Oswego Rd., Chatsworth, NJ 08019

Highbush blueberry (Vaccinium corymbosum) and cranberry (V. macrocarpon) are long- lived perennial crops. However, yields have begun to decline in older fields in New Jersey and replanting does not solve the problem, suggesting that degenerating soil health is associated with decline. The community of soil microbes affects soil health and specific organisms in the community may directly affect plant health. Establishing the core rhizosphere microbial community is required before those that specifically affect plant health can be determined. Soil samples (30 from blueberry; 30 from cranberry) Page 90 of 112

were collected across both crops throughout the growing regions in New Jersey. The bacterial and eukaryotic communities were determined by sequencing a portion of the 16S for bacteria and the ITS for eukaryotes. Fragments were sequenced on the MiSeq (Illumina) platform and taxonomically classified using BLASTn. Regarding fungi, the blueberry and cranberry soils were dominated by ascomycetes and basidiomycetes. Five hundred thirty-eight genera were identified, but most were below 1%. Symbionts, known pathogens and saprobes contributed to the community. Some differences between the blueberry vs. cranberry fungal communities were identified. The bacterial communities were nearly identical between the crops. They were dominated by Protobacteria and six other phyla. These data lay the foundation for determining what organisms are positively or negatively associated with crop health.

Characterizing the Symbiodinium microbiome Allison H. Kerwin1, F. Joseph Pollock1, Alaina Weinheimer1, Todd LaJeunesse1, Mónica Medina1 1Pennsylvania State University, 208 Mueller Lab, University Park, PA 16802

Changing ocean conditions and increasing thermal stress events affect a wide variety of marine organisms, including reef-building corals which exist due to a delicate and obligate symbiosis with photosynthetic Symbiodinium. In an effort to more fully understand this symbiosis efforts to characterize the coral microbiome have revealed a complex and diverse bacterial and archaeal community. Recent work has demonstrated that Symbiodinium spp. have their own closely-associated bacterial community, including core members found across all cultured clades. To examine this association in more depth we conducted next generation sequencing of the V4 region of 16S rRNA on Symbiodinium spp. from six different clades. We found that the bacterial community composition clustered by clade, and that Clade B had the highest level of alpha diversity, both in richness/evenness and in phylogenetic diversity. However, the strongest cluster pattern was based upon the collection of origin, with Symbiodinium originally isolated by Trench clustering apart from those isolated by Coffroth, regardless of being removed from those original culture conditions for a number of years. By focusing on Symbiodinium Clade B, most of which originate from the Coffroth collection, we were able to find a signal of original host species for Symbiodinium psygmophilum (B2). In the future we hope to explore the Clade B radiation of Symbiodinium in more depth, and to use microscopy to localize various members of the microbiome. Our understanding of the various microbial partners found in cnidarian symbioses will only increase in importance as corals experience more frequent and widespread mortality events. Page 91 of 112

Wheat and Brassica plants influence the structure and function of their rhizosphere communities but do not impact those of neighboring crops

Ann M. Klein1, Caroline Melle1, Catherine L. Reardon1 1USDA-ARS, Soil & Water Conservation Research Unit, 48037 Tubbs Ranch Rd. Adams, OR 97810

Wheat-fallow rotation is the primary cropping system of the dryland (non-irrigated) region of the Pacific Northwest. Diversification of the cropping system with oilseed Brassicas has the potential for economic and environmental benefits through reduced fallow periods, less wind erosion, and improved air quality. Canola and mustards, both oilseed Brassicas (oilseeds), are purported to provide rotational benefits in-field. When applied as plant residues or seed meals, Brassicas “biofumigate” the soil through the production of isothiocyanates, which reduce the abundance of plant pathogens and alter microbial communities to produce disease-suppressive soils. Significant value can be added to crop rotations if beneficial communities are promoted in-field by growing oilseed crops and if benefits are transferrable to the primary wheat crop. In this study, we evaluated the structure and function of rhizosphere communities of wheat, canola and mustard and whether impacts of oilseeds on wheat communities vary over the spatial distance between the two types of plants. We found that microbial community structure and function varied across plant type. In general, the alpha diversity of bacterial and fungal communities was greater in wheat rhizosphere soil than either mustard or canola. Similarly, extracellular enzyme activity related to carbon-, nitrogen-, and phosphorous- cycling was also greater in wheat versus oilseeds. When the plants were co-cropped, there was no apparent effect of spatial proximity to oilseeds on wheat rhizosphere community composition. Although oilseeds did not affect microbial communities in the rhizosphere of wheat plants grown in adjacent rows, wheat planted directly row on row to a previous oilseed crop may show observable differences in community composition and function as will be evaluated in a future study.

Maximizing multifunctionality of soil microbial communities via broad and fine- scale microbial diversity

Richard Lankau1, Diane Xue1, Rachel Christenson1, Alissa Erickson1, Isabelle George1 1Department of Plant Pathology, University of Wisconsin-Madison, Madison, WI 53706 USA

Healthy plants depend on the simultaneous provisioning of multiple ecosystem functions from soils. While levels of ecosystem functions are often increased in more diverse communities, functions may respond to diversity on different scales. For instance, functions that depend on niche complementarity tend to be maximized by greater diversity at higher phylogenetic scales, while exclusion of invading species is likely to be maximized by fine-scale diversity of species ecologically similar to the invader. We tested whether three ecosystem functions for potato plants depended on the broad (phyla- level) or fine (genera-level) scale diversity of soil microbial communities. In a Page 92 of 112

greenhouse study, we standardized abiotic soil conditions and inoculated with microbial communities from 13 potato fields throughout Wisconsin that varied in broad and fine- scale diversity (determined through sequencing of bacterial 16S and fungal ITS2 marker genes). Pots were grown with either high nutrients, low nutrients, or high nutrients plus inoculation with Streptomyces scabies, the causative agent of potato common scab disease. We repeated this study in two years using distinct sets of microbial community sources to confirm that relationships between microbial diversity and plant growth and disease were robust to variation in microbial composition. Consistent with ecological theory, we found that potato tuber yields under high nutrient conditions were independent of microbial diversity, tuber yields under nutrient-limited conditions correlated with broad-scale microbial diversity, and that suppression of common scab disease correlated with fine-scale diversity within related taxa (diversity of Actinomycete genera). These results suggest that maximizing microbial diversity may enhance plant health, but it may be challenging to simultaneously promote functions that respond to different aspects to soil communities.

Allelopathy of Brassica rapa in drought

Hansol Lee1, Steve Franks1 1Department of Biological Sciences, Fordham University, Bronx NY 10458

Allelopathy is one of important mechanism in competition and several species in Brassicaceae are known as an allelopathic plant inhibiting other species growth. However, little is known about the interaction between allelopathy and drought adaptation. We treated three different co-occurring species with soil that Brassica rapa was grown in water control. We also conducted drought experiment on Brassica rapa in soil that Brassica rapa was grown. Brassica rapa soil reduced germination proportion and delayed germination of all four species. Brassica rapa soil significantly The final height of Galium aparine, one of co-occurring species, was significantly decreased. The Brassica rapa soil has a significantly negative effect on cotyledon size of Brassica rapa. The Brassica rapa soil delayed flowering time of Brassica rapa in drought.

Novel roles of fungal volatile compounds in inter-species interactions and biocontrol

Ningxiao Li 1, Alsayed Alfiky 2, Wenzhao Wang 3, Md Islam 2, Yongho Jeon 2, Khoshnood Nourollahi 2, Xingzhong Liu 3 and Seogchan Kang 1,2 1 Intercollege Graduate Degree Program in Plant Biology and 2 Department of Plant Pathology & Environmental Microbiology, The Pennsylvania State University, University Park, PA, USA 3 State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China

Volatile compounds (VCs) can travel through air, liquids and porous soils and mediate diverse forms of intra- and inter-species interactions in animals and plants. However, Page 93 of 112

compared to well-known diverse and critical roles of semio-VCs in animals and plants, relatively little is know about if microbial VCs perform similar roles. Given the high likelihood that water may not be readily available to mediate organismal interactions in the soil, we investigated if and how VCs participate in fungal-fungal interactions with a focus on their potential roles in biocontrol. Many Trichoderma strains protect plants from diverse pathogens using multiple mechanisms. Our work on VC-mediated interactions between several Trichoderma spp. and Fusarium oxysporum, a diverse species complex of soilborne fungi, suggested novel mechanisms that potentially play an important role in determining the efficacy of Trichoderma-based biocontrol as well as fungal ecology. One mechanism is VC-mediated pathogen recognition and response by Trichoderma. Both T. virens and T. viride significantly increased the production/secretion of anti-fungal metabolites in response to VCs released by 13 strains of F. oxysporum, suggesting that they recognize the presence of other fungi without physical contact or water-mediated molecular interactions. Whereas T. asperellum did not respond to any, and T. harzianum responded to VCs from only three strains. Gene expression analysis via qPCR showed up-regulation of several biocontrol-associated genes in T. virens in response to F. oxysporum VCs. All four Trichoderma species suppressed F. oxysporum growth using their VCs, suggesting their role as chemical weapons. F. oxysporum also recognized and inhibited Trichoderma using VCs, suggesting that both forms of VC-mediated inter- species interactions are common among fungi. Implications of our findings to biocontrol and fungal ecology and ongoing research will be presented.

Soil indigenous microbe and plant genotypes cooperatively modulate soybean rhizosphere microbiome assembly

Fang Liu1a*, Meg E. Staton1a, Sarah Lebeis1b, Tarek Hewezi1c, Ernest C. Bernard1a 1 Department of Entomology and Plant Pathologya, Microbiologyb, Plant Sciencec, University of Tennessee, Knoxville TN, 37996 The increasing understanding of microbiome importance for plant growth, health and stress tolerance is providing a promising new way to approach sustainable agriculture. To effectively utilize microbes to influence plant and soil health, we need to develop a comprehensive understanding of plant microbiome assembly process and verify factors that are consistently involved in this recruitment. The rhizosphere as an interface between plant and soil is characterized by a diverse microbial community, intensive microbe- microbe interactions and complex signal communication between plant and microbes. In this study, we comprehensively characterized the soybean rhizosphere microbial community and evaluated the relative contribution of soil types (agriculture vs forest soil) and soybean genotypes on rhizosphere microbiome assembly based on 16 rDNA amplicon sequencing. The microbial community composition is significantly different between bulk soil and rhizosphere. Gammaproteobacteria and Actinobacteria were consistently enriched in the rhizosphere, while Acidobacteria and Verrucomicrobia were steadily depleted in rhizosphere. However, the recruitment pattern of microbiota is not always consistent between soil type, with Firmicutes, Alphaproteobacteria, Rhizobium, Page 94 of 112

Bacillus, Novosphingobium, Pseudomonas and Pantoea being specifically enriched in soybean rhizosphere when growing in agriculture soil while Bacteroidetes, Betaproteobacteria, Burkholderia being preferably enriched in that of forest rhizosphere samples. Soil type had the largest impact on microbial community composition, indicating the important driving force of the indigenous microbe pool for rhizosphere microbe assembly. The influence of the soybean cultivar in rhizosphere microbiome assembly was also found to be significant though smaller, with a drought tolerant cultivar and a wild type accession (Glycine soja) showing greater differences from other genotypes. Overall, the alpha diversity of the soybean rhizosphere was significantly reduced in comparison to bulk soil, and similarly, the co-occurrence network in rhizosphere featured with less complexity. To explore the difference of microbe function potential between rhizosphere and bulk soil, Tax4Fun software were used to predict microbial functional and metabolic capacities. Antibiotic producing pathways were significantly higher in bulk soil microbial community, while flagellar assembly, bacteria secretion, and phosphotransferase systems were more enriched in the rhizosphere microbial community. This again confirmed active microbe communication and sugar degradation capacity in response to rich root exudates present in the rhizosphere. This comprehensive comparison of microbial communities in the soybean rhizosphere could help to expand our understanding of rhizosphere microbe assembly in general and provide information of soybean as a legume- specific model for this assembly process.

The lifestyles, hosts and sources of Colletotrichum fioriniae and their relevance in causing bitter rot on apple

Phillip Martin1,2, Teresa Krawczyk2, and Kari A. Peter1,2 1Penn State University, Buckhout Lab, University Park, PA 16802 2Penn State Fruit Research and Extension Center, PO Box 330, 290 University Drive, Biglerville, PA 17307

Bitter rot of apple is caused by fungi in the genus Colletotrichum, with the dominant species being C. fioriniae of the C. acutatum species complex. In addition to causing disease on hosts as diverse as strawberries, peaches, almonds, cranberries, and scale insects, C. fioriniae has been isolated as an epiphyte from a broad assortment of mostly broadleaf plants, ranging from dandelions to garlic mustard to hemlocks. While it is thought that most bitter rot on apple come from C. fioriniae isolates that cycle within the apple tree canopy, infections sources could be from any of the aforementioned plants. To detect C. fioriniae in the environment we designed and tested a quantitative (q)-PCR protocol with C. fioriniae specific primers using SYBR Green as the fluorophore. Non- specific binding at higher cycle numbers kept the limit of detection in the thousands of conidia, which was much higher than our goal. After excluding all samples with non- specific binding, C. fioriniae was unexpectedly detected in forested areas at numbers that equaled the C. fioriniae inoculated positive control apple trees. Various forest understory plants were sampled for endophytic colonization by C. fioriniae. Abundant colonization was found on poison ivy and wild grape leaves, with moderate colonization on Japanese Page 95 of 112

honeysuckle. These findings are discussed in the context of C. fioriniae lifestyles and hosts and its applicability to bitter rot on apple.

Phylogenetic analysis of Pseudomonas sequences obtained from mushroom caps (Agaricus bisporus) with blotch symptoms

Samuel J Martins1, Ryan V. Trexler1, Fabrício R. Vieira1, John A. Pecchia1, Terrence H. Bell1, Kevin L. Hockett1, Carolee T. Bull1 1Department of Plant Pathology and Environmental Microbiology, Penn State University, University Park, PA 16802

Mushroom growers frequently cite bacterial blotch of mushrooms as one of the most economically important production problems they face. The disease results in a rapid browning or blotching deterioration of mushroom caps and is caused by a variety of fluorescent Pseudomonas species. We assessed the bacterial component of the microbiome in mushroom caps with blotch symptoms, collected in a commercial organic farm in Pennsylvania. In this analysis, we focused on a subset of all of the samples sequenced in this project (25 symptomatic mushroom caps), for which total DNA was extracted, and a portion of the 16S rRNA gene region was amplified using the universal 515f/806r primer set that targets the V4 hypervariable region. The resulting pool was sequenced on the Illumina MiSeq. Operational taxonomic units (OTUs) were generated by clustering sequences at 97% similarity and were classified with the GreenGenes (13.8) database. Twenty-two OTU representative sequences were found to be related to Pseudomonas spp. For this study, we compared the OTUs identified as Pseudomonas spp. with 162 Pseudomonas type strains and members of 265 putative Pseudomonas spp. (clique) downloaded from Joint Genome Institute (JGI) (https://jgi.doe.gov) using CLC Workbench 11. The 22 OTU sequences were located throughout the trees, suggesting the presence of a broad diversity of Pseudomonas spp. in mushrooms with blotch symptoms. Eleven OTUs grouped together with the fluorescent Pseudomonas species and three clustered closely to P. tolaasii, a known mushroom pathogen. Moving forward, we will compare our database to single sequence variants to determine Pseudomonas diversity at a finer scale.

Ancestry and morphology determine leaf and root microbial communities of Rhododendrons, with implications for disease resistance

Juliana S. Medeiros1, Michael Mann1, Jean H. Burns2, Sarah R. Carrino-Kyker*1, and David J. Burke1,2 1The Holden Arboretum, Kirtland, OH 44094 2Department of Biology, Case Western Reserve University, Cleveland, OH 44106

The plant-associated microbiome plays an important role in plant health, including through nutrient uptake and disease resistance. However, whether there are general rules that govern the community structure of the plant microbiome is currently unknown. We addressed the following questions using 12 species of Rhododendron Page 96 of 112

growing in a common garden at the Holden Arboretum: 1) How does phylogeny shape plant traits and the microbiome of roots and leaves? 2) What plant morphological traits are associated with components of the plant microbiota (e.g. bacteria, fungi)? 3) Do plant traits and the microbiome correlate with disease resistance? We used terminal restriction fragment length polymorphism to characterize fungal and bacterial communities on leaves and roots and we compared this with leaf and root morphology as well as with data on disease resistance obtained from the literature. There was a phylogenetic signal on bacterial and fungal community composition both in leaves and roots; as such, the phylogenetic generalized least squares method was used to test for effects of disease resistance and plant morphology on plant-associated microbial communities. We found that plant traits, especially specific leaf area, were predictors of microbial community structure, but not related to disease resistance status. Microbial communities in bulk soil, rhizosphere soil, and on roots were significantly predicted by resistance to pathogens in the genus Phytophthora, which cause root rot for many plants. Our data suggest the plant microbiome could be governed by phylogeny, plant morphology, and disease resistance status, but that plant-associated microbiota aboveground are structured in response to plant traits, while resistance to disease may be more important for shaping belowground communities.

Examining the potato (Solanum tuberosum) response to rhizosphere microbiomes across an evolutionary gradient

Max Miao1 and Richard Lankau1 1Department of Plant Pathology, University of Wisconsin-Madison, Madison WI 53706

Over millions of years plants have co-evolved with their soil microbes to overcome challenges, relying on them for non-innate immunity, stress tolerance, and nutrient acquisition. The modern potato, (Solanum tuberosum sp. tuberosum), has undergone many changes from its wild and landrace progenitors. Selection for greater yield in higher input soils may have led to the loss or alteration of plant traits related to microbial interactions. By inadvertently changing the diversity, assembly, and function of the potato microbiome, we may have reduced the potential for positive plant-microbe responses. Here, we examined the structure and function of potato rhizosphere microbiomes across an evolutionary gradient. Our experiment compared modern potato to more ancestral wild species (Solanum berthaulti), along with Andean and Chilean landraces grown in the presence of microbial communities from natural prairies or agricultural fields. The results found differences in biomass and yield in landraces versus modern potato varieties across different microbial inocula in low nutrient conditions. This may stem from potential similarities and differences in microbial recruitment, diversity, and function across S. tuberosum’s evolutionary history. Coupled with amplicon sequencing, we will compare plant physiological differences to the plant rhizosphere microbiomes among potato taxonomic groups, initial microbial inocula, and nutrient conditions. By using this community centric approach, we aim to link the potential evolutionary changes in the potato rhizosphere microbiome to functionality in terms of plant health and physiology. Page 97 of 112

Seed microbiome of agricultural crops

Zayda Morales1*, Bobbi L. Helgason2, James J. Germida3 1Dept. of Food and Bioproduct Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5A8, Canada, [email protected] 2Saskatoon Research Centre, Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan, S7N 0X2, Canada 3Dept. of Soil Science, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5A8, Canada

Microbial communities play a significant role in host fitness, contributing to plant health and sustainable crop production and yield. Most of the plant microbiome originates from the seed and soil. The seed acts as an initial source and reservoir of microbes, which will continue to interact with the plant throughout its life cycle. Cereals, oilseeds and legumes are staple crops on the Canadian Prairies and are important for world food security. A better understanding of the microbiomes of these crops is necessary to elucidate their role in plant development. The objective of this study was to characterize the seed microbiome in wheat, canola, and lentil to determine whether these microbiomes are unique or variable among different genotypes. Seeds from five wheat, canola, and lentil lines were subjected to DNA extraction, high-throughput amplicon sequencing, reads processing and OTU clustering. As a first step, the endophytic bacterial microbiome was analyzed using the 342F and 806R 16S rRNA universal primers; however, >80% of the sequences obtained originated from plant plastid or mitochondria. Thus, initially we have focused on the epiphytic seed microbiome. Information related to the microbial community composition inhabiting the seed surface among different lines was determined. All epiphytic microbiomes contained taxa of Proteobacteria and Actinobacteria. The most dominant bacterial genera were Erwinia in wheat and Pseudomonas in canola and lentil. In each of the three crops there were substantial differences in microbial composition and abundance among genotypes and in some cases between biological replicates. Future work will focus on the analysis of the endophytic microbiome using peptide nucleic acid (PNA) PCR clamps to suppress plant host plastid and mitochondrial 16S contamination.

Plant microbiome succession and assembly modulated by endogenous signal peptides in soybean (Glycine max)

Itumeleng Moroenyane1, Charlotte Giard-Laliberté1, Julien Tremblay2 and Etienne Yergeau1

1Institut National de la Recherche Scientifique, Centre Institut Armand-Frappier, 531 Boulevard des Prairies, Laval, Quebec, H7V 1B7, Canada

2 Energy, Mining and Environment, National Research Council Canada, 6100 Royalmount Avenue, Montréal, Québec, H4P 2R2, Canada Page 98 of 112

Managed agricultural ecosystems are unique systems where crops and microbes are intrinsically linked and selected to promote crop health and increase production. Advances in plant breeding and a broader understanding of the ecology of microbes in these systems have led to better management practices in these dynamic ecosystems. This study focuses on successional development of the plant microbiome and assembly processes that delimit them, and aims to 1) tests for evidence of niche differentiation and to what extend plant signal peptides influence community assembly and 2) create a successional model that can predict these niche shifts as they occur during plant development. To this end, Glycine max var. Pioneer plants were grown in an environmental chamber till seed maturation. Microbiome and xylem sap samples were collected at the various developmental stages: emergence, growth, flowering, and seed maturation. Microbial community structure and abundance were assessed with amplicon sequencing and qPCR using taxon specific primers. The sap was purified and signal peptides were identified using nano LC-MS/MS. Abundance was highest in the epiphytic and rhizosphere communities, and community structure varied according to- plant organ, plant developmental stage, and life history (epiphytic or endophytic). Machine learning models correctly predicted the abundance and distribution of key taxa. Phylogenetic niche shifts were detectable across plant organs and developmental stages. Interestingly, these niche shifts were in part related to turnover in signal peptide diversity and abundance. Lastly, niche-based processes delimited distribution and assembly of endosphere and rhizosphere communities. We demonstrate that by understanding the mechanistic processes that assemble microbiome communities, it becomes not only possible to identify key microbes that are essential for promoting plant growth and health, but also key intervention points where the microbiome community can be engineered to be composed of microbes that can mitigate plant stresses and influence long-term management practices.

Impact of commercial management practices on soil microbiomes of HLB-affected citrus trees

Andrea Nuzzo1, Bryce Meyering1, Ute Albrecht1, Sarah L. Strauss1 1Southwest Florida Research and Education Center - University of Florida, Institute for Food and Agricultural Sciences (IFAS/SWFREC), Immokalee, FL 34142

Root health is a major factor contributing to yields and disease protection in agriculture, and depends on several environmental interactions, including microbial communities in the soil. Some bacterial taxa exhibit plant-growth promoting (PGP) effects, while many fungal species colonize root tissues to reinforce and protect from diseases (i.e. Mychorrizae). Root health, and therefore beneficial microbiomes in soil, is particularly relevant to prevent or alleviate diseases that impact the root structure, such as Huanglongbing (HLB, aka citrus greening) which is a major epidemic disease of citrus in Florida and other citrus producing areas around the world. The aim of this work was to promote root health by enhancing soil microbial communities through five different treatments containing either seaweed, fulvic acids, beneficial microbes or combinations of the previous. The treatments were applied in two commercial citrus groves, approximately 10 miles apart, each with the same soil characteristics but with different Page 99 of 112

grove management programs. One grove had minimal insecticide/fungicides and nutritional applications (Grove V), while the other had a larger input of crop production chemicals (Grove D). A statistical modelling approach was used to go beyond classical distance-based analysis to assess the impact of treatment and management over the soil microbial communities (Bacteria and Fungi combined) and the potential interaction with root health. Differential abundance analyses showed that no treatment significantly altered the microbial communities after 6 months of application. Conversely, significant differences existed between communities in the two groves (adj p < 0.001), mostly occurring at the fungal level. While both groves were significantly infected by Fusarium solani (a pathogen), Grove D showed higher concentration of plant-pathogenic taxa (Saltozyma podzolica, Ramichlodrium cucurbitaceae, Exophiala pisciphila) than Grove V, potentially outweighing the presence of beneficial taxa (Trichoderma asperellum, Penicillum semplicissimus). Metabolic predictions based on taxa assignments suggested higher frequencies of carbon-cycling pathways and lower frequencies of saprotrophic fungi in Grove V, suggesting an overall higher metabolic activity of the community. The impact of treatments and management practices on root health and general plant physiology was positive for some parameters in Grove V, but not in Grove D after 6 months of applications. This work illustrates that grow management, including pesticides/fungicides and nutritional applications, has a profound impact over the indigenous microbiomes, and that none of the treatments significantly affected the microbial community structure, even when applying foreign species. Additionally, this work also shows how novel statistical modelling can identify single species with significantly different abundances in multipartite microbiomes.

Do plant nutrient demand and availability influence rhizosphere microbiome assembly and functioning?

Oladapo Olayemi1, Matthew Wallenstein1 1Natural Resource Ecology Laboratory and Department of Soil and Crop Sciences, Colorado State University, Fort Collins CO 80534

In order to meet their nutrient demands, plants attract beneficial microorganisms to their rhizosphere. Plants can influence rhizosphere microbiome assembly through root exudate chemistry, root architecture, and other mechanisms. As a result, microbiomes differ in structure and function among plant species and even genotypes. But, we do not yet know the degree to which plant influence on microbiome assembly is plastic, and whether plants can modulate these processes depending on environmental conditions. We hypothesize that plants recruit different microbiomes depending on soil N availability to support their demand for N. We predict that under low N availability, plants will support more diverse microbiomes with a higher potential for N mineralization. In a greenhouse experiment, we will grow two cultivars of a C3 (Wheat) and a C4 (Corn) plant in a common soil under three different inorganic nitrogen application rates. We will measure the microbiome structure, plant-derived 13C uptake pattern of the microbiome, extracellular enzyme activity as well as the functionality of the assembled microbiome Page 100 of 112

with respect to N cycling. This study will improve our understanding of how plants engineer their rhizosphere microbiome based on their N needs and level of N supply.

Phosphorus addition shifts the microbial community in the rhizosphere of Blueberry (Vaccinium corymbosum L.)

Hugo A. Pantigoso1, Daniel K. Manter2, Jorge M. Vivanco1 1 Center for Rhizosphere Biology and Department of Horticulture and Landscape Architecture, Colorado State University, Fort Collins, CO 80523-1173, USA 2 United States Department of Agriculture – Agricultural Research Services, Soil-Plant- Nutrient Research Unit, Fort Collins, CO 80526, USA

Phosphorous fertilization is critical to enhance plant P uptake and proper growth. However, it is presently unknown if its application may have detrimental effects on the soil microbiology. Blueberries are sensitive to fertilization hinting to close relationships with beneficial microbes related to soil nutrition. Our study explored the effect of P fertilization amendments on soil bacterial community composition in two varieties of commercial blueberry (Vaccinium sp. var. “Misty” and “Biloxi”). Shifts in microbial community composition and diversity were found in response to P amendment. The microbiome was influenced by P level and differentiated based on low P (0 and 45 lb/acre) and high P (90 and 171 lb/acre) rates. Additionally, predictive microbial acid phosphatase activity decreased with increasing levels of P solubilization. Thus, our results suggest that P fertilization decreases the ability of P–solubilizing microbes to naturally provide P to the plant.

Characterization of microbial communities between healthy and diseased Caribbean corals

Roitman, Sofia1, Collin Closek2, F. Joseph Pollock3, Eric Jordán-Dahlgren4, Marilyn Brandt5, Monica Medina1 1 Pennsylvania State University, Department of Biology, 208 Mueller Lab, University Park, PA 16802, USA 2 Stanford University, Department of Civil and Environmental Engineering, Stanford, CA 94305, USA 3 The Nature Conservancy, Eastern Caribbean Program, 3052 Estate Little Princess, Christiansted, St. Croix, U.S.V.I. 00820 4 Universidad Nacional Autonoma de Mexico, Instituto de Ciencias del Mar y Limnologia, Puerto Morelos, Mexico 5 University of the Virgin Islands, College of Science and Mathematics, Charlotte Amalie West, St. Thomas, U.S.V.I., 00802, USA

The decline of coral reefs worldwide has been attributed in part to the rise of disease outbreaks, often on the heels of increases in temperature. Asides from physiological changes such as the appearance of lesions or bleaching, infected corals can also exhibit Page 101 of 112

changes in their microbiome composition. A coral’s bacterial associates can play a key role in the health of the holobiont through metabolic complementarity, by producing antibacterial compounds and nutrient cycling, or by serving as an indicator for coral stress. The coral microbiome has also been shown to be sensitive to environmental stressors and changes in the health of the host, however we have yet to gain a full understanding of the breadth of microbial diversity across coral species as well as how microbial associations change under varying conditions. Determining the identity of corals’ microbial partners and their subsequent changes in abundance during periods of stress can provide us with a greater understanding of the interactions that take place in the coral holobiont and the role these microorganisms play, especially with regards to coral health and resilience against disease. In this study, over 350 samples of varying health conditions were collected from two Caribbean reef-building corals Orbicella faveolata and Acropora palmata. Preliminary analyses indicate differences in the microbial community composition between healthy and diseased samples from both species across four diseases (yellow band disease, white band disease, white pox disease, and white plague disease). These results suggest potential identification of pathogenic groups and the creation of predictive models and the use of certain bacterial taxa as bioindicators for specific disease outbreaks.

Interspecies interactions in the rhizosphere may influence the functions of plant growth promoting rhizobacteria

Amanda Rosier1, D. Janine Sherrier1, Harsh Bais1 1Delaware Biotechnology Institute and Department of Plant and Soil Sciences, University of Delaware, Newark, DE 19716

Agriculture systems disrupt microbial communities and reduce their diversity, leading to declines in ecosystem functions. Microbial diversity in the rhizosphere is critical to maintaining functional ecosystems. The use of plant beneficial organisms derived from the plant microbiome is gaining interest in agriculture as a solution to restore plant microbial ecosystems and decrease dependence on pesticide and fertilizer use. It is understood that the application of individual plant growth promoting rhizobacteria (PGPR) increases plant health. However, limited research efforts have investigated complex interactions occurring between multiple species of plant beneficial bacteria in the rhizosphere, and how the outcomes of those associations influence their ability to benefit the plant. Bacteria exert their influence on other species in the rhizosphere in multiple ways, including the production of a wide array of extracellular signal and defense molecules. Sinorhizobium meliloti is well documented to participate in a unique form of inter-species interaction with legumes and relies on a complex intra-species communication system call ‘quorum sensing’ (QS) for efficient nodulation of the plant. Using a tri-trophic model system of the legume Medicago truncatula A17 Jemalong, its mutualistic symbiont S. meliloti strain Rm8530 (hereafter Rm8530), and the PGPR Bacillus subtilis UD1022 (hereafter UD1022), we show that interactions between the PGPRs may influence their individual associations and activities on the plant root. Expression and functional analysis of Rm8530 suggest that UD1022 produces Page 102 of 112

extracellular compounds that impact the components involved in the Rm8530 quorum sensing (QS) system. At the same time, Rm8530 may be influencing the functional plant association strategies of UD1022. This interaction could have greater implications in altering the ability of the PGPRs to positively affect plant health and may allow for more targeted approaches in developing novel, sustainable solutions in agriculture.

Cheating and punishment in plant bacterial symbiosis

Joel Sachs1 1Department of Biology, UC Riverside, Riverside, CA, USA

Nitrogen-fixing rhizobia transform how legumes interact with their environment by providing a reliable and cheap source of fixed nitrogen for the host plant. But theory predicts an evolutionary conflict in symbiosis. Rhizobia have a tremendous evolutionary advantage over their legume hosts in terms of generation time and population size, and thus can rapidly generate mutants that exploit hosts without providing benefits. In turn, legumes must intensely select against exploitation in rhizobial populations in order to maintain the key service of nitrogen fixation. We have genotyped and phenotyped Bradyrhizobium across a natural metapopulation of Acmispon strigosus hosts in California. Phylogenetic reconstruction was used to analyze the origins and spread of non-fixing rhizobial genotypes. We sequenced whole Bradyrhizobium genomes to examine the genetic basis of ineffective (nonfixing) rhizobia. We found that non-fixing Bradyrhizobium have evolved at least 5 times across the sampled sites, apparently through multiple independent mechanisms of genome evolution. Host defense mechanisms were investigated using single and mixed infection experiments in Acmispon and its sister taxon Lotus. In both hosts, plants inoculated with ineffective rhizobia exhibited evidence for a cell autonomous and accelerated program of senescence within nodules. In plants that received mixed inoculations, only the plant cells harboring ineffective rhizobia exhibited features consistent with programmed cell death, including collapsed vacuoles, ruptured symbiosomes, and bacteroids that are released into the cytosol. These features were consistently linked with ultrastructural evidence of reduced survival of ineffective rhizobia in planta. Our data suggest an elegant cell autonomous mechanism by which legumes can detect and defend against ineffective rhizobia even when nodules harbor a mix of effective and ineffective rhizobial genotypes.

The ecology of bacterial invasions

Joana Falcao Salles1, Cyrus A. Mallon1, Xavier Le Roux2 1Groningen Institute for Evolutionary Life Sciences, University of Groningen, The Netherlands 2INRA, CNRS, Université Lyon 1, Université de Lyon, Laboratory of Microbial Ecology (LEM), Villeurbanne, France Page 103 of 112

Biological invasions have the potential to alter the ecological and evolutionary trajectory of the Earth’s ecosystems. Although less studied, microbial invasions are a constant in many fields of microbiology, including agricultural, medical, and environmental, where the entrance of a foreign microorganism into a resident community of microbes (a microbial invasion) is a common phenomenon. Despite the commonality, these fields view microbial invasions as a mere snapshot of a longer frame rather than a process. The latter is however crucial to develop general principles to foster cross- comparisons and enhance the understanding, interpretation and development of future strategies to facilitate or hamper invasions. In my talk I will look at bacterial invasions from continuum framework, in which invasion processes are divided in introduction, establishment, spread, and impact phases. Using the invasion of pathogenic E. coli strains in soils as example, in the first part of my talk I will examine the patterns and mechanisms associated with invasion resistance and create a mechanistic synthesis of how species diversity and resource availability influence invasion resistance. In the second part I will discuss the outcome of an invader’s introduction on the resident community. So far, only successful invasions have been explored, and it remains unknown to what extent an unsuccessful invasion can impact resident communities. I will thus explore how unsuccessful invasions impact soil functioning and generate shifts in native bacterial communities, both in terms of community composition and niche structure. I will conclude by exploring the advantages of using this theoretical invasion framework in the context of applied phytobiome research, such as the application of biological control agents to enhance plant resistance to biotic and abiotic stress.

Soil Depth and Landscape Position Structure Microbial Communities and Co- occurrence Networks in No-Till Wheat Systems

Daniel Schlatter1 and Timothy Paulitz1 1USDA-ARS, Wheat Health, Genetics and Quality Research Unit, Pullman, WA 99164- 6430

No-till agriculture is increasingly adopted by growers in the Palouse region of eastern Washington state as a means to limit erosion, build soil health, and reduce fuel costs. In no-till, soils are not disrupted by tillage, residue is left on the surface, and seeds are planted directly through the residue layer. Moreover, the Palouse is characterized by rolling, steeply sloping hills. As a result, soil physical, chemical, and biological characteristics become highly stratified with soil depth and differ with landscape position. However, the composition, diversity, and inter-relationships of soil microbial taxa differ among soil depths or landscape characteristics are poorly understood. To shed light on how microbial communities and their co-associations vary with soil depth and landscape positions, we took 4.4 cm diameter soil cores across a transect in a long-term no-till wheat field at the Cook Agronomy Farm near Pullman, WA. Soil cores were sampled at 0, 10, 25, 50, 75, and 100 cm from the soil surface, DNA was extracted, and 16S rRNA and ITS1 genes were amplified and sequenced (Illumina MiSeq) to characterize microbial communities. The composition and diversity of bacterial and fungal communities differed significantly with soil depth, where deeper soils tended to harbor Page 104 of 112

less diverse communities dominated by a small number of taxa. Differences in bacterial and fungal communities were strongly correlated with soil characteristics, such as pH, C, and N. Moreover, co-occurrence networks among fungal taxa became smaller, less dense, and more clustered with increasing depth. In contrast, there were few differences in microbial communities between north- and south-facing slopes. Together, these data shed important light on how microbial communities and their potential interactions with each other and with plant roots are stratified with soil depth in no-till systems.

Field study shows that phylogenetically redundant root microbiota is recruited by the common beans (Phaseolus vulgaris L.) across US bean growing regions

Nejc Stopnisek1,2, Ashley Shade1,2,3 1Department of Microbiology and Molecular Genetics 2The Plant Resilience Institute 3Department of Plant, Soil and Microbial sciences, Michigan State University, East Lansing MI 48824

The common bean (Phaseolus vulgaris L.) is the primary grain legume for human consumption and it represents a rich source of protein, vitamins, minerals, and fiber, especially in the developing nations of Africa and Latin America. However, yield estimates and production areas for common bean are predicted to decrease in this century due to the impacts of global change. One possibility for improving common bean resistance to global changes is to harness beneficial members of its microbiome, but there are many unknowns concerning the major biotic and abiotic factors shaping bean microbiota under relevant field conditions. Here, we assessed the common bean core microbiota by quantifying the diversity and composition of the bacterial, archaeal, and fungal communities in the rhizosphere of two common bean genotypes with distinct evolutionary background: California early light red kidney (CELRK) and Eclipse. Genotypes were grown under similar field management in 4 major regions of bean production in the US (Michigan, Nebraska, Washington, and Colorado). We used 16S rRNA gene and fungal ITS amplicon sequencing at the flowering stage to assess the rhizosphere microbiome of mature plants. In total, we identified 7054 bacterial and archaeal taxa (~3250 per sampling location), and 2321 fungal taxa (~380 per sampling location). Proteobacteria comprised 39.3% of detected bacterial and archaeal taxa, followed by the Acidobacteria (13.4%), Bacteroidetes (13%), and Actinobacteria (10.1%). In the fungal community, Ascomycota comprised 32.7% of the community, followed by the Basidiomycota (17%), and Mortierellomycota (11.7%). Even though sampling location had influence on the microbial composition, a large proportion of identical microbial taxa was observed across all sites. These consistently detected taxa were also highly abundant, comprising 10% of the total microbial diversity (~350 taxa) and 37% of total relative abundance. A core microbiome for common bean with such extent was unexpected, especially considering the large geographical distances between growing regions and their variability in climate and soil. This core also was conserved between bean genotypes, indicating that these taxa generally are important players for common bean. Rhizobiales, Xanthomonadales, Burkholderiales, Sphingobacteriales, and Sphingomonadales were the top bacterial orders found in the core Page 105 of 112

microbiome, and these typically contain many taxa reported to benefit pants by providing nutrients, defend against pathogens and promoting growth. These results suggest that though there are environmental nuances, a conserved core microbiome is consistently selected by common bean despite different soil environments and genotypes.

The WELL: Examining routes of entry for the foliar endophytic fungi of Magnolia grandiflora

Brian W. Tague1, Lexie Wang1, Tayler Stander and DJ Parker1 1Department of Biology, Wake Forest University, Winston-Salem NC 27109

The Wake Endophyte Legacy Lab (The WELL) is a long-term undergraduate-based research project examining the mycobiome of Magnolia, an ancient plant genus. We are primarily focused on Magnolia grandiflora, the iconic tree of Wake Forest University. M. grandiflora is an evergreen, allowing experimentation throughout the academic year. Magnolia leaves harbor a wide diversity of cultivable fungal endophytes, dominated by Diaporthe eres. Colletotrichum acutatum is a rarer, but easily identifiable member of this endophyte community. In all, we have cultivated over 40 different fungal morphotypes from magnolia leaves, and identified many by PCR bar-coding. It was remarkable (to us!) that demographically-matched leaves from consecutive nodes could contain a different community of endophytes. This prompted us to ask about the factors that shape this community, particularly routes of entry. Fungal endophytes do not appear to be vertically transmitted in magnolia. Arils and seeds are relatively devoid of endophytic fungi. Endophytes are not detected in leaves of lab-grown plants. Spore fall under the canopy is very high and allowed us to capture C. acutatum as a spore. Additionally, our potted endophyte-free plants can acquire endophytes under the canopy. These data indicate entry at the level of the leaf through stomata, hydathodes or penetrative entry as expected for many endophytes. We will also report on the foliar endophytes of root sprouts from our study tree and of volunteer magnolia seedlings. To ask about endophyte entry through the vascular system, we examined about 60 petioles from a branch of our magnolia tree for cultivable endophytes. The endophyte population appeared to be somewhat restricted and was dominated by two fast growing species. Both D. eres and C. acutatum were recovered from petioles, but more rarely. Terminal leaf buds have been examined to ask about “developmental” entry. In an analysis of several terminal leaf buds only Diaporthe eres has been isolated, but rarely. Taken together, these experiments begin to define the routes of entry that define the community of foliar fungal endophytes of Magnolia grandiflora. Page 106 of 112

Exploring a new approach to the reproducible transfer of microbiomes between soils

Ryan V. Trexler1, Emily M. Grandinette1, Idalys Bonet1, Terrence H. Bell1 1Department of Plant Pathology and Environmental Microbiology, Penn State University, University Park PA 16802 USA

The reproducible transfer of microbiomes between soils would greatly facilitate controlled investigations of 1) soil microbial ecology, 2) microbiome impacts on soil function and health, and 3) plant-microbiome interactions. Although a variety of approaches to soil microbiome transfer are described in the literature, a common method involves directly diluting the source soil into a sterile recipient soil at various concentrations. In addition to transferring microorganisms, this approach also transfers soils particles and compounds from the source soil, proportional to the amount of soil transferred, which can make it difficult to determine the impact of soil characteristics on the resulting microbiome structure or plant-microbiome interactions. Transferring a low percentage of source soil also results in low microbial diversity in the recipient soil. In this study, we explored the use of prolonged contact between source and recipient soils across membrane filters as an approach to microbiome transfer, and contrast this with the direct transfer of soil at two concentrations from three different source soils. We expect prolonged contact to limit the transfer of soil compounds, while allowing multiple waves of microbial succession into a recipient soil. After 6 weeks of incubation, distinct recipient microbiomes were produced by each transfer method, and the similarity between microbiomes transferred by each method and the source soil microbiome varied by soil type. We demonstrate that a direct contact approach results in a diverse microbiome, comparable to other commonly used methods, and is a viable option for soil microbiome transfer.

Endornaviruses in plants: mutualists or parasites?

Rodrigo A. Valverde1, Cesar Escalante1, Surasak Khankhum1 1Dept. of Plant Pathology and Crop Physiology, Louisiana State University Agricultural Center, Baton Rouge, LA 70803

Based on the type of relationship with the host, plant viruses can be grouped as acute or persistent. Acute viruses cause symptoms and plant diseases. In contrast, persistent viruses do not appear to affect the phenotype of the host. The viral family Endornaviridae includes persistent viruses that infect plants without causing visible symptoms. Infections by endornaviruses have been reported in many economically important crops, such as avocado, barley, common bean, melon, pepper, and rice. However, little is known about the effect they have on their plant hosts. We hypothesize that these viruses are in a mutualistic relationship with the host and may provide tolerance to unknown biotic or abiotic factors. To increase our knowledge on the nature of this interaction we conducted comparative studies between endornavirus-infected and endornavirus-free common bean (Phaseolus vulgaris) and bell pepper (Capsicum annuum) plants. Page 107 of 112

We evaluated physiological characteristics of eight lines of common bean, four of which were endornavirus-infected and four of which were endornavirus-free. Plants of all eight lines were morphologically similar and did not show statistically significant differences in plant height, wet weight, number of seeds per pod, and anthocyanin content. However, the endornavirus-infected lines had higher values of seed germination, radicle length, and weight of 100 seeds. In the case of bell pepper, we developed two near-isogenic lines of the cultivar Marengo, one infected with bell pepper endornavirus (BPEV) and the other endornavirus-free. The BPEV-negative line consistently yielded higher percentage of fruit weight and total dry matter than the BPEV-positive line; however, only the fruit weight value was statistically significant.

Studies on differential gene expression between endornavirus-infected and endornavirus- free common bean lines were conducted. RNAseq data revealed that a total of 132 genes were differentially expressed. In the endornavirus-infected line 84 genes were down- regulated while 48 genes up-regulated. Gene ontology distribution showed that oxidation- reduction (redox) processes were the main processes associated with endornavirus infection. Redox changes have been reported to be associated with plant response to pathogen infection, environmental stresses, development, and acclimation. It is worth mentioning that among the list of differentially expressed genes, one gene, Myzus persicae- induced lipase 1 (MPL1), was up-regulated 8-fold in the endornavirus-infected line. In Arabidopsis, this gene has been shown to play an important role in defense against the green peach aphid (Myzus persicae). Validation of the MLP1 expression may lead to future investigations on the role of endornaviruses on aphid feeding behavior.

The results of these investigations suggest that the type of symbiotic relationship between endornaviruses and the host depends on the character being evaluated and can range from mutualistic to parasitic.

The effects of host and environment on the maize microbiome

Jason G. Wallace1, Karl A. Kremling2, Tony Walters3, Nicholas K. Lepak5, Ruth E. Ley3, and Edward S. Buckler2,4,5 1Department of Crop and Soil Sciences, The University of Georgia, Athens, GA 2Department of Plant Breeding and Genetics, Cornell University, Ithaca, NY 3Department of Microbiome Science, Max Planck Institute for Developmental Biology, Tuebingen, Germany 4Institute for Genomic Diversity, Cornell University, Ithaca, NY 5United States Department of Agriculture – Agricultural Research Service, Ithaca, NY

Every maize plant has billions of microscopic organisms living in, on, and around it. These microbial communities—collectively called a “microbiome”—have great potential to influence plant growth, but the extent of their influence and the degree to which the host plant shapes them is unknown. We address the question of how much influence the host and the environment have on microbiomes by looking at bacteria in the maize Page 108 of 112

rhizosphere (roots) and phyllosphere (leaves). These two communities show very different makeups: the rhizosphere is highly complex, while the phyllsophere is dominated by <20 bacterial taxa. Environment is the largest driver of the rhizosphere community; host genetics has little direct effect but does show strong gene-by- environment interaction. Broad- and narrow-sense heritability analyses in both communities show that a subset of microbes are moderately affected by host genetics (heritability of 0.3-0.6), while the majority show little effect of the maize genotype. We also identify several metabolic pathways in the phyllosphere that may be shaped by host genetics. Taken together, these results indicate that the maize host exerts only partial control over the makeup of its rhizosphere and phyllosphere communities, and in many cases the environment and/or stochastic chance play a larger role. In addition, the metabolic capacity of these communities may be more important than their taxonomic identity. More work needs to be done to determine to what extent these communities affect their host plant, and to determine if and how manipulating them can benefit agriculture.

N-P fertilization did not reduce AMF abundance and diversity but altered AMF composition in an alpine grassland infested by a root hemiparasitic plant

Xue-Zhao Wang1, Xiao-Lin Sui1, Ai-Rong Li1,2 1 Department of Economic Plants and Biotechnology, Yunnan Key Laboratory for Wild Plant Resources, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China 2 Department of Biology, Pennsylvania State University, University Park PA 16802

Fertilization has been shown to have suppressive effects on arbuscular mycorrhizal fungi (AMF) and root hemiparasites separately in numerous investigations, but its effects on AMF in the presence of root hemiparasites remain untested. In view of the contrasting nutritional effects of AMF and root hemiparasites on host plants, we tested the hypothesis that fertilization may not show strong suppressive effects on AMF when a plant community was infested by abundant hemiparasitic plants. Plants and soil samples were collected from experimental field plots in Bayanbulak Grassland, where N and P fertilizers had been applied for three continuous years for control against a spreading root hemiparasite Pedicularis kansuensis. Shoot and root biomass of each plant functional group were determined. Root AM colonization levels, soil spore abundance, extraradical hyphae length density were measured for three soil depths (0-10 cm, 10-20 cm, and 20-30 cm). Partial 18S rRNA gene sequencing was used to detect AMF diversity and community composition. In addition, we analyzed the relationship between relative abundance of different AMF genera and environmental factors using Spearman's correlation method. Our results support the hypothesis that fertilization did not significantly influence the abundance and diversity of AMF in plant community infested by P. kansuensis. Further investigations are required to unravel the regulation processes and relevant mechanisms for the underground multi-species interactions. Page 109 of 112

Pseudomonas syringae and the sweet cherry leaf microbiome in Michigan orchards

Tammy Wilkinson1, Ashley Shade, George Sundin, and Gregory Lang 1Dept. of Horticulture, Michigan State University, East Lansing MI 48824

Bacterial canker caused by the gram-negative bacteria, Pseudomonas syringae pv. syringae (Pss), is a serious disease of sweet cherry (Prunus avium) in Michigan. Pss is ubiquitous in the orchard environment and can become problematic as populations increase during cool, wet weather conditions. Pss colonizes leaf and bark surfaces and is rain-splash dispersed. Severe Pss infections result in reduced or total yield loss and meristem, branch, or tree death. Management strategies for this disease are limited to applications of copper and restricted timing of cultural practices. Pss biology, virulence, and the abiotic conditions required for infection have been well studied. Little is known about the bacterial communities that thrive with, and potentially influence, Pss populations on sweet cherry leaves. Understanding these epiphytic communities in the phytobiome may lead to new strategies for Pss disease management, including possible biological control options. Leaves from 'Gold', 'Benton', and 'Sweetheart' sweet cherry were collected from orchards in two regions of Michigan in Fall 2017 at 10-25% leaf drop when Pss populations are typically high. Leaves were washed in a sterile 0.5% phosphate-buffered saline and sonicated to recover epiphytic bacteria. Bacterial DNA was extracted and then sent to be sequenced at the MSU Research Technology Support Facility using the 16S V4 rDNA conserved region with the Illumina MiSeq v2 standard

flow cell 2x250 bp paired end sequencing platform. Sequences were analyzed using Mothur open-source software (Schloss et al. 2009). Operational taxonomic unit (OTU) classification, rarefaction curves for species richness and the inverse Simpson Index for diversity are being analyzed and will be reported to establish the composition of the typical sweet cherry leaf microbiome in managed orchards. Additional sampling and analyses of the leaf microbiome will be conducted in spring and summer to determine potential seasonal changes as well.

Arbuscular mycorrhizal fungi facilitate the competitive growth of two invasive plant species Ambrosia artemisiifolia and Bidens pilosa by affecting N and P nutrient use efficiency

Ellen H. Yerger1, Fengjuan Zhang2 1Department of Biology, Indiana University of Pennsylvania, Indiana, PA 15701, USA 2College of Life Science, Hebei University, Baoding, Hebei, 071002, China

The significance of arbuscular mycorrhizal fungi (AMF) in the invasion success of exotic plant species has been widely recognized because of their ability to promote growth and influence competitive interactions between species. However, there is less information on how competition changes the soil AMF species diversity and extent of colonization on invasive and native species, and then how these feedbacks during competition change the accumulation of nutrient resources. Two exotic Asteraceae Ambrosia artemisiifolia and Bidens pilosa were monitored during competition with the native grass Setaria viridis. Page 110 of 112

This plant species replacement is currently occurring in invaded natural areas around the study site in China. From the rhizosphere soil of these plants, maintained in a field plot continuously for 5 years, AM fungal types were cloned and identified. Also AM fungal spores were isolated from field rhizosphere soil of the two invasive species and used as inoculum in manipulative greenhouse experiments to compare growth and nutrient accumulation during competition. The results showed that although the diversity of AMF communities in the rhizosphere soil of A. artemisiifolia and B. pilosa were different, the 3 most abundant fungal species were identical. In the greenhouse experiments the addition of AMF inoculum changed the competition between the plants resulting in both invasive species accumulating more biomass while the native species accumulated less. Our data support a mechanism during competition of increased percent AMF root colonization of the invasive and decreased of the native. When specific measurements of net photosynthetic rate, leaf N, and leaf P were taken, in competitive mixtures the invasive plant performed better with AMF inoculum added and the native plant performed worse.

Metabolic synchronization: How plant root exudation and microbial substrate preferences interact to shape rhizosphere microbiomes

Kateryna Zhalnina,1 Yuan Wang,2 Nameer Baker,3 Joelle Schläpfer Sasse,1 Eden Tepper- Fobes,1 Katherine B. Louie,1 Ulas Karaoz,1 Dominique Loque,4 Benjamin P. Bowen,1 Kelly Craven,2 Jennifer Pett-Ridge,5 Mary K. Firestone,1,3 Eoin L. Brodie1,3 and Trent R. Northen1 1Lawrence Berkeley National Laboratory, Berkeley, CA 94720 2Noble Research Institute, Ardmore, OK 73401 3University of California, Berkeley, CA 94720 4INSA de Lyon, CNRS, UMR5240, Microbiologie, Adaptation et Pathogénie, Université Claude Bernard Lyon 1, Villeurbanne, France 5Lawrence Livermore National Laboratory, Livermore, CA 94551

It has been known for more than a century that plants interact with soil microorganisms through their exudates, however the mechanisms of how plants shape their rhizosphere microbiome are poorly understood. We have examined how dynamic root exudate chemistry and microbial substrate preferences mediate rhizosphere community assembly to select for specific bacteria that are beneficial to the host plant. Our studies are focused on microbial community analysis in native systems, including both a Mediterranean grassland soil and a marginal soil. We performed comparative genomics of isolated rhizosphere bacteria and mass spectrometry based exometabolomics to characterize plant exudation and metabolites taken up by rhizosphere bacteria. The resulting data allow us to examine metabolic coupling of plant exudates to microbial substrate preferences and gain insights into the mechanisms driving rhizosphere microbiome assembly. Specifically, mass spectrometry-based metabolomics (LC-MS) was used to analyze exudation patterns at different developmental stages (and with nutrient limitation) of the annual grass Avena barbata and perennial grass Panicum virgatum. We then constructed media based on the plant exudates and used this media for cultivation of Page 111 of 112

relevant rhizosphere isolates. Substrate-use preferences of these bacteria were determined by comparing exometabolite profiles of the uninoculated vs. media inoculated with bacteria. All the bacteria we analyzed consumed primary plant metabolites, such as amino acids, sugars and nucleosides. However, bacteria that were preferentially stimulated by plant growth and bacteria involved in potential nitrogen stress elimination appear to preferentially use aromatic organic acids. These aromatic acids were largely released by plant during periods of active plant growth and under nitrogen depleted conditions. We suggest that substrate specialization of rhizosphere bacteria, together with changing composition of root exudates during plant development/stress conditions, helps plant recruit beneficial bacteria from soil to mitigate stress.

Full presenter list for the Wild and Tamed Phytobiomes Symposium Kenneth Acosta Beth Gugino Drew May Imtiaz Ahmad Ariel Heminger Marco Mechan Llontop Viridiana Avila Tory Hendry Max Miao Trudi Baker Adam Hoke Zayda Morales Mary Barbercheck Kelli Hoover Norma Morella Gwyn A. Beattie Mia Howard Itumeleng Moroenyane Arnab Bhowmik Alejandra Huerta Jessica Myrick Ari Fina Bintarti Dileepa Jayawardena Andrea Nuzzo

Mary Ann Bruns María del Mar Jiménez-Gasco Oladapo Olayemi Chris Burgess Asher Jones Andrea Ottesen Hugo A. Pantigoso Thomas Butzler Laura Kaminsky Guevara John E. Carlson Prem Kandel James Polashock Lindsay Chamberlain Allison Kerwin Kevin Rice Jedaidah Chilufya Linda Kinkel Sofia Roitman Mara Cloutier Ann Klein Amanda Rosier Gordon Custer George Kowalchuk Joel Sachs Elizabeth Davidson-Lowe Kurt P. Kowalski Dan Schlatter Christine Jade Dilla-Ermita Jasna Kovac Ashley Shade Hanareia Ehau-Taumaunu Gretchen Kuldau Nejc Stopnisek Joanne Emerson Sarah Kyker Brian Tague Bryan Emmett Richard Lankau Ryan Trexler Paul Esker Jesse Lasky Rodrigo Valverde Joana Falcao Salles Sarah Lebeis Jason Wallace Gary Felton Hansol Lee David Weller Mary Firestone Johan Leveau Tammy Wilkinson Brianna Flonc Airong Li Noah Winters Claudia Tatiana Galindo Martinez Ningxiao Li Corlett Wood Karen A. Garrett Fang Liu Etienne Yergeau Page 112 of 112

Sarah Gilbert Keerthi Mandyam Ellen Yerger Leland Glenna Phillip Martin Kateryna Zhalnina Andie Gonzales Samuel Martins Glen Groben Jennifer Martiny