The 9th International of the Microbial Ecology of Plant ...people.oregonstate.edu/~mahaffew/MeetingProgramandAbstracts.pdf · of the Microbial Ecology of ... Ninth International Symposium

The 9th International Symposium of the Microbial Ecology of

Aerial Plant Surfaces

August 15‐18, 2010 Oregon State University, Corvallis, OR

Phyllosphere 2010: Ninth International Symposium of the Microbial Ecology of Aerial Plant Surfaces

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Organizing Committee Convener and local arrangements

Walt Mahaffee USDA Agriculture Research Service, Corvallis, Oregon and Department of Botany and Plant Pathology, Oregon State University

Scientific Program Committee

Maria Brandl USDA Agriculture Research Service, Albany, California

Gwyn A. Beattie Iowa State Univ, Plant Pathology Dept, Ames, Iowa

Barry J. Jacobsen Montana State Univ, Plant Sciences & Plant Pathology Dept, Bozeman, Montana

Johan Leveau Department of Plant Pathology, University California, Davis

Steve Lindow Department of Plant and Microbial Biology, University California, Berkley

Cindy E. Morris INRA, Unite de Pathologie Végétale, Montfavet – France

Vicky Toussaint Agriculture and Agri-Food Canada, Horticulture Research and Development Centre, Quebec, Canada

Julia Vorholt Institute of. Microbiology, Swiss Federal Institutes of Technology in Zurich, Zurich, Switzerland

Special Thanks to: Maya Perez and Carly Weber of OSU Conference Services for web site, site planning,

and registration.


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Table of Contents Maps………………………………………………………………………………………………………………….. 4

Program Schedule……………………………………………………………………………………………… 6

Oral Abstracts (in order of presentation)…………….…………………………………………….. 13

Poster Abstracts………………………………………………..………………………………………………. 63

Directory of Participants……………………………………………………………………………………. 88

Sponsors

United States Department of Agriculture

Agriculture Research Service

Steve Lindow Family Fund

Pacific Gas and Electric

Agriculture and Agriculture et Agri-Food Canada Agroalimentaire Canada

Tuesday’s Wines Contributed by:

Annie Amie Winery

Argyle Winery

Chehalen Winery

Lemelson Winery

Lumos Winery

Soter Vineyards

Willamette Valley Winery

Oregon State University Campus Map

Bloss Hall

Hilton Garden Inn

LaSells Stewart Center

Downtown Corvallis 1 mile

Numerous restaurants, shops

and bars

Avery Park

Weatherford

Hall


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LaSells Stewart Center

Oral Presentations

Meals, Breaks, and posters


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Program Schedule LaSells Stewart Center, Construction and Engineering Hall

Sunday Aug 15 7:00 AM Hosted Breakfast ‐ Giustina Gallery

8:00 AM Meeting opening ‐ Construction and Engineering Hall

Interactions between the phyllosphere and the atmosphere Moderator: Steve Lindow, Department of Plant and Microbial Biology, University California, Berkley

8:15 AM Mapping the world’s VOC emissions

Christine Wiedinmyer

National Center for Atmospheric Research, Boulder, Colorado

8:55 AM Plants as sources of micro‐organisms for clouds – mechanisms and processes

P. Amato1,2,*, Leyronas, C.2, D. Courault1 and C. Morris2

1INRA, UMR1114 EMMAH, Avignon, France; 2INRA, UR407 Plant Pathology Unit, Montfavet, France: * Now at SEESIB – UMR6504 CNRS‐Clermont Université

9:35 AM Epidemic waves and dispersal of phyllosphere microorganisms

Christ Mundt

Oregon State University, Dept. Botany and Plant Pathology

10:15 AM Break ‐ Giustina Gallery10:30 AM Modeling the dispersion and deposition of microorganisms in sparse canopies

Rob Stoll1, Walt Mahaffee2 and Eric Pardyjak1

1Dept Mechanical Engineering, University of Utah, Salt Lake, UT; 2Hort Crops Research Lab, USDA‐ARS and Dept. of Botany and Plant Pathology, Oregon State University, Corvallis, OR

11:15 AM Trichomes and structured epicuticular waxes on plant surfaces – an optimality approach to the prevalent aerosol regimes?

J. Burkhardt, M. Hunsche, S. Pariyar

University of Bonn, Institute of Crop Science and Ressource, Bonn Germany

Ecological processes dependent on the open habitat of the phyllosphere Moderator: Johan Leveau, Department of Plant Pathology, University California, Davis

11:30 AM A bacterial effector manipulates the host phenotype to enhance insect vector reproduction

A. Sugio, H. N. Kingdom, V. M. Nicholls and S. A. Hogenhout

The John Innes Centre, Colney Lane, Norwich Research Park, Norwich, NR4 7UH, UK.


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12:10 PM Hosted lunch ‐ Giustina Gallery1:10 AM An archetypical epiphyte in the hydrosphere: Biogeography of Pseudomonas syringae in

river source waters.

Cindy E. Morris1,2 and David C. Sands2

1INRA, UR407 Pathologie Végétale, Montfavet, France; 2Dept. Plant Sciences and Plant Pathology, Montana State University

1:50 PM Insect vectoring of phyllosphere microbes

J.L. Shipp1, P.G. Kevan2, J.C. Sutton2, J.P. Kapongo1, .S. Al‐Mazra’Awi3, A.B. Broadbent4 & S. Khosla5

1Agriculture and Agri‐Food Canada, Greenhouse and Processing Crops Research Centre, Harrow, Ontario, Canada; 2Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada; 3Biotechnology Department, Al‐balqa’ Applied University, Assalt, Jordan; 4Agriculture and Agri‐Food Canada, Southern Crop Protection and Food Research Centre, London, Ontario, Canada ; 5Ontario Ministry of Agriculture, Food and Rural Affairs, Harrow, Ontario, Canada

2:30 PM The effects of species diversity and propagule density on fungal establishment and coexistence in the phyllosphere

Shannon S. Nix

Dept. of Biology, Clarion University, Clarion, PA

3:10 PM Break ‐ Giustina Gallery3:25 PM Multi‐trophic level interactions on the grass phyllosphere: Fungal endophytes confer

selection for a specific alkaloid toxin‐consuming bacterial microflora

Elizabeth Roberts and Steven Lindow

Department of Plant and Microbial Biology, University of California, Berkeley

4:05 PM Biodegradation and stress in the phyllosphere

Tanja R. Scheublin1, Johan H.J. Leveau1,2

1 Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO‐KNAW), Heteren, The Netherlands

4:20 PM Impact of site and plant species in structuring Methylobacterium communities in the phyllosphere

Claudia Knief, Alban Ramette, Julia Vorholt

Institute of Microbiology, ETH Zurich

4:35 PM Population dynamics of Pseudomas syringae in leaf litter and snow pack in the southern french Alps

Caroline L. Monteil1, Caroline Guiland1, Catherine Glaux1, François Lafolie2 and Cindy E. Morris1

1INRA, Unité de Pathologie Végétale, Domaine St Maurice, Montfavet cedex; 2INRA, Unité Climat‐Sol‐Environnement, Domaine Saint‐Paul, Site Agroparc, 84914 Avignon, France.

4:50 PM Poster Session ‐ Giustina Gallery5:45 PM Break 6:00 PM Board buses in front of LaSells Stewart Center

*Make sure to bring a jacket 6:30 PM Hosted Dinner at Tyee Winery9:00 PM


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Monday 16 August 7:00 AM Hosted Breakfast ‐ Giustina Gallery

Managing Phyllosphere Microorganisms for Plant Health and Food Safety Moderator: Maria Brandl, USDA Agriculture Research Service, Albany, California

8:00 AM Risks of contamination by human pathogens and challenges for food safety

Trevor Suslow

University of California, Davis

8:40 AM Treats and Tricks: Adaptation of Salmonella to the phyllosphere

Yulia Kroupitski,1 Dana Golberg,1 Eduard Belausov,2 Riky Pinto,1 Dvora Swartzberg,3David Granot,3 and Shlomo Sela1

1Microbial Food‐Safety Research Unit, Department of Food Quality & Safety, Institute for Postharvest and Food Sciences,

2 Confocal Microscopy Unit, and 3 Institute for Plant Sciences, Agricultural Research Organization, The Volcani Center, Israel

9:20 AM Interactions of Microbial Pathogens of Insects and Mites with the Phyllosphere

Donald C. Steinkraus

Dept. of Entomology, University of Arkansas, Fayetteville, AR

10:00 AM Break ‐ Giustina Gallery10:15 AM Existence of Salmonella Typhimurium on growing leafy greens as dictated by level of water

contamination, irrigation method and type of produce

Kisluk G, Yaron S.

Faculty of Biotechnology and Food Engineering, Technion‐Israel Institute of Technology, Haifa, Israel

10:30 AM The human pathogen Salmonella Typhimurium inhibits tobacco immune response

Natali Shirron and Sima Yaron

Department of Biotechnology and Food Engineering, Technion, Haifa, Israel

10:45 AM Relevance of the phyllosphere microbiota to E. coli O157:H7 persistence on lettuce

Tom Williams1, Anne‐Laure Moyne1,2, Linda J Harris1,2, and Maria L Marco1

1 Food Science & Technology, University of California, Davis;

2 Western Center for Food Safety, Davis,

California

11:00 AM Leaf Surface Interactions between Phakopsora pachyrhizi, the Soybean Rust Pathogen, and the Mycoparasite Simplicillium lanosoniveum

N. A. Ward, R. W. Schneider, M. C. Aime, and C. L. Robertson

Department of Plant Pathology and Crop Physiology,Louisiana State University Agricultural Center, Baton Rouge, LA

11:15 AM Phylloplane microfungal metabolites induce systemic acquired resistance in Hordeum vulgare var. jagriti against Drechslera graminea

Bulbul Khare, Joyeeta Mitra, V.Bhuvaneswari and P.K.Paul

Amity Institute of Biotechnology; Amity University, Uttar Pradesh, Noida, Uttarpradesh, India

11:30 AM Impact of the in vitro highly effective peptide antibiotic APV found in Pantoea agglomerans 48b/90 on the biocontrol of bacterial plant pathogens

Ulrike Sammer1, Dieter Spiteller2 and Beate Völksch1

Institute of Microbiology, Microbial Phytopathology, Neugasse 25, Jena


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11:45 AM Analysis of the surface properties of wheat spikelet components and their role in colonization by the biocontrol antagonist Cyptococcus flavescens OH 182.9.

Christopher A. Dunlap* and David A. Schisler

Crop Bioprotection Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture

12:00 PM The development and architecture of biofilms formed by the biocontrol agent Burkholderia pyrocinnia FP62 on Geranium.

Patricia Wallace1,Tara Neil2, Bruce Arey3, and Walter Mahaffee2

1Dept. Botany and Plant Pathology, Oregon State University, Corvallis, OR; 2USDA‐ARS Horticulture Crops Research Lab, Corvallis, OR; 3Pacific Northwest National Laboratory‐Environmental Molecular Sciences Laboratory, Richland, WA

12:15 PM Phyllo‐ecology of virginia and california grown tomatoes

Ottesen A. R., Strain E. A., Brown E.A.

Department of Plant Science and Landscape Architecture, University of Maryland, College Park, Maryland

12:30 PM Pick up boxed lunch and board buses1:00 PM Depart for Oregon Coast , includes hosted dinner

*Make sure to bring a comfortable pair of walking shoes and a jacket or sweater 10:00 PM

Tuesday 17 August 7:00 AM Hosted Breakfast ‐ Giustina Gallery

Impact of the plant as a habitat on the phyllosphere microflora and their ecological processes Moderator: Cindy Morris, 1INRA, Unité de Pathologie Végétale, France

8:00 AM Host effects on the composition of the microbial community

Ann E. Stapleton, Peter Balint‐Kurti, Susan J. Simmons, James E. Blum, Carlos L. Ballaré

Department of Biology and Marine Biology, University of North Carolina at Wilmington

8:40 AM Microbial populations of the Tamarix phyllosphere: some like it hot, dry, and saline

Omri M. Finkel1, Adrien Y. Burch2, Steven E. Lindow2, Anton F. Post3 and Shimshon Belkin1

1Department of Plant and Environmental Sciences, Hebrew University of Jerusalem, Edmond J Safra Campus, Givat Ram, Jerusalem, Israel; 2Dept of Plant and Microbial Sciences, University of California‐Berkeley; 3Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biology Laboratory, Woods Hole, MA, USA

9:20 AM Solute permeability of the plant cuticle and its significance for epiphyllic microorganisms

Lukas Schreiber

Department of Ecophysiology, Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, D‐53115 Bonn, Germany

10:00 AM Break ‐ Giustina Gallery


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10:15 AM Influence of the plant as a reservoir of airborne pollutants and quaternary ammonium compounds on phyllosphere microbes

Chiliang Chen, Amarjyoti Sandhu, Dana R. McKeever and Gwyn A. Beattie

Dept of Plant Pathology, Iowa State University, Ames, IA, USA

10:55 AM Genomic diversity of biocontrol strains of Pseudomonas spp. isolated from plant surfaces

Joyce Loper1, Ed Davis1, Kent Lim2, Karl Hassan2, Sasha Tetu2, Neil Wilson2, Sierra Hartney1, Brenda

Shaffer1, Virginia Stockwell1, Marcella Henkels1, Kedy Shen1, Rachel Blumhagen1, Dmitri Mavrodi3, Judith van Mortel4, Chunxu Song4, Diana Radune11, Jessica Hostetler11, Daniel Kluepfel5, Patrick Wechter6, Anne Anderson7, Young Cheol Kim8, Leland Pierson III9, Elizabeth Pierson9, Steve Lindow10, Jos Raaijmakers4, David Weller3, Linda Thomashow3, Andrew Allen11, and Ian Paulsen2

1USDA‐ARS and Oregon State University, USA; 2Macquarie University, Australia; 3USDA‐ARS and Washington State University, USA; 4Wageningen University, The Netherlands; 5USDA‐ARS, Davis, CA, USA; 6USDA‐ARS, Charleston, SC, USA; 7Utah State University, USA; 8Chonnam National University, Korea; 9Texas A&M University, USA; 10University of California at Berkeley, USA; 11J. Craig Venter Institute, USA.

11:10 AM Adaptations of lactic acid bacteria to plants

Henry Low, Thomas Williams, Sybille Tachon, Benjamin Golomb and Maria L Marco

Food Science & Technology, University of California, Davis

11:25 AM Very‐long chain aldehydes promote prepenetration processes of Blumeria graminis in a dose and chain‐length dependent manner.

Anton Hansjakob, Markus Riederer, Ulrich Hildebrandt

University of Wuerzburg, Julius‐von‐Sachs‐Institute for Biosciences, Dept. Botany II

11:40 AM The cusper bioreporter ‐ measuring reproductive success of individual bacterial in simple and complex environments

Mitja N. P. Remus‐Emsermann1 and Johan H. J. Leveau1,2

1NIOO‐KNAW, 6666 GA Heteren, The Netherlands; 2UC Davis, CA, USA

11:55 PM Hosted lunch ‐ Giustina Gallery

1:00 PM Explaining bacterial patterns in the phyllosphere: a modeling approach

Annemieke van der Wal1, Robin Tecon1,2, Jan‐Ulrich Kreft3, Wolf Mooij4, Mitja Remus‐Emsermann1 and Johan Leveau1,2

1NIOO‐KNAW, 6666 GA Heteren, The Netherlands; 2UC Davis, CA, USA; 3University of Birmingham, Birmingham B15 2TT, UK; 4NIOO‐KNAW, AC Nieuwersluis, The Netherlands

1:15 PM Comparative phylogenomics of phyllosphere associated Escherichia coli Isolates

Guillaume MERIC, Elizabeth J. SAGGERS, Tim F. BROCKLEHURST1 Sacha LUCCHINI

Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, United Kingdom

1:30 PM Bacterial communities in the phyllosphere of trees are associated with plant phylogeny

M.R. Lambais1 and D.E. Crowley2

1Department of Soil Science, University of São Paulo, Piracicaba, SP, Brazil. 2Department of Environmental Sciences, University of Cali

1:45 PM Bacterial growth in the apoplast is limited by nutrient availability

María Eugenia RAMOS, Steve LINDOW

Plant and Microbial Biology Department. University of California, Berkeley. USA

2:00 PM Environmental influence on motility in Pseudomonas syringae pv. syringae B728a and implications for plant‐microbe interactions.

Kevin Hockett and Steven Lindow



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2:15 PM Traits contributing to epiphytic and endophytic growth of Pseudomonas syringae revealed by global gene expression analysis

Russell Scott2, Xilan Yu1, Steven Lund1, Jessica Williams3, Steven Lindow2, Dan Nettleton1, Dennis Gross3 and Gwyn Beattie1.

1Department of Plant Pathology, Iowa State University; 2Department of Plant and Microbial Biology, University of California‐Berkeley; 3Department of Plant Pathology and Microbiology, Texas A&M University

2:30 PM Exploring the environment sensed by Pseudomonas syringae in the phyllosphere and the regulons involved in this sensing based on whole genome transcriptional profiling

Xilan Yu1, Russell Scott2, Steven Lund1, Jessica Williams3, Steven Lindow2, Dan Nettleton1, Dennis Gross3 and Gwyn Beattie1.

1Department of Plant Pathology, Iowa State University; 2Dept of Plant and Microbial Sciences, University of California‐Berkeley; 3Dept of Plant Pathology and Microbiology, Texas A&M University

2:45 PM Break ‐ Giustina Gallery3:00 PM Conservative production of biosurfactant in the phyllosphere

Adrien Burch, Briana K. Shimada, Patrick J. Browne and Steven E. Lindow

Department of Plant and Microbial Biology, University of California‐Berkeley

3:15 PM The contributions of swarming motility to Pseudomonas syringae behavior on plants

Juliana Cho, Steven Lindow


3:30 PM An individual‐based approach to surface colonization by bacteria

Robin Tecon1,2, Annemieke van der Wal1, Mitja N. P. Remus‐Emsermann1 and Johan H. J. Leveau1,2

1NIOO‐KNAW, 6666 GA Heteren, The Netherlands; 2UC Davis, CA, USA;

3:45 PM What’s in a Seed?

Mark A. Holland Department of Biology, Salisbury University, Salisbury, MD, USA;

4:15 PM Discussion

5:15 PM Poster session

6:30 PM Hosted Dinner ‐ walk to Avery park

Make sure to bring a jacket


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Wednesday, 18 August 7:00 AM Hosted Breakfast ‐ Giustina Gallery

Deciphering community and cellular processes in the phyllosphere at the microscopic and molecular scale Moderator: Gwyn Beattie, Iowa State Univ, Plant Pathology Dept, Ames, Iowa

8:00 AM Physiology of phyllosphere microbiota as revealed by community proteogenomics

Julia Vorholt

Institute of Microbiology, ETH Zurich, Switzerland

8:40 AM Cross talk among bacterial members of the phyllosphere

Glenn Dulla, Ksenia Krasileva, Beatriz Quinones, and Steve Lindow


9:20 AM Microbe interactions and functions in the phyllosphere as revealed by bioreporters and index organisms

Johan Leveau

Department of Plant Pathology, UC Davis

10:00 AM Break ‐ Giustina Gallery10:15 AM Membrane receptors functioning in the epiphytic and pathogenic interactions in the

phyllosphere

Matthieu Arlat1, Guillaume Dejean1, Alice Boulanger1, Servane Blanvillain‐Baufume1,2,Anne‐Laure Girard3, Martine Lautier1, Claudine Zischek1, Armelle Darasse3, Marie‐Agnès Jacques3 and Emmanuelle Lauber1.

1 Laboratoire des Interactions Plantes‐Microorganismes INRA/CNRSCastanet‐Tolosan, France; 2 Present address: Department of Plant‐Microbe Interactions, Max‐Planck Institute for Plant Breeding Research, Köln, Germany;

3 UMR077 PaVé, INRA, Beaucouzé, France

10: 55AM Variation in the TonB‐dependent outer‐membrane proteins in plant‐associated strains of Pseudomonas fluorescens

Sierra L. Hartney and Joyce E. Loper

Dept. Botany and Plant Pathology and USDA‐ARS, Oregon State University, Corvallis OR, USA.

11:10 AM Perspectives on the next challenges in phyllosphere ecology

Maria Brandl1, Steve Lindow2, Cindy Morris3, Walt Mahaffee4

1 Produce Safety and Microbiology; USDA Agriculture Research Service, Albany, California; 2Dept of Plant and Microbial Sciences, University of California‐Berkeley;3INRA, UR407 Plant Pathology Unit, Montfavet, France; 4USDA‐ARS Horticulture Crops Research Lab, Corvallis, OR

12:30 PM Hosted lunch ‐ Giustina Gallery


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Abstracts

Oral

presentations


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Mapping the world’s VOC emissions: Exchanges of organic compounds between the biosphere and the atmosphere

Christine Wiedinmyer

National Center for Atmospheric Research, Boulder, Colorado

The terrestrial biosphere emits significant amounts of organic compounds to the atmosphere. These compounds include isoprene, monoterpenes, sesquiterpenes, and a host of oxygenated compounds. Global emissions of biogenic volatile organic compounds (VOCs) are far greater than those released from anthropogenic sources. These VOCs contribute to atmospheric processes that control air quality and climate. The variables that determine the type and rate of organic emissions from vegetation include land cover characteristics, such as ecosystem type, vegetation density, and plant species distribution. Environmental conditions, including temperature, solar radiation, and water availability, also exert strong controls over emission rates. Estimates of terrestrial VOC emissions are produced using laboratory and field observations combined with maps of land cover characteristics from ground, areal, and satellite observations. These estimates from vegetation are used in regional and global model applications to explain atmospheric chemistry, including ozone formation and aerosol formation.

This presentation will discuss current practices to measure and quantify emissions of VOCs from vegetation. In the future, as climate, land cover, and anthropogenic emissions change, biogenic VOC emissions may be quite different, leading to large changes in atmospheric chemistry and climate. Land cover disturbances, such as urbanization, insect infestations, and fires, can dramatically alter emissions, resulting in substantial changes in regional chemistry and climate. Large uncertainties associated with the interactions between insects, microbes, and VOC emissions remain.


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Plants as sources of micro‐organisms for clouds – mechanisms and processes

P. Amato1,2,*, Leyronas, C.2, D. Courault1 and C. Morris2

1INRA, UMR1114 EMMAH, Avignon, France 2INRA, UR407 Plant Pathology Unit, Montfavet, France * Now at SEESIB – UMR6504 CNRS‐Clermont Université

The detection of living microorganisms up to several kilometers above the ground has aroused questions about their impact on processes such as the formation and behavior of clouds and the chemical reactivity of the atmosphere. Bacteria and fungi are present at concentration of ~103 to 105 cells mL‐1 in cloud water at ground‐based mid‐altitude sampling sites such as the puy de Dôme summit. Most of the microorganisms isolated from cloud water were found to be capable of transforming atmospheric organic compounds at low temperature, and inferred estimations suggested that microbial activity could be largely involved in the carbon chemistry in clouds. In addition, ice‐nucleation active microorganisms have been isolated from clouds and other aquatic environments and were hypothesized to participate to the water cycle, notably as “rain‐makers”. Since bacteria are in the size range of particles that have the longest life time in the atmosphere (~1 µm), maintaining a metabolic activity in clouds and causing their own redeposition onto the ground with precipitations would constitute a great advantage for disseminating and colonize new environments. A large fraction of those microorganisms isolated from clouds, and from the atmosphere in general, is related to species associated to vegetation, like Pseudomonas spp., Microbacterium phyllosphaerae, Botrytis sp., Fusarium sp…. In an effort to better apprehend and predict the dissemination of microbes and their role in atmospheric processes, documenting the rate at which they are lifted up from plant canopies into the atmosphere remains a need that has to cope with experimental difficulties. Physics theorizes that cells, like other aerosols, are carried up by the sensible heat flux. This has been verified experimentally by measuring fluxes of bacteria emitted from plant canopies along with microclimatic characteristics: maximum positive fluxes were found to evolve during the day in correlation with the sensible heat flux. Thus, numeric tools capable of predicting microclimatic conditions associated to surface characteristics (roughness, dryness, type of vegetation,…) could be directly used to predict the fluxes of bacteria from a surface. However, a critical parameter is missing: the “detachment factor”, i.e. the proportion of cells that are released from a surface (a leave for example) per unit of time. This characterizes the strength of a source in emitting bacteria and this is thought to depend notably on predictable meteorological variables such as wind speed, air humidity or temperature. This “detachment factor” and its dependences to environmental conditions must be determined in order to characterize the strength of sources of airborne microbes in association to surface and microclimatic characteristics. This key parameter is intended to be used for parameterizing coupled surface‐atmosphere models and investigate the impacts of land use on microbial‐mediated atmospheric physico‐chemical processes.


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Epidemic Waves and Dispersal of Phyllosphere Microorganisms.

Chris .C. Mundt.

Department of Botany and Plant Pathology, Oregon State Univ., Corvallis, OR, USA

Biological invasions have traditionally been described as traveling waves with a constant velocity determined jointly by the invading organism’s reproductive capacity and dispersal ability. However, aerially dispersed organisms capable of long‐distance dispersal often have dispersal gradients with extended tails that can result in acceleration of the invasion front. This is likely to be the case for many phyllosphere organisms that are aerially dispersed long distances. We have evaluated empirical disease data with a simple model of spread that incorporates logistic growth in time with an inverse power function for dispersal. The scale invariance of the power law dispersal function implies its applicability at any spatial scale. Indeed, the model successfully described epidemics ranging over six orders of magnitude, from experimental field plots to continental‐scale epidemics of both plant and animal diseases. Regardless of spatial scale, we found that the distance traveled by epidemic fronts approximately doubled per unit time, velocity increased linearly with distance (with a slope of ½), and the exponent of the inverse power law was approximately 2. The scale invariance of the inverse power law also is enabling development of simple scaling relationships between pathogen spread and landscape heterogeneity variables that have been measured in both experimental plots and in natural epidemics at the continental scale. An implication of our results in total is that initial conditions may be the dominant factor influencing subsequent spread of microorganisms, and this conclusion is supported by computer simulation analyses. Results are likely to be similar for saprophytic organisms as well as pathogens.


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Turbulent particle dispersion in sparse plant canopies

Rob Stoll1, Walt Mahaffee2 and Eric Pardyjak1

1Dept Mechanical Engineering, University of Utah, Salt Lake, UT; 2Hort Crops Research Lab, USDA‐ARS and Dept. of Botany and Plant Pathology, Oregon State University, Corvallis, OR

Turbulent dispersion is one of the most important transport mechanisms in the life cycle of some bacteria and most fungi, particularly fungal plant pathogens. Without turbulent dispersion airborne microorganisms would have limited spread beyond leaves adjacent to colonization or infection sites. For plant pathogens this would result in disease epidemics with strong aggregation and limited severity on field or regional basis. Thus, understanding the mechanisms that influence and control dispersion gradients from foci are of primary importance towards understanding epidemic development and improving our ability to prevent and respond to disease outbreaks. In sparse canopy environments, the influence of canopy geometry (row spacing, canopy height, and plant density) on turbulent fluxes results in highly intermittent transport. This intermittency can be problematic for traditional dispersion modeling techniques that rely on assumptions of steady or horizontally homogeneous velocity fields. The different turbulent transport processes that impact particle dispersion and primary particle deposition functions can be examined using high‐resolution 3 dimensional numerical simulations of wind flow and particle dynamics in plant canopies. The link between canopy geometry, turbulent fluxes and particle dispersion gradients in sparse agricultural canopies was explored to identify the length and time scales relevant to fungal pathogen spreading. As a particular case. we studied particle dispersion from point and line sources in grape plant canopies with geometry characteristic of vineyards at the single row, individual crop and regional scales. The concepts used to examine sparse agriculture canopies are also applicable to natural ecosystems with heterogeneous plant canopy structures.


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Trichomes and structured epicuticular waxes on plant surfaces – an optimality approach to the prevalent aerosol regimes?

J. Burkhardt, M. Hunsche, S. Pariyar,

University of Bonn, Institute of Crop Science and Ressource Conservation, Karlrobert‐Kreiten‐Str. 13, D‐53115 Bonn, Germany

The focus of this contribution is the hypothesis that the microstructure of plant surfaces can beinterpreted as an adaptation to aerosol regimes. Global concentrations of fine aerosols (< 2.5μm) have been stable over evolutionary time‐scales. Large ecosystems are known to depend on the nutrient input from aerosols originating from other continents. Deposited atmospheric aerosols on leaf surfaces can benefit both the plant and the phyllosphere by their nutrient content. In addition and due to their hygroscopicity, particles may be donors but also competitors for liquid water.

It has long been known that trichomes are preferential deposition sites for fine aerosols. However, wind tunnel experiments indicate a similar role of structured epicuticular waxes not protruding from the laminar sublayer, which has only been explained recently by a turbophoresis effect. Deposited aerosols can become deliquescent by plant transpiration, which may attract the deposition of additional water soluble gases to the leaf surface. The spatial extension of salts on hydrophobic cuticles can be shown by drying/wetting cycles using an environmental scanning electron microscope (ESEM).

It has been shown that the uptake of nutrients by leaves may happen along the stomatal path which is a contradiction to long established concepts. However, an activation process affecting individual stomata is necessary and may be established by the help of microorganisms and/or dynamical effects of salts.

It has been noted that some plants minimize the accumulation of particles on the leaf surface by extreme hydrophobicity (“Lotus effect”). As many of these plants live in humid environments, this might be a way of preventing the establishment of an extensive phyllosphere by a reduction of nutrients. In drier environments and on poor soils, phyllosphere establishment will be hindered by low humidity, and plant surfaces may represent adaptations to attract fine aerosols and water condensation for subsequent stomatal uptake. While leaf surface micro‐roughness can possibly be interpreted as an optimality approach to nutritional and phyllosphere aspects of aerosols, climate change and the strong increase of aerosol concentrations could make it dysfunctional.


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A bacterial effector manipulates the host phenotype to enhance insect vector reproduction

A. Sugio, H. N. Kingdom, V. M. Nicholls and S. A. Hogenhout

The John Innes Centre, Colney Lane, Norwich Research Park, Norwich, NR4 7UH, UK. Email: [email protected]

The bacterial phytoplasmas induce morphological changes in their plant hosts and affect plant‐insect interactions. We studied transmission of Aster Yellows phytoplasma strain Witches’ Broom (AY‐WB) by the aster leafhopper Macrosteles quadrilineatus. We found that the fecundity of M. quadrilineatus increases by approximately 60% on AY‐WB‐infected Arabidopsis thaliana. In addition, the maize specialist leafhopper Dalbulus maidis, which does not reproduce on healthy Arabidopsis plants, produces nymphs on AY‐WB‐infected plants. In order to identify proteins responsible for the changes in leafhopper interactions with Arabidopsis, the fully sequenced AY‐WB genome (Bai et al., 2006. J. Bacteriology 188: 3682‐3696) was mined for genes encoding secreted proteins. These proteins are candidate virulence factors (effectors) that may manipulate the plant or insect hosts. Fifty‐six secreted AY‐WB proteins (SAPs) were identified and one of these, the effector SAP11, carries a nuclear localization signal and accumulates in plant cell nuclei (Bai et al., 2009. MPMI 22: 18‐30). We found that 35S:SAP11 lines have severely crinkled leaves and produce more stems. In addition, M. quadrilineatus fecundity increases by in average 25% on 35S:SAP11 Arabidopsis lines compared to wild type Col‐0 plants. D. maidis did not produce progeny on the 35S:SAP11 lines indicating that other AY‐WB effectors may enhance susceptibility of Arabidopsis to this leafhopper species. We have also revealed the mechanism of SAP11 action. We found that SAP11 instigates the down regulation of genes required for the Arabidopsis defense response to M. quadrilineatus through its interaction with specific Arabidopsis transcription factors that regulate plant development. In conclusion, we show how vector‐transmitted pathogens can have far‐reaching phenotypic effects on their hosts by manipulating transcriptional and signaling systems in such a way as to enhance vector and pathogen reproductive success.


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An archetypical epiphyte in the hydrosphere: biogeography of pseudomonas syringae in river source waters.

Cindy E. Morris1,2 and David C. Sands2

1INRA, UR407 Pathologie Végétale, Montfavet, France 2Dept. Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT, USA

Over the past few years there has been an explosion of interest in the interaction of air‐borne micro‐organisms with atmospheric processes. This interest has been founded on the unique capacity of a few micro‐organisms such as P. syringae to catalyze the freezing of super‐cooled water at temperatures warmer than those of the numerically dominant mineral‐based ice nucleators in the atmosphere. Furthermore, P. syringae has been detected by several authors in clouds and has recently been shown to be prevalent in niches and substrates associated with the water cycle. As will be illustrated in the presentation at this meeting by P. Amato, the important contribution of the phyllosphere microbial community to the composition of aerosols – both in terms of numbers and functional groups – is that it opens novel research questions on a range of impacts that plant canopies can have on climate processes.

In light on the potential link that P. syringae has with the water cycle – as a participant and possibly as a critical player – we explored its biogeography as if it were principally an aquatic bacterium rather than an epiphyte. From a study of the biogeographical relationships of P. syringae in river headwaters upstream from agricultural regions on three continents, we have obtained unique insight into the evolutionary history of this bacterium. A collection of 236 strains from 11 sites in the USA, in France and in New Zealand was characterized for genetic diversity based on housekeeping gene sequences and for phenotypic diversity based on pathogenicity and ice nucleation activity. Water harbored several new genetic clades not previously observed among strains from crops or wild plants. Populations on all continents were dominated by a few haplotypes and hence population structure was not significantly influenced by geographic location. However up to 70% of the haplotypes in the populations on each continent were unique to each site suggesting a role for endemism. Comparison with 87 strains from crops revealed that the metapopulation of P. syringae is structured into three genetic ecotypes: a crop‐specific type, a water‐specific type, and an abundant ecotype found in both habitats. Aggressiveness of strains was significantly and positively correlated with ice nucleation activity. Furthermore, the ubiquitous genotypes (found in all substrates investigated in this and other studies) were the most aggressive on average. This work reveals that there is considerable exchange of populations between freshwater and agricultural habitats and that the former contribute considerably to the diversification of P. syringae. Furthermore, given the overall abundance and genetic diversity of P. syringae outside of its association with diseased plants, it could be argued that pathogenicity per se has a relatively minor role in the fitness of this species and hence in its overall life history.


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Insect Vectoring of Phyllosphere Microbes

J.L. SHIPP1, P.G. KEVAN2, J.C. SUTTON2, J.P. KAPONGO1, M.S. AL‐MAZRA’AWI3, A.B. BROADBENT4 & S. KHOSLA5

1Agriculture and Agri‐Food Canada, Greenhouse and Processing Crops Research Centre, Harrow, Ontario, Canada N0R 1G0, [email protected]; 2Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1, [email protected], [email protected]; 3Biotechnology Department, Al‐balqa’ Applied University, Assalt, Jordan 19117, [email protected]; 4Agriculture and Agri‐Food Canada, Southern Crop Protection and Food Research Centre, London, Ontario, Canada N5V 4T3, [email protected]; 5Ontario Ministry of Agriculture, Food and Rural Affairs, Harrow, Ontario, Canada N0R 1G0, [email protected]

Microbial control agents are traditionally applied to the phyllosphere of crops for arthropod pest control using chemical spray technology. Over the last 10 – 15 years, pollinator bees have been investigated as vectors for microbial control agents (fungi, viruses and bacteria). The initial studies were conducted using honey bees to delivery microbial control agents to the fruit and leaves of small berry crops for control of plant diseases, such as grey mould. The approach was expanded to bumble bees and honey bees for insect pest control. This presentation reviews the development of the Pollinator Biocontrol Vector Technology and how pollination of crops can be combined with arthropod pest management. This technology can potentially be used now for simultaneous pest control and plant disease suppression and extrapolated for use with other insects.


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The effects of species diversity and propagule density on fungal establishment and coexistence in the phyllosphere

Shannon S. Nix

Dept. of Biology, Clarion University, Clarion, PA

Establishment and coexistence in multi‐species communities is the result of numerous complex factors of which competitive interactions often play a significant role. In extreme and variable environments such as the phylloplane, interspecific interactions between inhabitants that result in differential mortality have long been suspected as a likely mechanism for the patterns of species density and diversity reported in both in vitro and in vivo studies. Yet, in order to account for the extent of microbial diversity and abundance measured, differential survival strategies would also have had to evolve that would allow phylloplane microbes to establish and coexist in spite of high environmental and competition pressures. In an effort to explain the ecology of phylloplane microbes, some have attempted to describe the life history of these organisms using three primary survival strategies: a ruderal strategy (R‐selection), a competitive strategy (C‐selection), and a stress‐tolerant strategy (S‐selection). It has been proposed that phylloplane fungi are essentially S‐strategists that endure multiple, sustained environmental stresses (temporally and spatially), however, it is unlikely that any successful group would rely on a single strategy for survival. In the case of habitat‐adapted, stress‐tolerant phylloplane fungi, there may be primarily S‐strategists that have R or C characteristics as well as primarily R or C strategists with underlying S capabilities. Therefore, the key to the establishment and coexistence of phylloplane fungal species may lie in the ability of these organisms to employ multiple resource allocation and reproductive strategies. While these strategies may provide a measure of predictive value for successful establishment in the phylloplane environment, the extent to which the members of the phylloplane influence each other’s ecology is not directly assessed or accounted for. In an attempt to begin to fill this gap in knowledge, the community‐level attributes of species diversity and propagule abundance will be used to illustrate the impact that competitive interactions have on the ability of select phylloplane fungi to establish and coexist in in vitro model systems.


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Multi‐trophic level interactions on the grass phyllosphere: Fungal endophytes confer selection for a specific alkaloid toxin‐consuming bacterial microflora Elizabeth Roberts and Steven Lindow Department of Plant and Microbial Biology, University of California, Berkeley 94720

Neotyphodium endophytes are asymptomatic fungal colonizers of the aerial tissues of cool season grasses, and have a mutualistic relationship with their hosts which is in part supported by the production of loline (saturated 1‐aminopyrrolizidine) alkaloids. While lolines are known for inhibiting herbivory by insects, we show that N‐formylloline on the surface of tall fescue (Festuca arundinacea) and meadow fescue (Festuca pratensis) leaves selects for a unique epiphytic bacterial microflora. Population sizes achieved by a loline‐catabolizing Burkholderia sp., are about 10‐fold higher on fescues infected with a loline‐producing Neotyphodium strain than on loline‐free plants. Likewise, a Burkholderia mutant incapable of using loline as a sole Carbon and Nitrogen source attained similar population sizes when inoculated onto loline‐containing and loline‐free plants and which were about 10‐fold lower than that achieved by the WT loline‐catabolizing strain. Additionally, Burkholderia sp. constitutes a large proportion (>70%) of the culturable tall fescue phyllosphere bacterial community while they are rare or absent from the surfaces of plant species which are not infected with Neotyphodium endophytes. Terminal restriction fragment length polymorphisms of loline enrichment cultures inoculated with bacteria recovered from tall fescue revealed a simple community of strains capable of using loline as a sole carbon source. While less than 6% of the bacterial taxa recovered from tall fescue could consume lolines, they comprised over 70% of the total population size. These results indicate that loline catabolism is specialized amongst bacterial colonists of loline‐producing grass‐fungal mutualists and that loline derivatives are the major source of carbon and energy on such plants. Therefore, fungi play a role in determining the composition of the bacterial community in the phyllosphere of Neotyphodium‐infected grasses. The selectivity of loline catabolism might be exploited to establish particular bacteria on plants for beneficial purposes.


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Biodegradation and stress in the phyllosphere

Tanja R. Scheublin1, Johan H.J. Leveau1,2 1 Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO‐KNAW), Heteren, The Netherlands; 2 Department of Plant Pathology, University of California, Davis, USA

In soil, aquatic and marine environments, bacterial degradation of organic pollutants has been well studied. However, via air particulate deposition or volatile absorption, these pollutants can also accumulate on or in the waxy cuticle layer of plant leaves, where they become accessible for degradation by bacteria that colonise the phyllosphere. The phyllosphere is an environment that evokes high levels of bacterial stress due to rapid changes in humidity, temperature and solar radiation. In this project, we investigated how bacteria cope with phyllosphere stress in relation to their catabolic activity towards pollutants. For this, we used two different approaches. Firstly, we tested the phyllosphere performance of several model strains with different catabolic capabilities. These included 4‐chlorophenol degrading strain Arthrobacter chlorophenolicus A6, toluene degrading strain Pseudomonas putida KT2440 and dioxin degrading strain Sphingomonas wittichii RW1. Of these, only strain A6 exhibited a phyllosphere fitness that was comparable to our control bacterium, leaf coloniser Erwinia herbicola 299R. This finding is surprising given that A. chlorophenolicus A6 was originally isolated from soil, while Pseudomonas and Sphingomonas species have been reported to occur on leaf surfaces more commonly than Arthrobacter species. Ongoing studies are exploiting the available genomic resources for A. chlorophenolicus A6 to obtain a transcriptional profile of this strain under phyllosphere conditions and expose any hardwiring between genes involved in stress resistance and pollutant degradation. Secondly, we isolated pollutant‐degrading bacteria from two phyllosphere environments in The Netherlands: one was an apple orchard that was treated with the foliar pesticide triadimenol, the other a vegetated shoulder along a major highway. Enrichment cultures were checked for growth on triadimenol and 4‐chlorophenol (a degradation product of triadimenol), and toluene and benzene, which are common traffic‐related pollutants. Several 4‐chlorophenol degrading strains were recovered from the apple orchard phyllosphere. These strains had three different 16S rRNA sequence identities and were all confirmed to belong to the genus Arthrobacter. Several toluene degrading phyllosphere bacteria from near the highway were identified as Rhodococcus sp.. Phyllosphere fitness and stress resistance were variable between isolates. One of the Arthrobacter isolates was an excellent leaf coloniser that reached up till ten times higher numbers than A. chlorophenolicus A6 or E. herbicola 299R in standardised phyllosphere tests that included drying stress in the form of low air humidity. The presence of a competing strain did not negatively affect phyllosphere performance of this isolate. We are currently investigating the influence of 4‐chlorophenol on phyllosphere performance of Arthrobacter strains in order to find out whether the presence of pollutants can improve phyllosphere fitness and competitiveness of pollutant degrading bacteria.


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Impact of site and plant species in structuring Methylobacterium communities in the phyllosphere

Claudia Knief, Alban Ramette, Julia Vorholt

Institute of Microbiology, ETH Zurich

Members of the genus Methylobacterium are omnipresent colonizers of plant leaves. Different factors will affect the dispersal of these bacteria on plants. In order to evaluate the importance of plant species, site and time on the Methylobacterium community composition in the phyllosphere, samples were collected from Arabidopsis thaliana and different adjacently grown plant species at five different sampling sites in two consecutive years. These samples were analyzed by cultivation‐independent Methylobacterium‐specific automated ribosomal intergenic spacer analysis (ARISA). A variation partitioning analysis based on the obtained ARISA patterns revealed that sampling site and plant species explained 50% of the variation seen in the Methylobacterium communities of the collected sampling material. The site at which a plant grew had a stronger impact on the Methylobacterium community composition than the plant species. Differences in the community composition on plants grown at different sites were mainly due to the presence/absence of distinct ARISA peaks, while the plant species specific differences seen within individual sites were a consequence of variation in the abundance of ARISA peaks that were detected on diverse plant species at that site. 16S rRNA gene sequence analysis of representative Methylobacterium isolates, which were obtained from the sampling material, revealed that the studied plants were colonized by 23 different genotypes, of which 10 were found at more than half of the sampling sites. Taken together, these data suggest that plant colonization is not highly host‐plant specific. Plants are rather colonized by strains of those Methylobacterium species that are present at the respective site where the plant is growing.


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Population dynamics of Pseudomonas syringae in leaf litter and snow pack in the southern French alps.

Caroline L. MONTEIL1, Caroline GUILBAUD1, Catherine GLAUX1, François LAFOLIE2 and Cindy E. MORRIS1

1‐INRA, Unité de Pathologie Végétale, Domaine St Maurice, BP. 94, 84140 Montfavet cedex, France; 2‐INRA, Unité Climat‐Sol‐Environnement, Domaine Saint‐Paul, Site Agroparc, 84914 Avignon, France.

The recent revelation that the life cycle of the plant pathogen Pseudomonas syringae is closely associated with the water cycle raises novel questions about the role of non agricultural niches of plant pathogens in disease epidemiology. In addition to cultivated plants, P. syringae is frequently found in rain and cloud water, in alpine rivers, in wild plants and in annual snow pack. In temperate zones, snow pack is the major source of river water during the season of crop growth. Prior to its melting, snow covers the remains of alpine vegetation (both litter and senescent plants) and insulates the soil from sub‐zero temperatures. Thus this environment could play an important role in the survival and diversification of P. syringae. In this light, our objective is to elucidate the dynamics of population size and structure for P. syringae in leaf litter and the covering snow pack in the Southern French Alps. We estimated abundance and genotypic diversity (rep‐PCR) of P. syringae in the leaf litter and the snow cover at four sites in the Southern French Alps during two months in 2010. At each site we also sampled freshly‐fallen snow to determine the sizes of incoming populations. P. syringae was detected in snow pack and the covered plant material (“leaf litter”) regularly at all sites during the sampling period, and occasionally in precipitations. Abundance and population structure was stratified from the leaf litter to the top of the snow pack. The whole of our field and microcosm studies at these sites indicates that in the winter, P. syringae arrives in the catchment with the first snow falls. Strains from early snow falls constitute a part of the populations in the 10 cm‐layer of snow pack in contact with the soil. The other part of this population is composed of strains from the leaf litter. Rapidly after the early snow falls, bacteria from leaf litter emigrate into the snow pack where they survive, multiply and die during the winter as a function of organic matter availability, humidity, the low conductivities and the temperature (always 0°C in the snow cover). To better understand how selection pressures influence the population structure of P. syringae, phenotypic (ice nucleation activity, production of toxins, and pathogenicity) and genetic (housekeeping gene sequencing) characterization is in progress.


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Risks of contamination by human pathogens and challenges for food safety

Trevor Suslow

University of California, Davis


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Treats and Tricks: Adaptation of Salmonella to the phyllosphere

Yulia Kroupitski,1 Dana Golberg,1 Eduard Belausov,2 Riky Pinto,1 Dvora Swartzberg,3 David Granot,3 and Shlomo Sela1 1Microbial Food‐Safety Research Unit, Department of Food Quality & Safety, Institute for Postharvest and Food Sciences, 2 Confocal Microscopy Unit, and 3 Institute for Plant Sciences, Agricultural Research Organization, The Volcani Center, P.O. Box 6, Beth‐Dagan 50250, Israel

Outbreaks related to consumption of contaminated fresh produce have been increasingly reported and result in high morbidity and substantial economical losses. Plants might become contaminated in the field through the use of contaminated irrigation water, or the application of animal manures for fertilization. Contamination can also occur at various postharvest stages, including processing and packaging. Human pathogens can colonize the phyllosphere at sites inaccessible to sanitizers, which may lead to contamination of the produce. The fresh‐produce industry currently lacks an efficient control mean to assure complete removal or killing of foodborne pathogens in fresh or minimally processed fruits and vegetables. Therefore, gaining knowledge on the contamination routes and the interplay between foodborne pathogens and plant tissues is essential in order to develop new intervention strategies for assuring the safety of fresh produce.

To investigate Salmonella interactions with leafy vegetables, iceberg lettuce leaves were experimentally inoculated with green‐fluorescent protein‐tagged Salmonella enterica serovars Typhimurium and bacterial colonization was assessed using confocal microscopy. Inoculation under light conditions resulted in aggregation of bacteria near open stomata and invasion into the inner leaf tissue. In contrast, incubation in the dark resulted in scattered attachment pattern and very poor stomatal internalization. Forcing stomatal opening in the dark by fusicoccin had no significant effect on Salmonella internalization. These results imply that the pathogen is attracted to nutrients produce de‐novo by photosynthetically active cells in internal leaf tissues. To examine this hypothesis, we have tested the internalization of Salmonella mutants deficient in motility and chemotaxis. Indeed, entry of these mutants into stomata was severely affected. Furthermore, pre‐incubation of Salmonella with sucrose, glucose and fructose also inhibited bacterial internalization. Taken together, these findings imply that entry of Salmonella into lettuce stomata involves chemotaxis toward chemo‐attractants present in the plant's apoplast and suggest that either Salmonella antigens are not well recognized by the stomatal‐based innate immunity, or that this pathogen has evolved means to evade it. Internalization of leaves may provide a partial explanation for the failure of sanitizers to efficiently eradicate foodborne pathogens in leafy greens.


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Interactions of Microbial Pathogens of Insects and Mites with the Phyllosphere

Donald C. Steinkraus

Professor of Entomology, Dept. of Entomology, University of Arkansas, Fayetteville, AR 72701

Insects and mites have major beneficial and negative effects on plants as herbivores and pollinators, and as predators and parasitoids of the herbivores and pollinators. Insect and mite microbial pathogens are extremely important agents affecting the abundance of pest and beneficial arthropods. Microorganisms infecting arthropods include viruses, bacteria, fungi, protozoa, and nematodes. Many of these microorganisms are affected by the phyllosphere either positively or negatively. While a substantial body of knowledge indicates that plants are able to use predatory and parasitic insects as “bodyguards” to protect themselves from herbivores, more recently evidence is indicating that plants may also utilize entomopathogens as “bodyguards”. The phyllosphere has major effects on entomopathogen survival and infectivity through physical, microbial, chemical and other mechanisms. Very little research has been conducted on the interactions of microorganisms of the phyllosphere with entomopathogenic microbes. Clearly the interactions of entomopathogenic microbes with the phyllosphere is an understudied area that deserves more attention.


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Existence of Salmonella Typhimurium on growing leafy greens as dictated by level of water contamination, irrigation method and type of produce

Kisluk G, Yaron S.

Faculty of Biotechnology and Food Engineering, Technion‐Israel Institute of Technology, Haifa 32000, Israel

Fresh produce has been documented as a vehicle of foodborne pathogens and as such it is the subject of increased concerns. Contaminated irrigation water may be a source of leafy greens contamination via roots and aerial tissues. The purpose of this study was to evaluate the impact of common irrigation regimes with water containing different Salmonella concentrations on persistence of Salmonella enterica serovar Typhimurium on parsley and basil. Parsley and basil plants were subjected to spray and drip irrigation during days or nights with water containing 4 to 8 Log CFU ml‐1 of Salmonella and its abundance on leaves, stems, roots and in soil was determined. Results revealed that existence of S. Typhimurium on plants during preharvest contamination significantly depends on the type of produce, method of irrigation and the time irrigation is applied. While S. Typhimurium was abundant in all parts of spray and drip irrigated parsley, it was not detected in basil leaves following drip irrigation with water containing less then 8 Log CFU ml‐1 Salmonella. Conversely, survival of the pathogen on basil aerial parts was high when applied by spray irrigation. The pathogen was detected on basil leaves at levels of 2.3 Log CFU g‐1 following irrigation by water containing 5 Log CFU ml‐1 Salmonella. Still, spray irrigations of basil and parsley indicated that bacterial counts were up to 3.5 Log CFU g‐1 higher on parsley aerial parts and roots. Basil drip vs. spray irrigation reveals that bacterial survival in soil and the subsequent adhesion to roots were up to 2.5 Log CFU g‐1 higher when applied by drip irrigation. However, uptake of bacteria from the soil via roots and the mobilization of the pathogen to edible portions of the plant were heavily hindered. When basil was spray irrigated at night, the pathogen survival on aerial parts and the adhesion to roots were up to 2 Log CFU g‐1 higher in comparison to irrigations at day times, whereas bacterial survival on parsley did not differ between day and night irrigations. Studies are currently aimed at determining the causes of these significant differences. The findings provided by this research may lead to a reassessment of current field methodologies and ultimately affect postharvest applications and interventions in order to improve microbiological safety of fresh ready to eat leafy greens.


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The human pathogen Salmonella Typhimurium inhibits tobacco immune response

Natali Shirron and Sima Yaron

Department of Biotechnology and Food Engineering, Technion, Haifa, Israel

The persistence of human pathogenic bacteria on plants has been studied extensively in recent years, mainly due to the potential hazard of human pathogens being able to invade plants without causing disease symptoms. Salmonella spp. survive and grow on plants and can exceed levels of contamination that may cause disease. Although plant pathogens and human pathogens share many similarities in their virulence determinates, the colonization in plants requires different strategies. Recent evidence has pointed the way to specific host‐bacteria interactions between plants and Salmonella enterica. Researches have demonstrated Salmonella’s ability to penetrate the roots, transfer to other parts, and survive for a long time in/on the plant. In line with the recent evidence of Salmonella persistent on plants, we were interested in testing the possibility of plants being a true host for Salmonella and whether Salmonella affects plant physiology and triggers the immune response. The plant immune system functions at different levels during pathogen attack. The entry of a bacteria into the plant is followed by the recognition of a pathogen‐associated molecular patterns (PAMPs), which activate the hypersensitive response (HR), used by plants, to prevent the spread of infection. To explore the early steps of immune response of plants to Salmonella, we conducted a detailed analysis on the ability of Salmonella to elicit or evade immune response by the plant. We tested HR both by testing oxidative burst and pH changes in Nicotiana tabacum cells suspensions and in tobacco plants, following triggering the plant with Salmonella enterica serotype Typhimurium. Controls were the nonpathogenic bacterium Pseudomonas fluorescens and the pathogenic incompatible bacterium Pseudomonas syringae. Interestingly, we found that Salmonella actively inhibits pH changes and the production of an oxidative burst by the plant. This inhibition is contact dependant and most likely depends on secreted factors, since a stronger response was observed with heat killed bacteria, with chloramphenicol treated Salmonella or with purified LPS. Since invA mutant also abolished the ability of Salmonella to suppress HR, we demonstrated that a functional SPI‐1 TTSS is required for the alteration of HR response. Moreover, although salmonella does not promote disease symptoms in the plant it causes significant cell death in N. tabacum cells suspension after only two hours of co‐incubation. Based on these results we suggest that plant colonization by S. typhimurium is indeed an active process. Salmonella's ability to escape HR which usually enables the plant to recognize and response to the entry of a wide range of bacteria, plus its ability to cause cell death on a cell suspension model, gives directions to the establishment and the characterization of a novel type of interaction between Salmonella and plants.


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Relevance of the phyllosphere microbiota to E. coli O157:H7 persistence on lettuce

Tom Williams1, Anne‐Laure Moyne1,2, Linda J Harris1,2, and Maria L Marco1

1 Food Science & Technology, University of California, Davis, 2 Western Center for Food Safety, Davis, California

Outbreaks of enterohemorrhagic Escherichia coli O157:H7 (EcO157:H7) illness have been increasingly associated with the consumption of leafy green vegetables. Preharvest contamination of plants in the field is likely the main point of entry of the pathogen into lettuce supply chains. Although E. coli O157:H7 is generally not a good colonist of plants, it is able to survive and persist in low amounts on lettuce in the field (Harris et al). This project aims to identify the contributions of the indigenous phyllosphere microbiota to the survival and persistence of EcO157:H7 on lettuce. In 2009, two field trials (spring and fall) were performed whereby an attenuated, rifampicin‐resistant, non‐pathogenic strain of EcO157:H7 (Stx I and II negative) was applied onto young Romaine lettuce plants in the Salinas Valley which were exposed to either overhead (sprinkler) or ground (drip) irrigation. Lettuce plants were then collected weekly for the following month to enumerate the culturable amounts of the inoculated strain and indigenous bacteria associated with the lettuce. Levels of viable EcO157:H7 diminished rapidly upon inoculation and this organism was found by selective‐enrichment methods in only a fraction of the inoculated plants within 7 d after the start of the experiment (Harris et al). In the spring field trial, the amounts of culturable phyllosphere microbiota ranged from 3.8 to 5.5 log10 cfu/g plant on tryptic soy agar and differed significantly as a function of sampling time, irrigation method, and prior inoculation of EcO157:H7. In comparison, higher amounts of bacteria were cultivated from the plant surfaces (between 5.5 to 6.5 log10 cfu/g) in the fall field trial and no significant differences were found between the plant treatment groups. Notably, the total culturable population sizes of bacteria were directly (fall) and inversely (spring) correlated with the detection of viable EcO157:H7 by enrichment on the same plants, thereby indicating potential relationships between the total amounts of bacteria and persistence of EcO157:H7 in the phyllosphere. In addition, direct antagonism against the pathogen might occur on plants in the field as was shown for a collection of >20 bacterial isolates from the lettuce which inhibited the growth of attenuated and pathogenic EcO157:H7 variants in laboratory culture medium. To identify diversity and abundance of the indigenous bacteria associated with lettuce, high‐throughput,16S rRNA‐based, culture‐independent approaches are being applied including pyrosequencing (454 Life Sciences) and DNA microarrays (Phylochips). These analyses are demonstrating that only a small fraction of the lettuce phyllosphere bacterial communities is typically cultivated on standard laboratory media. These data are assisting efforts to determine whether specific phyllosphere associated species and/or communities of bacteria are directly and/or indirectly correlated to E. coli O157:H7 persistence on lettuce. The results are intended to assist the leafy greens industry with developing agricultural practices aimed at reducing the risks of outbreaks associated with these economically valuable and otherwise healthy crops.


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Leaf Surface Interactions between Phakopsora pachyrhizi, the Soybean Rust Pathogen, and the Mycoparasite Simplicillium lanosoniveum N. A. WARD, R. W. Schneider, M. C. Aime, and C. L. Robertson Department of Plant Pathology and Crop Physiology, Louisiana State University Agricultural Center, Baton Rouge, LA 70803

Phakopsora pachyrhizi, causal agent of Asian soybean rust, has been documented to cause yield losses of up to 100% in subtropical soybeans. In 2007, an apparent mycoparasitic fungus was observed colonizing urediniospores within rust sori. Scanning electron microscope observations revealed intense colonization of uredinia and apparent trophic growth of hyphae of an unknown fungus from uredinium to uredinium and between urediniospores within a sorus. The fungus was recovered, single‐spored, and identified as Simplicillium lanosoniveum using morphological and molecular techniques. Inoculation onto field‐grown rust‐infected soybean leaves resulted in a significant reduction in production of hyaline urediniospores within 7 days of inoculation. The fungus colonized rust sori within 10 days, but it failed to establish on noninfected leaf surfaces. Colonized spores turned dark brown and did not germinate. Immunofluorescence microscopic observations indicated that S. lanosoniveum penetrated and colonized urediniospores within 5 days of inoculation. By staining colonized urediniospores with Calcofluor White and an antiserum specific to P. pachyrhizi, we observed hyphae of the mycoparasite entering and coiling within urediniospores. These findings indicate that S. lanosoniveum is a mycoparasite of urediniospores and not simply an inhabitant of sori. To evaluate S. lanosoniveum as a biological control agent, we conducted field tests in 2009 near Baton Rouge, LA. Treatments included foliar applications of conidia of S. lanosoniveum at various plant growth stages and rust severities. Leaf samples were collected weekly from late vegetative stages through senescence and were rated for disease severity and total number of sori. Next, we subjected total leaf genomic DNA, including all associated microorganisms, to qPCR analyses in order to quantify S. lanosoniveum and P. pachyrhizi. We found that S. lanosoniveum colonized and survived on leaf surfaces for 6 weeks after the earliest inoculation. This led to delayed disease development and a reduction in numbers of sori. We conclude that S. lanosoniveum has mycoparasitic properties that should be exploited for biological control of soybean rust and possibly other rust diseases


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Phylloplane microfungal metabolites induce systemic acquired resistance in Hordeum vulgare var. jagriti against Drechslera graminea.

Bulbul Khare, Joyeeta Mitra, V.Bhuvaneswari and P.K.Paul,

Amity Institute of Biotechnology; Amity University, Uttar Pradesh, Sec‐125, Express highway , Noida, Uttarpradesh‐201303, India.

Phylloplane microbes have been shown to interact with plants in many ways to affect its growth and development and protecting plants against pathogens. Such information has been utilized to develop a number of crop protection strategies using microbes as a biocontrol agent. In vitro studies have shown that microbial metabolites are inhibitory to many plant pathogens. However in both these strategies the biocontrol agent or its metabolites are pathogen specific. The target is to prevent the pathogen from infecting the host. However a more suitable strategy would be to induce hosts intrinsic defence mechanism so that the protection is broad spectrum against a wide range of pathogens. In the present study an effort was made to understand the impact of phylloplane mycoflora metabolites on expression and activity of two key defence enzymes and their ability to induce defence responses against Dreschlera graminea. Phylloplane mycoflora was isolated from pathogen infected and uninfected leaves. Four dominant microfungi (Aspergillus niger; A sydowii; Alternaria altenata; Trichoderma viridae) from uninfected leaves were selected for study. The microfungi were grown in liquid medium for metabolite production at 25 ±1°C for 7 days. The supernatant after centrifugation was used for treatment of plants. Barley plants were raised and maintained in sterlised soil rite in sterlised trays in a culture room with a temperature of 25°C, 75 % RH and 12L/12D light cycle. Three week old plants were used for the study. The plants were divided into groups and sprayed either with a dilution series of each microfungal metabolite. Control plants were sprayed with sterlised distilled water. Each group of plants was divided into three subgroups. Subgroup 1 plants were inoculated with spores of the pathogen (105 spores /ml) a week before spraying of metabolite; group 2 was inoculated simultaneously with metabolites and group 3, a week after metabolite treatment. Plants were also treated with combination of extracts. Activity of enzymes peroxidase and polyphenol oxidase and concentration of total phenols were estimated from all samples. Isoenzyme profiles were also analysed from each sample. The disease incidence was recorded from all samples 3‐weeks post pathogen inoculation. Result showed an enhancement of activity of both enzymes in samples treated with undiluted metabolites. The other two dilutions did induce an increase in activity but not as significant as by undiluted metabolite. Enhancement in activity was observed in plants treated with a combination of metabolites. The disease incidence on newly emerged leaves was lowest in metabolite mixture treated plants when pathogen inoculation was preceded by metabolite treatment. The metabolites also influence expression of genes as evidenced from variation in isoenzyme profiles. Though all the four metabolites could induce protection against the pathogen, there were significant variations in the patterns of isoenzyme expression and activities of the two defense enzymes. The results indicate that phylloplane microfungal metabolites can induce SAR in barley. Thus, the conservation and enhancement of population of such combination of microfungi could be a suitable strategy for disease control. Also such fungal metabolites could potentially be caused for inducing SAR in crop plants.


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Impact of the in vitro highly effective peptide antibiotic APV found in Pantoea agglomerans 48b/90 on the biocontrol of bacterial plant pathogens

Ulrike Sammer1, Dieter Spiteller2 and Beate Völksch1

1Institute of Microbiology, Microbial Phytopathology, Neugasse 25, D‐07743 Jena; 2Max Planck Institute for Chemical Ecology, Hans‐Knöll‐Str. 8, D‐07745 Jena; email: Ulrike.Sammer@uni‐jena.de

The strain Pantoea agglomerans 48b/90 (Pa48b) was isolated from a soybean leave. It attracted our attention because of its growth inhibition activity against Pseudomonas syringae pv. glycinea in vitro and in planta. Additional tests showed its ability to suppress also other important bacterial plant pathogens (i.e. Erwinia amylovora, Pseudomonas syringae pathovars, Agrobacterium tumefaciens) and the human pathogen Candida albicans.

This antibiotic compound produced by Pa48b was identified as 2‐amino‐3‐(oxirane‐2,3‐dicarboxamido)‐propanoyl‐valine (APV) and was first described for this species. Three out of 20 tested strains which all show an antibiotic activity produce APV like Pa48b; strain C9‐1 which is sold as commercial biocontrol agent, too. Closely related molecules were described for Streptomyces collinus and Micromonospora sp. strains.

In contrast to other well‐known Pantoea antibiotics the growth‐inhibiting effect of APV is not compensated by amino acids or casein hydrolysate. Only supplementing the media with N‐acetylglucosamine caused APV to become inactive.

Interestingly, the formation of APV in vitro is growth associated and strongly temperature dependent: its optimal production rate is between 8 °C and 12 °C. Therefore, we conducted plant experiments at moderate and low temperatures. We could show that Pa48b and its APV‐negative mutant establish stable populations in a wide temperature range on apple blossoms and on soybean leaves. The coinoculation of the APV‐negative mutant with the pathogens should highlight the role of APV within the biocontrol of the bacterial blight pathogen Pseudomonas syringae pv. glycinea and the fireblight pathogen Erwinia amylovora.

Surprisingly, independent from the temperature the difference in suppression of the symptoms between wildtyp and mutant was minimal. In coinoculation with Pa48b or its APV‐negative mutant the population size of Pseudomonas syringae pv. glycinea was 10‐fold lower at low temperatures and at least 2 orders of magnitude lower at moderate temperatures. Furthermore, the population size of Erwinia amylovora was decreased by 2 to 4 orders of magnitude in the coinoculation with Pa48b and its APV‐negative mutant, respectively. Consequently, other factors have to play a part in these in planta interactions.


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Analysis of the surface properties of wheat spikelet components and their role in colonization by the biocontrol antagonist Cyptococcus flavescens OH 182.9. Christopher A. Dunlap* and David A. Schisler Crop Bioprotection Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, United States Department of Agriculture, 1815 North University Street, Peoria, Illinois 61604, USA *corresponding author: PH: (309)861‐6339, email: [email protected]

Cyptococcus flavescens OH 182.9 (NRRL Y‐30216) is a biocontrol antagonist which has been shown to be effective in managing Fusarium head blight in wheat. C. flavescens works by colonizing the wheat spikelet and competing with potential pathogens for the limited resources available. Knowledge of the parameters that influence microbial colonization of the wheat spikelet is important in developing successful control strategies. The surface of the aerial parts of wheat heads are covered by a waxy cuticle which mediates the interactions between the plant and the environment. The chemical and physical properties of the plant surface determine the nature of these interactions. The physicochemical surface properties of wheat spikelet components were determined using contact angle methods. The contact angles of several solvents were measured for the glumes and lemmas of two wheat cultivars. The results demonstrate that the surface chemistry and ultrastructure of glume and lemma tissues changes around the time of anthesis for both of the wheat cultivars tested, with the structures reaching a minimum in hydrophobicity immediately after flowering. These results are reported in conjunction with microbial colonization data of C. flavescens to understand the role of the physiochemistry of infection court tissues in wheat‐microbe‐pathogen interactions on wheat heads. Further elucidating the nature of these interactions in the phyllosphere of wheat head tissues will lead to improved methods of controlling microbial pathogens such as the causal agent of Fusarium head blight of wheat.


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The development and architecture of biofilms formed by the biocontrol agent Burkholderia pyrocinnia FP62 on Geranium.

Patricia Wallace1,Tara Neil2, Bruce Arey3, and Walter Mahaffee2

1Dept. Botany and Plant Pathology, Oregon State University, Corvallis, OR; 2USDA‐ARS Horticulture Crops Research Lab, Corvallis, OR; 3Pacific Northwest National Laboratory‐Environmental Molecular Sciences Lab, Richland, WA

Once bacteria immigrate to the leaf surface they must withstand tremendous fluctuations in environmental stresses and secure nutrients in an environment relatively void of resources. Biofilm formation would aid in both endeavors. There is some debate as to whether biofilm formation, with complex three dimensional architecture, occurs in the phyllosphere. We used multiple microscopy techniques to examine the spatial and temporal colonization pattern and the surface subsurface structure of Burkholderia pyroccina FP62 on geranium leaves. Twice washed cells of FP62 were applied to 8 week old geranium seedlings and maintained in the greenhouse with bottom watering. Leaves were sampled for culturable populations on 5% yeast extract broth agar and microscopic examination at 0,1, 3, 7, and 14 days after inoculation. At the same time leaf samples were examined and the spatial‐temporal distribution and surface structure of bacterial aggregates were observed using fixed leaf samples with scanning electron microscopy techniques or unfixed samples using environmental scanning electron microscopy, confocal laser scanning microscopy and fluorescence stereomicroscopy techniques. The biofilm surface and subsurface architecture was examined using fixed samples with dual beam focused ion beam scanning electron and scanning ion microscopy techniques. The culturable populations of FP62 rapidly declined and reached a steady state by 7 days after introduction whereas the microscopy investigations suggested that populations were increasing. Observations made with a scanning electron microscopy and environmental scanning electron microscopy techniques established that there large populations of FP62 cells on the leaf surface that develop into large biofilms, encased in an exopolymeric matrix by 7 days after introduction. Biofilms had highly structured surfaces and were made up of large continuous cell aggregates or cell aggregates connected by exopolymeric matrices. FP62 cells underwent a phenotype change; cells at sampling days 0, 1 and 3 range in size from 1.0‐2.0 × 0.02‐0.05µm and at days 7 and 14 0.75‐1.0 × 0.2‐0.5µm. This is similar to what has been observed in saturated (high moisture and humidity) biofilms as a result of nutrient deprivation. Using fluorochromes for detection of viable bacterial cells, it was established that most of the cells in biofilms were viable. These observations also indicate that FP62 forms biofilms on leaf surfaces following a typical stepwise pattern similar to biofilms in water‐saturated environments which begins with bacterial cell attachment to a surface followed by the production of microcolonies or aggregates that then develop into structured biofilms. Investigations of the subsurface architecture with the dual beam focused ion beam scanning electron microscope techniques revealed that there are many empty spaces below the biofilm surface and many of these spaces are interconnected to form microchannels that run throughout the biofilm but mostly go from the surface to the inner layers of cells. These microchannels could serve for the translocation of gasses, nutrients and possibly water and wastes. These data suggest that unsaturated (low moisture and humidity) biofilms produced by FP62 have some commonality with saturated biofilms formed by other bacteria.


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Phyllo‐ecology of Virginia and California grown tomatoes

Ottesen A. R., Strain E. A., Brown E.A.

Department of Plant Science and Landscape Architecture, University of Maryland, College Park, Maryland

Of the eleven documented outbreaks of Salmonella associated with tomatoes grown in the continental United States during a period spanning (1998‐2008) where geographic growing region of origin could successfully be traced to a specific location, ten were associated with tomatoes grown in the Eastern United States. Hypothesizing that this might not be coincidence but instead a rare opportunity to explore the epidemiology of tomato contamination by Salmonella, a quest that with many cases of fresh produce contamination is confounded by a complexity of variables, we characterized bacterial and fungal species associated with surfaces of tomato plants of eastern and western grown tomatoes (VA and CA) to examine whether phyllosphere microflora may play a role in Salmonella’s ability to persist in eastern field environments. Characterization was achieved using ‘direct capture’ DNA recovered from surfaces of Virginia and California grown tomatoes and leaves at time of harvest. PCR product of 16S and 18S was cloned, sequenced and aligned to the Silva SSU reference database and NCBI Genbank to assign taxonomic identity. Not surprisingly, observed fungal and bacterial taxa from each region were almost completely different from east to west (P<.05). Only 3 bacterial OTUs at (D= .03) were shared between California and Virginia and only 7 were shared between fungal libraries. Dominant bacterial families in the Virginia libraries were members of Rhizobiaceae while California bacterial families were dominated by Enterobacteriaceae. California fungal libraries were dominated by Erisiphaceae consortia, while Virginia libraries were almost completely dominated by Alternaria spp. and other Pleosporaceae. Fungal species have been known to play a commensal role in the ability of bacterial species to survive in certain niches. It is possible that Alternaria spp. are contributing to the survival of Salmonella in eastern tomato phyllopsheres.


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Host effects on the composition of the microbial community

Ann E. Stapleton, Peter Balint‐Kurti, Susan J. Simmons, James E. Blum, Carlos L. Ballaré Department of Biology and Marine Biology, University of North Carolina at Wilmington

Plant leaves host a specific set of microbial epiphytes. Plant genetic and solar ultraviolet‐B radiation effects on the diversity of the phyllosphere were examined by measuring epiphytic bacterial ribosomal DNA diversity in a maize recombinant inbred mapping population. Several chromosomal loci (QTL) with significant effects on bacterial diversity were identified, some of which had effects only in the presence of ultraviolet‐B (UV‐B) radiation and others which had effects both with and without UV‐B. Candidate genes with allele‐specific effects were mapped to the bacterial diversity chromosomal regions. A glutamate decarboxylase candidate gene was located at a UV‐B‐specific chromosomal locus, and in a comparison between two recombinant inbred lines with contrasting bacterial diversity phenotypes, high bacterial diversity was associated with high levels of glutamate decarboxylase enzyme activity, a component of the gamma‐aminobutyric acid (GABA) pathway. The bacterial diversity loci exhibited a significant overlap with loci important for Southern leaf blight (SLB) susceptibility in the field. A SLB‐resistant inbred genotype had less beta bacterial diversity, and antibiotic treatment of inbreds increased this diversity. The co‐localization of QTL between low bacterial diversity and fungal‐blight‐resistance, and the increase in beta diversity in antibiotic‐treated leaves, suggest that occupation of leaf habitats by a particular set of suppressive bacteria may restrict phyllosphere bacterial variability and increase resistance to fungal infection.


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Microbial Populations of The Tamarix Phyllosphere: Some Like It Hot, Dry, and Saline

Omri M. Finkel1, Adrien Y. Burch2, Steven E. Lindow2, Anton F. Post3 and Shimshon Belkin1

1Institute of Life Sciences, Hebrew University of Jerusalem, Israel; 2Department of Plant and Microbial Biology, University of California, Berkeley, CA, USA; 3Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biology Laboratory, Woods Hole, MA, USA.

The leaf surfaces of Tamarix, a salt secreting desert tree, harbor a diverse community of microbial epiphytes. These microorganisms are exposed to very strong radiation, to long periods of desiccation, to extremely high and transient salinities, and in some cases to high pH. Furthermore, they continuously undergo an almost daily wetting/drying cycle. Nevertheless, the Tamarix tree enjoys a practically world‐wide distribution; in the US it is a highly successful invading species. Thus, the Tamarix island‐like phyllosphere is an excellent opportunity for the study of biogeographical effects on the microbial diversity of an extreme environment. In an attempt to understand the ecological gradients along which the structure of this microbial community varies, we have analyzed phyllosphere microbial richness and diversity on the leaves of three Tamarix species found in Israel (T. aphylla, T. nilotica and T. tetragina). Leaf samples were collected from four locations in Israel and from two locations in the US. DNA was extracted from these samples and 16/18S rRNA hypervariable tag sequencing was performed.

A total of over 200,000 V6 and V9 sequences were obtained, revealing a complex community, with over 400 bacterial, 80 eukaryotic and one archaeal genera. When clustered according to a 6% similarity cutoff, bacterial samples created more than 2500 clusters. Two main community types were detected. One, found on trees by the Mediterranean coast, is dominated by �‐proteobacteria (mostly Halomonas); the other, characteristic of trees in the inland Dead Sea region, is dominated by Actinomycetalles and Bacillales. Samples from Martha's Vineyard, MA, clustered with the former "marine" group, while samples from Davis, CA, were much closer in character to the latter.

Our findings demonstrate that while the microbial community composition on Tamarix leaves is stable on a local scale, trees of the same species that grow in different climatic regions can host microbial communities that are almost completely unrelated.


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Solute permeability of the plant cuticle and its significance for epiphyllic microorganisms

Lukas Schreiber

Department of Ecophysiology, Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, D‐53115 Bonn, Germany

Leaf and fruit surfaces are covered by the plant cuticle forming the interface between the plant and the environment. Plant cuticles protect land‐living plants from uncontrolled water loss. Due to the hydrophobic nature and the low permeability of the cuticle, water and nutrient supply from the leaf interior to epipyhllic microorganism living on the cuticle should be rather limited. Models established in the past measuring solute permeability of isolated cuticles, predicted that ions and polar compounds should not penetrate cuticles in significant rates supporting microbial growth on the cuticle. However, this conclusion must now be questioned to some extent. Experiments measuring cuticular permeability of ions and polar compounds across intact leaf surfaces have shown that the cuticle covering stomatal cells, trichomes and anticlinal epidermal cell walls is substantially more permeable to polar compounds than the cuticle covering regular periclinal epidermal cell walls. Thus, leaf surface permeability is characterized by a pronounced lateral heterogeneity due to the preferential occurrence of polar paths of transport at certain characteristic leaf surface structures. This conclusion agrees with frequent observations of leaf surface microorganisms often being located around stomata, on trichomes and along anticlinal cell walls, where nutrient availability should be favourable.

Further reading:

Schreiber L (2010) Transport barriers made of cutin, suberin and associated waxes. Trends in Plant Science (in press)

Schreiber L, Schönherr J (2009) Water and solute permeability of pant cuticles. Measurement and data analysis. Springer, Heidelberg pp. 1‐29


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Influence of the plant as a reservoir of airborne pollutants and quaternary ammonium compounds on phyllosphere microbes

Chiliang Chen, Amarjyoti Sandhu, Dana R. McKeever and Gwyn A. Beattie Dept of Plant Pathology, Iowa State University, Ames, IA, USA

We are interested in the chemical environment on leaves and its influence on leaf‐associated bacteria. We have recently focused on two components of this environment, volatile airborne pollutants and plant‐derived quaternary ammonium compounds (QACs). We evaluated whether leaf‐associated bacteria have access to airborne pollutants using the model compound phenol. Following exposure to airborne phenol, plant leaves accumulated phenol and accumulated it in sites that were accessible to leaf‐surface bioreporter bacteria. Furthermore, natural communities on leaves from phenol‐rich areas exhibited phenol degradation activities in planta, with multiple degradation‐active community members. These results support a model in which airborne pollutants are part of the chemical milieu of the phyllosphere relevant to the resident microbes. Plant‐derived QACs may also be available and important to the biology of the leaf‐surface microbiota. These QACs include choline, which is a component of the major membrane phospholipid phosphatidylcholine, glycine betaine, which is a compatible solute conferring protection against drought and salinity stress, and carnitine, which is a carrier of fatty acids during fatty acid oxidation. We have used bioreporter bacteria to demonstrate that one or more QAC compounds were released from bean seeds during germination and that QACs, primarily choline, were produced and available to Pseudomonas syringae during leaf colonization. In a comprehensive characterization of QAC transporters in the P. syringae strains B728a and DC3000, we found evidence that these strains are adapted for living in choline‐rich environments. One of the QAC transporters, Cbc, represents a novel ABC transporter that employs multiple substrate‐binding proteins, each of which is specific to a distinct QAC and thus allows P. syringae to discriminate among closely related QACs in uptake for catabolism. The ability of these binding proteins to discriminate among QACs may allow the bacteria to avoid the uptake of toxic QACs, such as those commonly used as sanitizing agents, as supported by the relatively high tolerance of Pseudomonas species to such agents. In B728a and DC3000, the QAC catabolic transporter Cbc is complemented by a second ABC transporter, OpuC, that has a single substrate‐binding protein with broad specificity for QACs; this transporter promotes uptake for osmoprotection. Field studies with mutants deficient in the three major QAC transporters Cbc, OpuC and BetT showed that the transport of QACs provided a clear fitness benefit to B728a during leaf colonization of host and nonhost plants, as well as to DC3000 during leaf colonization of nonhost plants. Recently, we have found that B728a exhibits chemotaxis toward carnitine and are exploring the hypothesis that carnitine helps direct the cells toward germinating seeds, which should have abundant carnitine associated with the high levels of fatty acid oxidation. Collectively, our results indicate that airborne pollutants and plant‐produced QACs are available to plant‐associated bacteria and that these bacteria have a multiplicity of adaptations to exploit and benefit from these compounds.


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Genomic diversity of biological control strains of Pseudomonas spp. isolated from plant surfaces

Joyce Loper1, Ed Davis1, Kent Lim2, Karl Hassan2, Sasha Tetu2, Neil Wilson2, Sierra Hartney1, Brenda Shaffer1, Virginia Stockwell1, Marcella Henkels1, Kedy Shen1, Rachel Blumhagen1, Dmitri Mavrodi3, Judith van Mortel4, Chunxu Song4, Diana Radune11, Jessica Hostetler11, Daniel Kluepfel5, Patrick Wechter6, Anne Anderson7, Young Cheol Kim8, Leland Pierson III9, Elizabeth Pierson9, Steve Lindow10, Jos Raaijmakers4, David Weller3, Linda Thomashow3, Andrew Allen11, and Ian Paulsen2

1USDA‐ARS and Oregon State University, USA; 2Macquarie University, Australia; 3USDA‐ARS and Washington State University, USA; 4Wageningen University, The Netherlands; 5USDA‐ARS, Davis, CA, USA; 6USDA‐ARS, Charleston, SC, USA; 7Utah State University, USA; 8Chonnam National University, Korea; 9Texas A&M University, USA; 10University of California at Berkeley, USA; 11J. Craig Venter Institute, USA.

The striking ecological, metabolic, and biochemical diversity of Pseudomonas has intrigued microbiologists for many decades. To explore the genomic diversity of biological control strains of Pseudomonas spp., we derived high quality draft sequences of seven strains known to suppress plant disease. The strains were isolated from the phyllosphere of pear (P. fluorescens A506), the rhizosphere of wheat (three strains of P. fluorescens and two strains of P. chlororaphis), or the rhizosphere of peach (P. synxantha BG33R). The sequences are in one to nine scaffolds, with each genome having 50 to 80 intrascaffold gaps. Genome size varies from ca. 5.9 MBases with 5500 open reading frames (ORFs) (P. fluorescens A506) to 7.0 MBases with 6400 ORFs (P. chlororaphis strain O‐6). Phylogenetic analysis of the seven strains and three previously‐sequenced strains of P. fluorescens (Pf‐5, Pf0‐1 and SBW25) defined three distinct clades. Within each clade, the genomes share 75 to 90% of their proteomes. Typically, 60 to 70% of the proteome is shared between strains in different clades. All seven strains share 2525 genes, representing a core genome, with each strain having approximately 600 to 1000 unique genes not found in the other genomes.

Bioinformatic analysis revealed genes with potential roles in the biology of each strain and its multitrophic interactions on plant surfaces. As expected, all strains have genes for pyoverdine biosynthesis and uptake. Four strains also have biosynthetic clusters for the siderophores pseudomomine or achromobactin. Type III secretion systems with operon structures different from those in P. fluorescens SBW25 or P. s. pv. syringae were detected in five of the sequenced genomes. In addition, one to three copies of type VI secretion system clusters were identified in each of the seven genomes. All strains contain putative insect toxin genes of either the Mcf or Tcc type. In addition to previously‐described quorum sensing systems, many strains contain one to four “luxR solos” similar to those responsive to exogenous signals in other bacteria. Many orphan gene clusters containing non‐ribosomal peptide synthetases, a polyketide synthase, or other genes characteristic of secondary metabolite biosynthesis are present in the genomes. Exploration of these gene clusters provides avenues for the future discovery of novel natural products, including those contributing to biological control of plant disease.


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Adaptations of lactic acid bacteria to plants

Henry Low, Thomas Williams, Sybille Tachon, Benjamin Golomb and Maria L Marco

Food Science & Technology, University of California, Davis

Lactic acid bacteria (LAB) are essential for many industrial and artisanal plant, dairy and meat fermentations where they contribute to the formation of taste, texture, nutritional, and aroma attributes of the food product. Certain LAB, specifically strains of Lactobacillus, also confer health benefits upon ingestion and entry into the mammalian gut. Although LAB are generally regarded to be ubiquitous in nature, they are most commonly isolated from either plants or mammalian digestive tracts. Assessments of LAB in the phyllosphere have generally found that these organisms colonize plants in low (<104 cfu/g) but persistent amounts. Upon plant harvest and incubation in anaerobic conditions, these bacteria grow rapidly to become the dominant organisms associated with the fermented plant materials (e.g. silage, olives, sauerkraut). This project is investigating the roles of plants as an ecological niche for LAB and the associations of LAB with plants which are relevant to human health. To initiate these studies, amounts of phyllosphere bacteria able to grow on MRS, a semi‐selective culture medium for LAB, were measured over time for lettuce grown in the Salinas Valley, CA. These plants were found to contain low but persistent quantities of LAB (an average of 1000 cfu/g plant), including members of the Leuconostoc, Lactococcus, and Enterococcus species. In growth‐chamber experiments, strains from three species of industrially‐relevant and/or probiotic LAB, Lactococcus lactis, Lactobacillus plantarum, and Lactobacillus casei, were grown on bean (Phaseolus vulgaris) leaf tissue macerates. Only the plant‐associated strains were found to grow to high levels (>108 cfu/g) within 24 h after inoculation onto the plant tissues. The adaptations of these LAB to plant environments are being investigated by gene expression analysis. These studies complement on‐going experiments in this laboratory aimed at understanding the effects of a plant‐based diet on health‐benefits conferred by Lactobacillus in the mammalian gut. Growth and survival of LAB in the phyllosphere is of interest to understand the ecological niche to which LAB are most adapted and the functional relevance of phyllosphere‐associated bacteria to human health


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Very‐long chain aldehydes promote prepenetration processes of Blumeria graminis in a dose and chain‐length dependent manner.

Anton Hansjakob, Markus Riederer, Ulrich Hildebrandt

University of Wuerzburg, Julius‐von‐Sachs‐Institute for Biosciences, Department of Botany II

Surface properties of aerial plant organs have been shown to affect the interaction of plant pathogens and their hosts. Conidial germination and differentiation of appressoria ‐ the so‐called prepenetration processes of the barley powdery mildew fungus (Blumeria graminis f.sp. hordei) ‐ are known to be triggered by n‐hexacosanal, a minor constituent of cuticular barley leaf wax. In order to assess the inducing capabilities of different barley leaf wax constituents, germination and differentiation behaviour of B. graminis conidia were analyzed by applying a Formvar resin based in vitro system. Alkanes (C24‐C33), primary alkanols (C20‐C30), fatty acids (C20‐C28) and aldehydes (C22‐C30) as most prominent wax constituents were analyzed. Among those exclusively very‐long chain aldehydes distinctly promoted fungal germination and differentiation in a chain length and concentration dependent manner. In addition, the primary alkanol n‐hexacosanol (C26) significantly enhanced conidial germination and early differentiation but not appressorium formation. Our results suggest a critical role of cuticular very‐long chain aldehydes in conidial morphogenesis during the prepenetration processes that are essential for fungal penetration and establishment.


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The cusper bioreporter – Measuring reproductive success of individual bacteria in simple and complex environments

Mitja N. P. Remus‐Emsermann1 and Johan H. J. Leveau1,2

1NIOO‐KNAW, 6666 GA Heteren, The Netherlands; 2UC Davis, CA 95616, USA

One of the challenges in the exciting and emergent field of individual‐based microbial ecology is the development of experimental tools that reliably quantify the individual contributions of microorganisms to population size and structure in natural environments. With CUSPER, we introduce a novel bioreporter tool that is capable of determining the reproductive success of individual bacteria in simple and complex environments. The concept of CUSPER is based on the dilution of pre‐formed stable green fluorescent protein (GFP) from dividing bacteria, which inversely relates GFP concentration to reproductive success (reproductive success, or repsuc reads CUSPER backwards). To assess the effect of environmental heterogeneity on the reproductive success of individual bacteria, we first exposed this bioreporter, based on the bacterium Erwinia herbicola 299R, to LB broth, which represents a condition of low environmental heterogeneity. Cells that were recovered from this environment and analyzed by epifluorescence microscopy and image cytometry, exhibited a normal distribution of reproductive success at all sampling times. This suggests that each individual bacterium in the broth culture contributed equally to the observed population increase. In contrast, cells that were exposed to leaf surfaces, which represent a more complex environment and which are the natural habitat for E. herbicola 299R, showed increasingly larger deviations from the normal distribution over time, suggesting that some initial colonizers of the leaf surface were more successful in creating offspring than others. These results demonstrate the usefulness of CUSPER as a tool to assess reproductive success of individual bacterial cells. The compatibility of CUSPER with other single‐cell interrogation techniques offers future promise for linking individual reproductive success to specific individual bacterial behaviors and/or environmental experiences.


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Explaining bacterial patterns in the phyllosphere: a modeling approach Annemieke van der Wal1, Robin Tecon1,2, Jan‐Ulrich Kreft3, Wolf Mooij4, Mitja Remus‐Emsermann1 and Johan Leveau1,2 1NIOO‐KNAW, 6666 GA Heteren, The Netherlands; 2UC Davis, CA 95616, USA; 3University of Birmingham, Birmingham B15 2TT, UK; 4NIOO‐KNAW, 3631 AC Nieuwersluis, The Netherlands

The phyllosphere represents a topographically highly diverse landscape for micro‐

organisms as it contains veins, trichomes, epidermal cells and stomata. This environmental heterogeneity at the micrometer‐scale may explain the non‐random spatial distribution of bacterial aggregates on plant leaf surfaces. It has been shown by others that the frequency distribution of aggregate sizes is skewed, with many small aggregates and only a few large aggregates, the latter usually associated with veins or trichomes. This may indicate that these sites on the leaf favor bacterial growth, e.g. by retention of water and/or enhanced concentration of sugars. In this study we try to explain the pattern of bacterial aggregation on plant leaf surfaces using a spatially explicit modeling approach. We hypothesized that the bacterial patterns can be explained by variation in the availability of water on the leaf, which in turn determines the availability of the growth limiting nutrient (assumed to be sugar). To test our hypothesis, we simulated three different ‘water’ landscapes on a 1‐cm2 patch of leaf surface by keeping the total volume of water constant (10 µl), but by varying the area coverage of water: 1) a landscape covered by a water film, 2) a landscape covered by water drops of the same size (volume) and 3) a landscape covered by water drops of different sizes (following a normal distribution). Next, bacteria were randomly dispersed on the leaf. The number of initial bacterial colonizers was constant (1×104), but two scenarios were simulated for initial bacterial aggregation: 1) all bacteria arrived as single cells or 2) bacteria arrived as aggregates of different sizes following a log‐normal distribution. Bacterial growth was determined by the concentration of sugar and their maximum growth rate. The concentration of sugar was determined by volume and area coverage of the water drops, and by bacterial uptake of sugars. The permeability of the leaf cuticle and the concentration of sugar in the plant’s interior were kept constant. These scenarios were simulated for 48 hours, with a time step of 0.25 hours and ignoring evaporation of water. In scenario 1, all bacteria formed aggregates at the end of the incubation period, no small (< 64 cells) aggregates were found, independent of the initial aggregate size distribution. In both water drop scenarios, only small bacterial aggregates were found when all bacteria arrived as single cells. When we introduced variation in initial aggregate sizes, we obtained the experimentally observed pattern of bacterial aggregation in both water drop scenarios. An increased variation in the degree of aggregation was found when different water drop sizes were introduced. Using this model we conclude that non‐uniform distribution of water, and concomitantly sugar availability, such as resulting from drops deposited as rain or as dew on leaf surfaces is necessary to explain the observed bacterial patterns. In addition, variation in initial bacterial aggregate sizes is necessary to achieve these patterns. This may likely occur as bacterial cells usually arrive in aggregates of more than one individual. Our predictions will be tested by experiments in the greenhouse.


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Comparative Phylogenomics of Phyllosphereassociated Escherichia coli Isolates

Guillaume MERIC1*, Elizabeth J. SAGGERS1, Tim F. BROCKLEHURST1 Sacha LUCCHINI1

1 Institute of Food Research, Norwich Research Park, Norwich NR4 7UA, United Kingdom; * Corresponding author: [email protected]

The Escherichia coli species is mostly represented by strains that are natural commensals of the gastrointestinal tract (GIT) of vertebrates. However, a few E. coli strains can cause various intestinal and extraintestinal diseases, resulting in more than 2 millions deaths per year. This serious health issue has driven medical research to understand the molecular mechanisms of E. coli colonization and virulence. While pathogenic E. coli infections are commonly linked to the consumption of animal products, multiple food‐related outbreaks have recently been related to the ingestion of strains associated to fresh fruits and vegetables, raising interest on the ecological links between E. coli and plants, and on the functions involved in this association. Indeed, little is known about how wild‐type E. coli persist and adapt to their living environment when outside the animal gut, despite the fact that E. coli can be easily isolated from multiple nonhost environments such as soils, fresh waters or vegetation, where it seems to persist very well and sometimes even replicate. In this study, E. coli isolates from the phyllosphere of field‐grown crops were compared to strains of the widely used ECOR collection. Using Bayesian analysis, we inferred the phylogenetic relationships between the tested strains using a microarray‐based comparative genomic hybridization (CGH) dataset. Additionally, we assessed the strains for various phenotypes related to life conditions on plants or in the GIT. Our results indicate that specific plant‐related metabolic abilities may be important for an increased fitness of E. coli on leaves. CGH did not reveal any correlation between phylogenetic relationship and sample origin (gut or plant surface). This could indicate that there is no specific adaptation to plants at the phylogenetic level, but that key functions acquired through lateral gene transfer may play an important role in the association of E. coli with plants.


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Bacterial Communities in The Phyllosphere of Trees Are Associated With Plant Phylogeny.

M.R. Lambais1 and D.E. Crowley2

1Department of Soil Science, University of São Paulo, Piracicaba, SP, Brazil. 2Department of Environmental Sciences, University of California, Riverside, CA, USA. ; E‐mails: [email protected] or [email protected]

We have previously shown evidence that tree species in the Atlantic Forest have unique bacterial communities, comprised mostly of unknown species. It can be hypothesized that each type of tree phyllosphere selects for its own bacterial community based on species‐specific differences in the phytochemicals available for microbial growth and/or with antimicrobial activities, and that plants phylogenetically related may have more similar bacterial communities in the phyllosphere. However, whether the bacterial populations inhabiting the leaf surfaces are related to the plant phylogeny has not been studied. To examine this hypothesis, we evaluated 9‐12 individuals of 7 tree species in a pristine Dense Ombrophilous Atlantic forest. The trees that were studied encompassed species from three orders and four families. The bacterial communities were characterized using 16S rRNA gene PCR‐DGGE and clone library sequencing. Our results showed that the bacterial communities varied among tree species, but were highly similar for individuals of the same species. Moreover, the grouping pattern observed by discriminate analysis of the bacterial communities consistently reflected the phylogenetic relationships among the tree species, based on the DGGE profiles. In general, approximately 50% of the bacterial 16S rRNA clones from the phyllosphere were assigned to Gammaproteobacteria, and 22% to Alphaproteobacteria. The vast majority of the species are new, but included approximately 35% that were assigned to the genus Pseudomonas. Our findings reveal the high selectivity of this habitat for unique bacterial communities. Their contribution to the ecology of forest ecosystems is unknown, but can be speculated to contribute to the degradation of terpenes and phenolics that are involved in plant defense responses or chemical signaling. In addition to comprising yet another large reservoir of genetic diversity in tropical forests, the phyllosphere‐inhabiting bacteria and their enzymes may have biotechnological applications.

Project funded by BIOTA‐FAPESP (São Paulo, Brazil) and CNPq (Brasília, Brazil).


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Bacterial growth in the apoplast is limited by nutrient availability María Eugenia RAMOS, Steve LINDOW


The presence of a functional type‐III secretion system (TTSS) is known to be a requirement for pathogenic growth inside plant tissues, since both saprophytes and hrp (type‐III secretion defective) mutants of phytopathogenic bacteria exhibit little growth in the apoplastic environment. However, we have observed high population levels of saprophytes and hrp mutants when they are co‐infiltrated into plants with a variety of carbon sources. The increase is also dose‐dependent. Metabolizable carbon‐sources such as succinate and sugars can induce very high population levels of saprophytes and hrp mutants that are comparable to wild type Pseudomonas syringae pv. syringae B728a in compatible bean plants. This effect is not seen with non‐metabolizable nutrients. Moreover, this major population increase is also evident when a hrp mutant of a normally incompatible pathogen such as P. syringae pv. tomato DC3000 (a pathogen of tomato and Arabidopsis) is infiltrated in bean leaves. Similarly high bacterial levels were found when water congestion in the apoplast was maintained after inoculation, even without nutrient addition; suggesting that inaccessible nutrients in the apoplast are made more available to bacteria by introduction of water to the intercellular spaces. This effect is transient and no population increase is seen in plants in which the introduced water is allowed to dry. Maintained wetting of the apoplast is thus sufficient to allow bacterial multiplication although some additional release of nutrients into the apoplast during the wetting process cannot be ruled out. Endophytic growth in leaves is thus limited by apoplastic nutrients and TTSS‐proficient pathogens can overcome this limitation with effectors that release water and/or nutrients into the apoplast.


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Environmental influence on motility in Pseudomonas syringae pv. syringae B728a and implications for plant‐microbe interactions. Kevin Hockett and Steven Lindow Plant and Microbial Biology Department. University of California, Berkeley. USA

Environmental factors, including light and temperature, affect the behavior of Pseudomonas syringae pv. syringae B728a (Pss), and its association with plant hosts. The effect of temperature on plant‐microbe interactions has been studied extensively in diverse bacterial genera. Generally, pathogenic determinants (secondary metabolite production, T3SS, etc…) are positively regulated by temperatures below the optimal growth temperature of the organism. Less well understood is the effect of temperature on the motility of plant‐associated bacteria. When Pss is incubated at 28‐30C or greater temperatures, it is inhibited in swarming motility (a form of social, surface‐associated motility). A fractal form of swarming (which is observed in the absence of functional flagella on 0.25% agar) is also inhibited at the same elevated temperature. Both forms of motility require the production of syringafactin, a biosurfactant produced by Pss. Using both an oil over‐overspray assay to quantify surfactant production, as well as a transcriptional fusion between the promoter of syfA (the gene encoding the non‐ribosomal polyketide synthase which produces syringafactin) and a GFP reporter gene, we show that syringafactin production is reduced at elevated temperature, and that this regulation occurs at least in part or in whole at the transcriptional level. Additionally, we show that flagellin production is thermoregulated in a similar manner—that is, expression is repressed at elevated temperatures. To identify thermoregulatory components, ca. 30,000 transposon mutants were screened for increased syfA expression at 30C. From this screen we recovered multiple, independent insertions in an ORF encoding a NUDIX hydrolase, as well as an ORF encoding an acyl‐CoA dehydrogenase. Both of these mutants exhibit significantly increased expression of syfA at 28C. Additionally, the NUDIX hydrolase mutant is able to swarm at elevated temperatures, compared to WT.

Light appears to be an additional, yet more subtle, environmental factor which has the ability to repress swarming motility in Pss. This repression does not appear to be mediated through reduction in flagellin expression or reduction in syringafactin expression. Pss possesses three putative photoreceptor homologs (two phytochrome homologs and one phototropin homolog). A bphP1 (bacteriophytochrome 1) mutant exhibits a hyper swarming phenotype under both light and dark conditions. Repression of swarming by light in this WT strain is inconsistent, indicating that other genetic or environmental determinants supersede light regulation of swarming, or that light regulates factors that indirectly affect swarming. Previous work by several groups has demonstrated that flagellar motility in pathogenic Pseudomonas spp. is an important factor that contributes to both invasion into as well as survival on leaf surfaces. Additionally, disease of plants caused by this group of pathogens tends to be more prevalent during cool, humid conditions. We are currently performing in planta experiments to address the effect of temperature and light on the epiphytic phase as well as the pathogenic phase (and the transition between the two phases) of Pss.


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Traits Contributing to Epiphytic and Endophytic Growth of Pseudomonas syringae Revealed By Global Gene Expression Analysis

Russell Scott2, Xilan Yu1, Steven Lund1, Jessica Williams3, Steven Lindow2, Dan Nettleton1, Dennis Gross3 and Gwyn Beattie1.

1Department of Plant Pathology, Iowa State University; 2Department of Plant and Microbial Biology, University of California‐Berkeley; 3Department of Plant Pathology and Microbiology, Texas A&M University

Pseudomonas syringae is an important plant pathogen with a prominent epiphytic phase that serves as inoculum for subsequent entry into the apoplast and infection. To obtain a comprehensive understanding of the traits contributing to its associations with the surface and interior of plants and their regulation, we are performing whole genome transcriptional profiling of P. syringae pv. syringae B728a, a pathogen of bean, using an ORF‐based microarray. Gene expression of the wild‐type strain and mutants of the global regulators ahlR and aefR are being assessed in seven conditions. Our in planta conditions consist of inoculation and incubation upon leaf surfaces, or within the leaf apoplast. In vitro treatments consist of culturing in a basal medium unmodified, or amended to achieve conditions of low iron, nitrogen and water availability or high oxidative stress. Though transcriptomes appear to be largely comparable between both in planta treatments, the transcriptional responses of B728a to the leaf surface are distinct from its responses to the apoplast. A subset of these genes appears to have functions independent of a known stress response. For example, genes involved in flagellar motility, phosphate transport, and, remarkably, 25 of 58 annotated chemotaxis sensory transducers were expressed more in epiphytic sites, whereas genes involved in syringomycin production, and genes within two separate phage regions of unknown function were expressed more in endophytic sites. Interestingly, genes for the production of the siderophores pyoverdine and achromobactin were up‐regulated in vitro under low iron conditions, yet were not upregulated in either in planta condition, indicating high iron availability. Additionally, both ahlR and ahlI components of the luxI/R type quorum sensing system appear to be active in planta, though these genes generally are more active in the leaf interior compared with epiphytic cells. This finding could be explained by 1) Lower overall production of 3‐oxo‐C6‐HSL within the oligotrophic leaf surface environment (nutrient availability as a super‐regulator of quorum sensing), 2) Larger overall population densities in the leaf interior (more and larger aggregates), 3) Less diffusional loss of signal or greater signal stability in the apoplast (an acidic environment, where colonies may be more compartmentalized), or any combinations thereof. Preliminary findings identify a surprisingly small quorum regulon in vitro, consisting of 10 positively ahlR‐regulated genes. These same genes appear to be positively regulated by quorum sensing in planta as well. In addition to autoregulation of ahlI and ahlR by themselves, an adjacent five gene operon likely involved in the modification and export of amino acid‐like compounds is quorum regulated. Two genes coding for putative analogs of post‐transcriptional regulators in Bacillus, and a marR‐like transcriptional regulator, are also positively quorum regulated. The relevance of these transcriptional responses to the process of plant niche colonization is being investigated.


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Exploring the environment sensed by Pseudomonas syringae in the phyllosphere and the regulons involved in this sensing based on whole genome transcriptional profiling

Xilan Yu1, Russell Scott2, Steven Lund1, Jessica Williams3, Steven Lindow2, Dan Nettleton1, Dennis Gross3 and Gwyn Beattie1.

1Department of Plant Pathology, Iowa State University; 2Dept of Plant and Microbial Sciences, University of California‐Berkeley; 3Dept of Plant Pathology and Microbiology, Texas A&M University.

We are performing whole genome transcriptional profiling to identify the environmental conditions sensed by Pseudomonas syringae during its growth in association with leaves as well as the role of various sigma factors during this growth. We have performed a multifactorial transcriptome analysis of Pseudomonas syringae pv. syringae strain B728a and four sigma factor mutants, rpoS, rpoE (algU), rpoN and hrpL, in each of seven distinct environmental conditions. These include the leaf surface and leaf apoplast and cells cultured in vitro and exposed to low iron availability, low water availability, oxidative stress, and nitrogen starvation. In the data that we have obtained thus far (a single replicate representing each of the 35 distinct transcriptomes), we have found that a large number of genes induced in leaf‐associated P. syringae cells were also induced by exposure of cells to water limitation or N starvation, but only a relatively few were induced by exposure to oxidative stress (hydrogen peroxide) or low iron availability. Approximately 1,300 genes were induced in association with leaves based on having ≥ 2‐fold higher transcript levels than in a basal medium, and of these ~25% were induced specifically on the leaf surface and another ~25% were induced specifically in the leaf apoplast. Exposure of leaf‐associated cells to low water availability was strongly supported by their high transcript levels of genes involved in the synthesis of compatible solutes, including NAGGN and trehalose, and uptake of osmoprotectant and alginate biosynthesis. Surprisingly, water limitation was greater in the apoplast than on the leaf surface based on the particularly high transcript levels in the apoplastic bacteria. Among the sigma factors examined, RpoE and RpoN were required for the induction of the greatest numbers of genes, with RpoS and HrpL influencing the expression of relatively few genes, in the leaf‐associated cells. Among the environmental stresses examined, RpoE influenced primarily genes responding to water limitation whereas RpoN was dramatic in its influence on genes responding to all of the environmental stresses. The microarray included 61 putative sRNA genes that were predicted based on other studies, bioinformatic analyses, and promoters identified in an IVET‐screen. Among these putative genes, half exhibited ≥ 2‐fold changes in transcript abundance under at least one of the environmental stress conditions in vitro, providing evidence that small RNAs were involved in these bacterial responses. Furthermore, many of these small RNAs were abundant in the leaf‐associated cells.


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Conservative production of biosurfactants in the phyllosphere

Adrien Burch, Briana K. Shimada, Patrick J. Browne and Steven E. Lindow


Microbial surfactant production has classically been characterized as a trait used by bacteria to access hydrophobic nutrient sources, and has since been found to play a role in swarming motility, biofilm structure, and antagonistic interactions against membranes. An unexplored arena where biosurfactants may prove particularly important is the colonization of waxy leaf surfaces, a water‐repellent environment where epiphytes endure daily fluctuations in moisture availability. In order to estimate the prevalence of epiphytic surfactant production, bacteria from a variety of environments were assessed using a novel atomized oil assay, and it was found that surfactant producers were significantly more common in the phyllosphere than in environmental water samples. Furthermore, this novel assay detected surfactant production in organisms which were not identified with the standard drop‐collapse assay, some of which may have unique surfactant properties. Biosurfactant production in the phyllosphere was further explored in Pseudomonas syringae pv. syringae B728a, a sequenced surfactant‐producing organism with a prominent epiphytic lifestyle. The genetic regulation of biosurfactant production was addressed by evaluation of random transposon mutants with the atomized oil assay. Two surfactants with different patterns of production were identified with this method. Syringafactin was found to be negatively regulated by the AlgT extracellular stress pathway; exposure of the strain to a variety of stresses which trigger the pathway resulted in repression of syringafactin production. Production of another surfactant, tentatively identified as HAA, was dependent on flagellar function. The behavior of these mutants on plants is being assessed. The regulation of these two surfactants points to a vital role of surfactants in motility in the phyllosphere, but only when conditions are permissive for motility.


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The contributions of swarming motility to Pseudomonas syringae behavior on plants

Juliana Cho, Steven Lindow

Department of Plant and Microbial Biology, University of California‐Berkeley; [email protected]

Pseudomonas syringae is a widespread gram‐negative bacterial pathogen of a broad range of agriculturally important crops, such as bean and tomato. Strain B728a is the causal agent of brown spot disease of bean, resulting in the development of necrotic lesions on leaves and pods after invasion and proliferation in the plant apoplast. The initial invasion into the plant is crucial for successful colonization and resultant plant disease, and appears to rely on flagella‐mediated swarming motility. However, relatively little is known about how motility contributes to the behavior of the pathogen in natural environments. We have shown that blockage of swarming motility through flagellar component deletions such as the hook‐filament junction gene FlgK results in greatly reduced virulence in Pseudomonas syringae B728a when applied on bean plants, even though the growth rate in vitro is similar to WT, highlighting the importance of motility in initial pathogen colonization of plants. Motility appears to be a highly selectable trait in Pseudomonas syringae. Comparison of various WT Pseudomonas syringae B728a isolates from four prominent labs studying this model organism demonstrate a high degree of variability in swarming motility, and segregate into two major lineages of swarming phenotypes, divided between “hypo‐swarmers” and “hyperswarmers”, with hypo‐swarmers covering less than 50% of the area that hyper‐swarmers cover 17 hours post inoculation on swarming plates. Increased swarming motility on swarming plates correlates to increased virulence in planta, with a 35% increase in lesion size, confirming the relevance of the in vitro swarming motility assays. This swarming phenotype is heritable and reproducibly maintained within each strain. This variability is surprising, because all of these strains derived from a single original stock. The degree of Pseudomonas syringae motility may be influenced by local environmental conditions or phase variation, as has been shown in Sinorhizobium meliloti and Campylobacter coli. Quorum sensing is a cell density and environmentally dependent system of bacterial communication, and has been proposed to repress swarming motility in Pseudomonas syringae, since regulatory mutants in this communication system exhibit a hyper‐swarming phenotype. Isogenic deletion mutants in quorum sensing regulatory factors (AefR, AhlR) across different strains exhibit varying motility. While production of AefR‐ and AhlR‐ mutants in isogenic lines can result in conversion of a hypo‐swarmer to a hyper‐swarmer, complementation of these mutations does not restore motility, suggesting that these mutations may be polar or secondary mutations. Comparison of various Pseudomonas syringae isolates in swarming assays suggest that other potential regulatory and environmental factors may play a role in determining the degree of swarming motility and plant pathogenicity. Iron‐limiting conditions cause changes in the morphology of social swarming “fingers” in Pseudomonas syringae, suggesting that iron availability may be one such environmental factor modulating the degree to which Pseudomonas syringae can initiate invasion and disease.


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An individual‐based approach to surface colonization by bacteria

Robin Tecon1,2, Annemieke van der Wal1, Mitja N. P. Remus‐Emsermann1 and Johan H. J. Leveau1,2

1NIOO‐KNAW, 6666 GA Heteren, The Netherlands; 2UC Davis, CA 95616, USA

Most microbial habitats are characterized by physical, chemical, and biological conditions that differ significantly along micrometer distances. This environmental heterogeneity at the scale of individual microbes may account for much of the nonrandom distribution of microorganisms observed in natural settings. Here we report on an individual‐based approach that combines spatially explicit modeling and bioreporter‐driven experimentation to improve our basic understanding of the impact of small‐scale heterogeneity on the bacterial colonization of surfaces. We used bioreporters of Erwinia herbicola 299R tagged with the green fluorescent protein (GFP) in two ways. One type of bioreporter constitutively expressed GFP which allowed for easy visualization of the location of bacteria in relation to one another. In the second type of bioreporter, preformed GFP was diluted from cells as they divided, rendering GFP content of daughter cells an inverse function of the reproductive success of their mothers. These bioreporter cells were inoculated on a defined agarose surface and analyzed by fluorescence microscopy to measure bacterial growth and colony formation during incubation. E. herbicola cells all grew at the same rate on the surface of the gel medium, regardless of the bacterial spatial density. This suggests that under these conditions, growth of bacteria was not influenced by the presence of others nearby. Fluorescence intensity and surface area measurements of the reproductive success were in good correlation. The pattern simulation matched the experimental data when it introduced initial variations in the cell cycle of colonizers. This approach of validating bioreporter output in simple environments with defined complexity are essential for our ability to explain their performance in more complex environments, such as the natural habitat of E. herbicola 299R, i.e. the plant leaf surface.


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What’s in a seed?

Mark A. Holland, Department of Biology, Salisbury University, Salisbury, MD 21801

[email protected]

What’s in a seed? A standard answer is that it is an embryonic plant with some stored nutrients:

a baby in a box with its lunch. But past experiences with seed‐transmitted phylloplane bacteria,

Methylobacterium spp., have demonstrated to us that this is too simple a definition. We have learned,

for example, that; 1) many (most?, all?) seeds carry the bacterium, 2) sometimes seeds fail to germinate

only because their bacteria have died, 3) plant development is can be shaped by bacterial populations,

4) the size of bacterial populations is directly correlated with the tissue concentration of certain of the

plant growth regulators, 5) mutations in bacterial metabolism have a measured effect on plant

metabolism (and vice versa), 6) mutations in the bacteria have the appearance of maternally inherited

genes in the plant host and 7) some plant phenotypes are directly attributable to the bacteria living with

them.

So‐ What’s in a seed? Our observations suggest that a seed is a consortium of organisms, a

collection of multiple genomes, reliably transmitted from one generation to the next as a unit and

traveling together though time and space in a package that we recognize as a plant propagule.

This definition suggests a different way of thinking about the nature of plants and raises

significant questions:

1) Is it possible to define a microbiome common to all, many, most plants?

2) Other than Methylobacterium, spp., what other bacterial species are regularly found in seed

consortia? How widespread are they?

3) What is the nature of the relationships among plant and seed‐transmitted bacteria? We

might expect that widespread seed‐transmitted bacteria are of particular significance.

4) Can knowledge of seed consortia be exploited to improve seed/plant performance?

Looking for answers to these and related questions is an ongoing concern of the lab. Feedback,

comments and collaboration always welcome!


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Physiology of phyllosphere microbiota as revealed by community proteogenomics

Julia A. Vorholt

Institute of Microbiology, ETH Zurich, Switzerland

Diverse bacteria have adapted to life on leaf surfaces and colonize the phyllosphere. Most of these are commensals that are not well studied so far and that may have important effects with regard to the global carbon and nitrogen cycles as well as plant health. While metagenomics allows determining which organisms are present in a certain habitat and gives insights into the potential of organisms, metaproteomics allows the identification of proteins synthesized under certain conditions. With the aim to identify and characterize the dominating phyllosphere bacteria in situ, we performed a culture‐independent analysis of the microbiota on soybean, clover, and Arabidopsis thaliana plants using a combination of metagenomic‐ and metaproteomic approaches. We found a high abundance of members of the Alphaproteobacteria and in particular of the genera Sphingomonas and Methylobacterium on the analysed plants. Comparative metaproteogenomics revealed that their proteomes showed a remarkable high consistency. The identification of more than 2,200 bacterial proteins sheds light on general adaptation mechanisms and specific adaptations of these and others bacteria and suggests new protein functions important in the environment. Known proteins expressed in Methylobacterium were related, to a large extent, to the ability of these bacteria to use methanol as source of carbon and energy. The abundance of a conspicuous protein, a methanol‐dehydrogenase‐like protein (XoxF), which is only hardly detectable under laboratory conditions, suggests a new important protein function under environmental conditions. Members of the genus Sphingomonas showed a remarkably high expression of different TonB‐dependent receptors. Since these outer membrane proteins are involved in transport of various carbohydrates, a particularly large substrate utilisation pattern can be assumed to occur in the phyllosphere. Overall, community proteogenomics offers new insights into the physiology of phyllosphere microorganisms, their mechanisms of adaptation to the habitat, and their coexistence.


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Cross talk among bacterial members of the phyllosphere

Glenn Dulla, Ksenia Krasileva, Beatriz Quinones, and Steve Lindow


Quorum sensing (QS) within the phyllosphere is affected by many biotic and abiotic elements. QS has been classically characterized as operative in assemblages of high cell density planktonic cells or within biofilms wherein signal of a sufficient concentration to trigger positive feedback loops is achieved. In these conditions, QS activation is defined by or limited to the large volumes of liquid into which signal diffuses away from cells. The plant pathogenic Pseudomonas syringae pv. syringae (Pss) exhibits quorum sensing utilizing the production of the diffusible signal molecules called N‐(acyl)‐L‐homoserine lactones (AHL). QS in Pss controls genes conferring extracellular polysaccharide production, motility, and factors contributing to virulence to bean only when cells reach a relatively high local concentration. QS deficient mutants are hypermotile and entered moist bean leaves more readily and incite formation of more lesions after topical application to bean leaves than the parental strain. We analyzed QS as affected by water availability in Pss in its natural habitat on leaf surfaces. Using confocal microscopy, we utilized the red fluorescent protein mRFP1 as an AHL‐dependent reporter of autoinduction in strains harboring a GFP marker gene allowing us to account for the total epiphytic cell population of Pss. On leaves, it is clear that cells can exhibit QS behavior quickly and when found in small groups on dry leaves where AHL signal diffusion is apparently restricted. QS in Pss is affected by about 18% of culturable epiphytic bacteria via production of small diffusible molecules. About 7% of bacterial epiphytes produced the same AHL, often in amounts more than 20‐fold higher, as Pss. While coinoculation of AHL‐producing strains with Pss reduced the number of lesions when sprayed together on leaves compared with that of plants inoculated with Pss alone, increased lesion number occurred on bean coinoculated with Pss and QS‐inhibiting strains. Premature induction of QS in Pss thus inhibits disease initiation and can be exploited for disease control. Mutants of QS‐interfering strains deficient in this phenotype did not increase disease when coinoculated with Pss. Analysis of mutants in QS‐inhibiting strains revealed interruptions in genes involved in iron‐uptake, such as corA, mvaT, and mucA, suggesting iron plays an inductive role in QS. A direct relationship between the ability of isogenic Escherichia coli strains to sequester iron via their production of different siderophores and their ability to suppress QS in Pss was also observed. QS induction was inversely related to iron availability in culture media supplemented with iron chelators or with FeCl3. Supplementation of iron increases the production of AHLs, affects downstream quorum regulated traits, and reduced the number of lesions on Pss‐inoculated bean.


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Microbe interactions and functions in the phyllosphere as revealed by bioreporters and index organisms

Johan Leveau,

Department of Plant Pathology, UC Davis

This presentation will focus on the bacterial diversity associated with the above‐ground parts of field‐grown crops. Specifically, I will present recent data on the phyllosphere microbiota of lettuce and grape, and focus on 1) the ecology of one particular functional guild, i.e. bacteria that can produce the plant hormone indole 3‐acetic acid (IAA) and 2) any evidence for a link between community composition and foliage contamination with unwanted microorganisms. Bacteria with the ability to synthesize IAA in vitro turned out to be quite abundant on lettuce leaves. Representative isolates were shown to belong to the genera Erwinia, Pantoea and Pseudomonas, and to the suborder Micrococcineae, which includes Arthrobacter, Curtobacterium, Microbacterium, and Pseudoclavibacter species. To reveal whether IAA production by these bacteria also occurs in vivo, a GFP‐based bioreporter is currently being used, which relies on the IAA‐inducibility of the recently identified iac genes for IAA degradation in Pseudomonas strain 1290. In preliminary experiments, it was demonstrated that application of IAA to detached leaves of Romaine lettuce in the dark results in the opening of stomata. Whether this is something that bacterial IAA producers can do too, and what advantage this constitutes to said bacteria or those that live in their vicinity will be a topic of discussion. In the second part of the talk, I will present preliminary data on the community composition of lettuce and grape leaves, as determined by high‐throughput sequencing of 16S rRNA gene amplicons. Of special interest in the analysis of these data are inter‐taxa relationships, i.e. addressing the question whether the occurrence of one microbial taxon is linked to or predicted by the presence or absence of one or more other bacterial taxa. The latter are referred to as index organisms, which might have practical use in forecasting the contamination of lettuce with Escherichia coli O157:H7 or the establishment of the grape powdery mildew pathogen Erysiphe necator in vineyards.


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Plant carbohydrate scavenging through TonB‐dependent transporters by the epiphytic plant pathogen Xanthomonas campestris pv. campestris.

Matthieu Arlat1, Guillaume Dejean1, Alice Boulanger1, Servane Blanvillain‐Baufume1‐2, Anne‐Laure Girard3, Martine Lautier1, Claudine Zischek1, Armelle Darasse3, Marie‐Agnès Jacques3 And Emmanuelle Lauber1.

1 Laboratoire des Interactions Plantes‐Microorganismes – UMR 2594 INRA/CNRS – F‐31326 Castanet‐Tolosan, France; 2 Present address: Department of Plant‐Microbe Interactions, Max‐Planck Institute for Plant Breeding Research, Carl‐von‐Linné Weg 10, 50829 Köln, Germany; 3 UMR077 PaVé, INRA, 42, rue George Morel, F‐49071 Beaucouzé, France

Xanthomonas campestris pv. campestris (Xcc) is a Gram negative bacterium that causes black rot disease on a wide range of Brassicaceae plants of economic interest, including cabbage, cauliflower and radish, as well as the model plant Arabidopsis thaliana. This epiphytic bacterium naturally infects host plants via hydathodes or wounds in the leaves.

Our analysis of Xcc genome revealed an overrepresentation of a particular family of receptors, named TonB‐dependent transporters (TBDTs). These proteins are located in the outer membrane of Gram‐negative bacteria and are mainly known to transport iron‐siderophore complexes and vitamin B12 into the periplasm. In Xcc, we showed that only 9 TBDTs out of 72 are directly associated with iron uptake. A genome context analysis combined with functional studies, allowed us to propose a new function for TBDTs: the scavenging of plant carbohydrates. We established that in Xcc several TBDTs belong to systems, named CUT systems (Carbohydrate Utilization containing TBDT systems), involved in the utilization of various plant compounds. They are defined by the presence of genes coding for carbohydrate degradative enzymes, inner membrane transporters and sugar related regulators beside TBDT genes. We studied 3 such systems, involved in sucrose, xylan or glycan utilization. The sucrose CUT system is clearly required for full pathogenicity on host plants whereas preliminary data suggest that the xylan CUT system might be involved in epiphytic life on cabbage leaves. The glycan CUT system is controlled by N‐acetylglucosamine and our data clearly show that this amino‐sugar is exploited by Xcc during host plant infection.

The overrepresentation of TBDTs and CUT systems is a feature conserved in Xanthomonas species but is not restricted to this genus. Indeed a genome survey and metagenomic studies identified such systems in human gut symbionts, in bacteria associated with plants or plant debris and in bacteria living in aquatic environment as oligotrophs or associated with marine snow, algae or crustaceans. These bacteria share the ability to degrade a wide variety of complex carbohydrates. We therefore propose that TBDT overrepresentation and the presence of CUT systems designate the ability to scavenge carbohydrates. Thus CUT systems, which seem to participate to the adaptation of phytopathogenic bacteria to their host plants, might also play a very important role in human health as well as in the biogeochemical cycling of organic carbon in various environments including phyllosphere.


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Variation in the TonB‐dependent outer‐membrane proteins in plant‐associated strains of Pseudomonas fluorescens

Sierra L. Hartney and Joyce E. Loper

Department of Botany and Plant Pathology and USDA‐ARS, Oregon State University, Corvallis OR, USA.

Nutrient acquisition is key to the ecological fitness of environmental bacteria such as P. fluorescens and TonB‐dependent outer‐membrane proteins are important components of the cellular machinery for the uptake of substrates from the environment. TonB‐dependent outer‐membrane proteins facilitate the uptake and transport of substrates, via association with the energy supplying protein TonB. The roles of TonB‐dependent outer‐membrane proteins as receptors for siderophores, vitamin B12, and certain phages have been recognized for decades, but their broader functions in the uptake of carbohydrates and other substrates have been recognized only recently. Most bacteria have less than 14 TonB dependent outer membrane proteins in their proteomes, but certain environmental bacteria have very large numbers of these receptors. Genomic sequences of ten strains of plant‐associated Pseudomonas spp. were surveyed for the presence of TonB‐dependent outer‐membrane proteins. The strains represent P. fluorescens (seven strains), P. chlororaphis (two strains) and P. synxantha (one strain) isolated from the phyllosphere (two strains) or rhizosphere (eight strains). In addition to published genomic sequences for three strains of P. fluorescens (Pf0‐1, Pf‐5, and SBW25), draft genomic sequences for seven well‐characterized biocontrol strains were also evaluated in this study. The number of TonB‐dependent outer‐membrane proteins range from 14 in the proteome of P. fluorescens Q2‐87 to 45 in P. fluorescens Pf‐5. Sequence motifs defining conserved trans‐membrane, plug, and receptor domains were identified in all of the TonB‐dependent outer‐membrane proteins. An N‐terminal signaling domain was identified in a subset of the proteins defined as transducers, which typically interact with a regulatory protein and ECF sigma factor to regulate expression of target genes. The TonB‐dependent outer‐membrane proteins from all ten genomes were aligned and subjected to maximum parsimony analysis generating two trees (one for transducers and one for receptors, which lack the N‐terminal signaling domain) with distinct, well‐supported clades. This phylogenetic analysis identified a set of transducers and receptors that are conserved across all genomes. Comparisons to proteins with known functions allowed the assignment of putative roles in heme, ferrichrome, B12, copper, and pyoverdine uptake to conserved TonB‐dependent outer‐membrane proteins present in all ten strains. There is further conservation among certain strains;for example, P. fluorescens A506 has 27 proteins in this family with orthologs in P. synxantha BG33R and P. fluorescens SS101. Some strains have TonB‐dependent outer membrane proteins that are not found in the other strains, indicating the possibility of acquisition by horizontal gene transfer. The identification of the core and unique sets of TonB‐dependent outer‐membrane proteins in these genomes will highlight functions conserved across the species as well as those specific to the distinctive lifestyles of each strain.


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Abstracts

Poster Presentations


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Influence of phylloplane microflora metabolites on gene expression in choloroplasts of barley .

Shweta Bhardwaj , Soundarya D , Sumi Jeevan , V. Bhuvaneswari and P.K Paul.

Amity Institute of Biotechnology ; Amity University , Uttar Pradesh ; Sector 125 , Express Highway ; Noida –

201303. Uttar Pradesh , India.

Plant surfaces are known to be colonized by an array of microbes. These microbes interact with their host plants in various ways to influence its growth and development. Though phylloplane microbes have been shown to effect host plants physiology but not much is known on impact of microbial metabolites on functioning of chloroplast. Since microfungal metabolites are known to enter host cells therefore it would be interesting to understand if these metabolites can affect expression of chloroplast genome.

In the present study an effort was made to study the impact of metabolites of four dominant phylloplane microfungi of Hordeum vulgare var. jagriti on expression of chloroplast genome.The fungi selected for the study included Aspergillus niger, Alternaria alternata , Curvularia lunata , Dreschslera spp.

Barley plants were raised and maintained in sterlised soil rite in sterlised trays (24”X 12”x 0.6”) in a culture room with a temperature of 250 C, 75 % RH and 12L/12D light cycle. Three week old plants were used for the study. The plants were divided into groups and sprayed either with undiluted, 1:10 or 1:100 dilutions of each microfungal metabolite.Control plants were sprayed with sterlised distilled water. The microfungi were grown in liquid medium for metabolite production at 25 ±1°C for 7 days.The supernatant after centrifugation was used for treatment of plants. Two dilutions of the extract , 1:10 and 1:100 were also used. Three samples each with three replicates were collected at intervals of 24 hours . Chloroplasts were isolated and purified from sampled leaves . Total DNA , total protein profiles and mRNA were extracted from all chloroplast samples.

Analysis of the results showed that there was a variation in concentration of total DNA between the metabolite treated and control leaves. Metabolite treated leaves had higher concentration of chloroplast DNA.However variation in DNA contents was observed among the various metabolite treated samples.Maximum DNA concentration was recorded from chloroplasts of plants treated with 1:10 dilution of extracts . Undiluted extracts was inhibitory. The effects of metabolites was highest after 48 hours of treatment.The protein profiles of chloroplasts from metabolite treated plants was highest after 72 hours in samples treated with 1: 10 dilution.All the four metabolites seemed to enhance the expression of chloroplast genes as evidenced from higher mRNA concentration in chloroplasts of treated plants.

The results demonstrates the potential of phylloplane microfungal metabolites in affecting the functioning of chloroplasts which could be crucial in affecting crop productivity.


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Salmonella enterica niche overlap in Dickeya dadantii plant lesions and human intestinal epithelia revealed by transcriptome analysis

Brandl, MT1, Goudeau, DM1, Parker, CT1, Sela, S2, and Zhou,Y1.

1Produce Safety and Microbiology Research Unit, USDA, ARS, Albany, CA. 2ARO, Volcani Center, Beth Dagan, Israel.

Human enteric pathogens can exploit the plant habitat and thereby affect public health, as evidenced by the emergence of food‐borne illness associated with leafy vegetables. We have investigated the fitness of Salmonella enterica on lettuce and cilantro leaves, and its interaction with the soft‐rot plant pathogen Dickeya dadantii (Erwinia chrysanthemi). The enteric pathogen achieved large population sizes and formed complex biofilms on leaves infected with D. dadantii. Microarray analysis of the gene expression profile in S. enterica cells colonizing lettuce and cilantro soft rot lesions revealed that the human pathogen deploys a large part of its transcriptional machinery for anaerobic metabolism and the utilization of propanediol and ethanolamine. These pathways, which function in the human intestine, are activated in lettuce and cilantro by the presence of substrates, such as rhamnose, fucose and ethanolamine, resulting directly from the degradation of plant cells by the soft rot pathogen. PduE‐, EutK‐ and CobS‐minus mutants of S. enterica had decreased fitness in the lesions compared to the parental strain. Genes involved in the transport of, or regulated by, quorum sensing autoinducers were induced also in soft rot lesions, indicating possible cross‐talk between the plant and human pathogens. Our observations suggest that D. dadantii contributes to creating a hospitable niche for Salmonella on plants by making available host‐related nutrients that are not abundant on healthy plant surfaces.


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Salmonella interaction with Aspergillus niger leads to biofilm formation and increased internalization into Hela cells

Brandl, MT1, Parker, CT1, Carter, MQ1, Huynh, S1, Zhou, Y1 and Hinton, JCD2.

1Produce Safety and Microbiology Research Unit, USDA, ARS, Albany, CA. 2Trinity College, Dublin, Ireland.

Salmonella enterica cycles between host and nonhost environments, where it may become an active member of complex microbial communities. We report here the rapid attachment and biofilm formation by S. enterica on Aspergillus niger hyphae. Several serovars of S. enterica engaged in a similar association with A. niger, whereas other bacterial species such as E. coli K12, Pantoea agglomerans, and Pseudomonas chlororaphis were unable to bind to the fungus. Bacterial attachment to chitin beads followed the same specificity. N‐acetylglucosamine, a major component of chitin and therefore, of fungal cell walls, inhibited S. enterica attachment to chitin beads by 723‐fold, as well as to A. niger hyphae, suggesting a role for chitin in the initial interaction between the pathogen and the fungus. A cellulose‐deficient mutant of S. enterica Typhimurium with a deletion in bcsB‐bcsA‐yhjQ did not bind to chitin beads nor to the fungus. Complementation of the mutant with the cellulose operon restored binding to both, as well as the ability to form biofilms, providing evidence that cellulose is involved in the attachment of the pathogen to A. niger via the chitin component of its cell wall. In addition, fluorescence microscopy with calcofluor white indicated the presence of cellulose in the biofilm matrix. Microarray transcriptional profiling revealed that curli‐encoding genes were among the most highly upregulated in S. Typhimurium biofilm cells on A. niger. Mutants deficient in AgfD and AgfBA, as well as in the curli regulators RpoS and MlrA, behaved similarly to the parental strain in the early phase of biofilm formation but appeared to contribute to the long‐term stability of the biofilm architecture. We observed also the upregulation of multiple genes involved in pathogenicity, such as SPI‐1, SPI‐2, and SPI‐5 genes, and of numerous fimbriae genes, some of which are virulence determinants. S. Typhimurium cells recovered from these mixed biofilms invaded Hela cells at greater densities than cells pre‐incubated in buffer and LB broth. The transcriptional signature of Salmonella mixed biofilm cells and their increased invasion of human cells suggest that the interaction of this pathogen with A. niger may enhance its virulence in eukaryotic hosts.


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Effects of nitrogen availability on lipid production in Neochloris oleoabundans

when co‐cultured with Methylobacterium

Davis, Charles F., and Holland, Mark

Department of Biology, Salisbury University, Salisbury, MD USA

In order to reduce Americas dependence on foreign oil, biodiesel has been suggested as

an alternative that can be produced domestically. Cellulosic and seed sources, however, require

vast acreage and influence the price of food. Lipid rich algae are a source of biodiesel that can

be produced in large quantities in a small area. In order to develop a strategy for increasing the

productivity of algae farms, one of two types of pink pigmented facultatively methylotrophic

bacteria (PPFMs), either wild type Methylobacterium mesophilicum or a B‐12 overproducing

mutant, were co‐cultivated with the microalga Neochloris oleoabundans. After measuring algae

growth, significant differences were noted between treatments with added bacteria and those

grown without, as well as between the two strains of bacteria. Analysis shows that the addition

of even a small number of PPFMs enhances the growth of algae. Fourteen days into the trials,

however, the chlorophyll content of all cultures decreased dramatically and cell size increased.

These are symptoms of depletion of nitrogen in the media. Without nitrogen, algae are unable

to synthesize chlorophyll or proteins. Without nitrogen the cells are forced to convert new

photosynthate into hydrocarbons and simply store them. It has been shown that under

nitrogen limiting conditions N. oleoabundans responds with increased lipid production. This

study analyzed the lipid production of N. oleoabundans under nitrogen limiting conditions while

being co‐cultured with Methylobacterium. Salisbury University


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The composition and diversity of microbial epiphytes on Tamarix, a salt secreting desert tree, is strongly affected by geography.

Omri Finkel, Adrien Y. Burch, Steven E. Lindow, Anton F. Post and Shimshon Belkin

Department of Plant and Environmental Sciences, Hebrew University of Jerusalem, Edmond J Safra Campus, Givat Ram, Jerusalem, Israel 91904.

The leaf surfaces of Tamarix, a salt secreting genus of desert tree, harbor a diverse community of microbial epiphytes. This island like ecosystem presents a unique set of biogeographic characteristics, where the environment provided by the host is extreme on one hand, and open to the surrounding environment on the other. Thus the Tamarix phyllosphere microbes are free‐living, host‐specific and extremophiles all at the same time. The objective of this study was to understand the ecological gradients on which the structure of the microbial community varies by analyzing the phyllosphere microbial richness and diversity on the leaves of three Tamarix species found in Israel (T. aphylla, T. nilotica and T. tetragina). Leaf samples were collected from four locations in Israel and from two locations in the US. DNA was extracted from these samples and 16/18S rRNA hypervariable tag sequencing was performed.

A total of over 200,000 V6 and V9 sequences were obtained, revealing a complex community, with over 400 bacterial, 80 eukaryotic and one archaeal genera. When clustered according to a 6% similarity cutoff, bacterial samples created more than 2500 clusters. Two main community types were detected. One, found on trees by the Mediterranean, is dominated by Halomonas and Halobacteria. The other, characteristic of trees around the Dead Sea, is dominated by Actinomycetalles and Bacillales. Our findings demonstrate that while the microbial community composition on Tamarix leaves remains steady on a local scale, trees of the same species that grow in different climatic regions, can host microbial communities that are almost completely unrelated.


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Characterization of the barley (Hordeum vulgare) phyllosphere

C. Gravouil, A.C. Newton, M.J. Dickinson, J. Fountaine

The phyllosphere (i.e. the above–ground part of plant) is a largely uncharacterised dynamic habitat where numerous genera (bacteria, fungi, oomycetes etc.) interact with each other, ranging from mutualists to parasites (Lindow and Brandl, 2003). Over the years, interest has been growing in understanding the interactions between leaf‐associated microbes (plant pathogens, biocontrol agents, human pathogens, plant‐growth‐promoting micro‐organisms etc.) and their leaf habitat. Most of the studies in the past were based on culturable techniques but more recently various molecular techniques have enabled us to monitor these micro‐organisms in more detail and the phyllosphere appears to be much more complex and species‐rich than previously described (Yang et al., 2001).

Rhynchosporium secalis (Rs) is a hemibiotrophic fungus and the causal agent of leaf blotch, one of the most important diseases on barley worldwide. Owing to its high genetic variability, Rs can rapidly adapt and overcome the conventional control strategies (Zhan et al., 2007). Newton et al. (2004) have described how resilient pathogenic inoculum (Pectobacterium atrospeticum, causal agent of potato blackleg) slowed the development of Rs on subsequent non‐host barley in the field. This interaction has been also observed under controlled conditions (unpublished data). Hence, it is important to determine the leaf‐associated microbial population of barley and identify how these microbes can interact with cereal foliar diseases and the host, in order to understand and improve control of Rs in the field.

This work has focused on identifying an efficient method to monitor microbial communities of the barley leaves from an experimental field through a growing season to provide an overview of the microbial diversity. A sonication extraction method has been shown to offer a reliable approach for the study of the leaf ecology, as it enables us to differentiate more efficiently the epiphytic communities from the endophytic ones. However, other factors such as the type of Taq polymerase and the primers sequences, which are known to influence the outcome of the PCR, have yet to be evaluated. Taxonomic identities and phylogenetic relationships of epiphytic bacteria have also been undertaken and show contrasting composition of the bacterial phylloplane depending on the isolation technique (either culturable or molecular).

Lindow SE, Brandl MT (2003) Microbiology of the phyllosphere. Appl. Env. Microb. 69: 1875‐1883.

Newton AC, Toth IK, Neave P, Hyman LJ (2004) Bacterial inoculum from a previous crop affects fungal disease development on subsequent nonhost crops. New Phytol. 163: 133‐138.

Yang C‐H, Crowley DE, Borneman J, Keen NT (2001) Microbial phyllosphere populations are more complex than previously realized. PNAS 98: 3889‐3894.

Zhan J, Fitt BDL, Pinnschmidt HO, Oxley SJP, Newton AC (2007) Resistance, epidemiology and sustainable management of Rhynchosporium secalis populations on barley. Pl. Pathol. 57: 1‐14.


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Genetic diversity and phylogeny of methylotrophic community from phyllosphere of tropical crop plants

Manish Kumar, Kamlesh K. Meena, and Dilip. K Arora

National Bureau of Agriculturally Important Microorganisms, Kusmaur, Mau Nath Bhanjan Uttar Pradesh, India‐275101

This study is the demonstration that a diverse methylotrophic microbiota exists in the phyllosphere of tropical crop plants. The methylotrophic communities were observed from phyllosphere by both culture dependent and culture independent assessment. The methylotrophic population abundant in the phyllosphere of the plants to utilize the reduced carbon substrates as a source of carbon and energy. Genes encoding the mxaF of methanol dehydrogenase, and the 16S rRNA genes were detected in all samples tested. mxaF gene amplification, diagnostic for methanol oxidisers was carried out with bacterial DNA (metagenomic DNA and culture DNA) from different tropical crop plants .Over 85 pure cultures of methylotrophs ,isolated on methanol as a corbon source, mxaF gene sequences corresponded to the dominant species Methylobacterium mesophilicum. The DGGE profile of mxaF gene amplified product were obtained from metagenomic DNA of phyllosphere corresponded to the dominant uncultured methylotroph sequences. The Species richness and the evenness was observed remarkably higher in the Pea phyllosphere.


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CONTROL OF COTTON PATHOGENS BY NATURAL ANTAGONISTS

R. N. MANNANOV*, R. K. SATTAROVA

Department of Agricultural Biotechnology and Phytopathology Tashkent State Agrarian University 100140 Tashkent, Republic of Uzbekistan; *Corresponding Author’s Address: Chilanzar 10‐9‐1, 100123 Tashkent, Republic of Uzbekistan e‐mail:[email protected]

Uzbekistan is the world fifth largest cotton grower and second, after USA, cotton exporter growing cotton on 1,35 million ha. In this connection, cotton disease control management is of serious economic importance. Chemical pesticides have been a traditional method used to protect the crop from diseases. However, growing public and scientific concern about presence of synthetic pesticides in food and in the environment has led to great interest in biological control as means of plant protection. In our research, the use of natural soil antagonists Pseudomonas fluorescens, Bacillus subtilis, Bacillus megaterium as means of pre‐sowing seed treatment had shown high biological efficacy by improving seed germination and growth of cotton plants, and by decreasing infection by major cotton pathogens: Xanthomonas malvacearum, Rhizoctonia solani, Fusarium oxysporum var. vasinfectum, Verticillium dahliae. Results from field trials had shown significant biocontrol action of cell suspension of antagonists: root rot infection was reduced from 97,6% to 35,2%, in case with pathogen of bacterial blight – from 92,5% till 37,5%. In result of the use of antagonist cotton yield was increased from 2,2% in control variant till 26,2% and 27,4% in case with strain B. subtilis 23 against bacterial blight and root rot pathogens respectively. The highest biological efficacy was recorded in case with the use of antagonist B. subtilis 23 against R. solani and was 63,9%. Obtained results prove scientifically the prospective from the use of B. subtilis for developing complex, ecologically non ‐ hazardous products for biological control of cotton and other agricultural plants.


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Role of light‐sensing in the Pseudomonas syringae disease lifecycle

Regina Nickel, Liang Wu, Seth Wenner and Gwyn Beattie. Department of Plant Pathology, Iowa State University.

Light‐sensing proteins have been studied in many photosynthetic bacteria; however, their function in non‐photosynthetic bacteria is poorly understood. Genes for three photosensory proteins are present in the genomes of the foliar pathogen Pseudomonas syringae. These three photosensory proteins all have an N‐terminal photosensory domain and a C‐terminal histidine kinase domain. One is a LOV (light, oxygen, voltage) histidine kinase (LOV‐HK), which senses blue light, whereas the other two, BphP1 and BphP2, are bacteriophytochromes, which respond to red light. Among the sequenced strains of Pseudomonas spp., the P. syringae strains are the only ones predicted to encode LOV‐HK proteins; however, a gene for a LOV‐HK protein is present in the genomes of several species of Xanthomonas foliar pathogens as well as the common phyllosphere resident Methylobacterium, suggesting an association between LOV‐HK proteins and adaptation to life in the phyllosphere. In contrast, bacteriophytochromes are not associated with the phyllosphere, as they are absent in Xanthomonas and only a single bacteriophytochrome gene is present in Methylobacterium. Interestingly, P. syringae is the only species of Pseudomonas with genes for two bacteriophytochromes, suggesting that it may be able to sense the ratio of red to far‐red light, similar to phytochromes in plants. Using splice‐overlap‐extension deletion mutagenesis applied to two strains, P. s. pv. syringae B728a and P. s. pv. tomato DC3000, we generated clean deletion mutants that lack the lov‐hk gene (PSPTO2896, Psyr2700), the bphP1 operon (PSPTO1901‐2, Psyr3504‐5), which includes the gene bphO encoding a heme oxygenase for generating a biliverdin cofactor, the bphP2 operon (PSPTO2651‐2, Psyr2384‐5), which includes a gene encoding a response regulator, and both the bphP1 and bphP2 operons. In evaluating the impact of light on the biology of B728a and DC3000, we found that both strains showed reduced growth in the light in multiple liquid growth media, with the reduction more pronounced for DC3000 than for B728a and greatest in low osmolarity media. Thus far, we have not found evidence for a role of any of the phytochrome genes in this light toxicity nor have we found evidence for a role in P. syringae epiphytic growth in a growth chamber, which provides conditions that poorly mimic natural solar radiation. Results will be presented from our initial studies aimed at evaluating the impact of the loss of these photosensory proteins on the behavior of B278a during various stages of its lifecycle in the field.


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In Vitro Antibacterial Activity of Fronds (Leaves) of Some Important Pteridophytes

Pradeep Parihar1, Leena Parihar2 And Achaleshwar Bohra3 1,2 Department of Biotechnology,Lovely Professional University, Phagwara‐144003, Punjab, India; 3Microbiology Laboratory, Department of Botany,Jai Narain Vyas University, Jodhpur‐342 001, Rajasthan, India 1E‐mail: [email protected], [email protected]

Aims: The main objective for this research work is to screen various unexploited plants for their antimicrobial activity as these unexploited or pteridophytic plants are being used ethanomedicinally but very little work has been done on antimicrobial aspects. So, to explore the efficacy of these plants the following research has been carried out.

Methods and Results: Bacterial strains of Agrobacterium tumefaciens, Escherichia coli, Salmonella arizonae, Salmonella typhi and Staphylococcus aureus were procured from the Institute of Microbial Technology (IMTECH), Chandigarh and the aqueous and alcoholic leaves extract of twelve important pteridophytic plants were prepared and tested for their antimicrobial activity against the bacteria selected by Disc diffusion method as suggested by Bauer et al.

It has been observed that nearly all the leaves extracts have shown inhibitory effect against the bacterial strains selected and some of the extracts were more competent than the selected antibiotic.

Conclusions: Our findings provide the novel insights with regards to antimicrobial agents and these could be further enhanced through in vivo studies and isolation and characterization of active constituents for human health.

Significance and Impact of Study: In the present scenario the use of herbs and herbal medicine is at its peak and majority of researchers are screening higher plants for the same but very few researchers are considering the lower plants for their antimicrobial potential. Since these pteridophytic plants are considered to be the disease free plants and are being used ethanobotanically by various tribal communities. These plants are to be further screened for their in vivo potential as well as for their drug properties.


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Phyllosphere of foxtail millet as a habitat for diazotrophic bacteria and facultative symbiotrophic associations.

M. Mohammed Rafi and P.B.B.N. Charyulu

Department of Microbiology, Sri Krishnadevaraya University, Anantapur‐515 003, India.

Leaf surfaces have been found to provide suitable conditions for the colonization by different microorganisms which also include diazotrophic bacteria that are capable of fixing atmospheric nitrogen. In the present study we isolated bacteria from the phyllosphere of foxtail millet (Setaria italica (L.) Beauv) (varieties Lepakshi and Chitra) at 15 day intervals, based on their morphology and pigmentation. Abundant population of bacteria was found on the leaf surfaces which ranged from 3.20 to 4.63 x 106 per gram dry weight of leaf material. A comparison was made on the bacterial population in the phyllosphere of the two varieties of foxtail millet, which revealed that the population in the phyllosphere of Lepakshi variety was slightly higher than the population in Chitra variety. The population of bacteria obtained from the phyllosphere samples at 45 DAS was higher in both the varieties which coincided with the flowering stage. The bacterial populations were tested for their nitrogen fixing abilities which varied in their efficiency to fix nitrogen. Of the 20 isolates selected, only 16 isolates were found to be diazotrophic, with 10 isolates exhibiting appreciable nitrogen fixation. Most of the axenic bacterial isolates were identified as Beijerinckia indica. Facultative symbiotrophic associations isolated from the phyllosphere also exhibited appreciable nitrogen fixing activity.


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The integrated management of foliar diseases of seed spices

T.S. Rajpurohit Agricultural Research Station, (S. K. Rajasthan Agricultural University), Mandor, Jodhpur‐ 342 304 (Rajasthan) India

Sesame (Sesamum indicum L.), suffer due to foliar diseases caused by fungi bacteria, viruses, and phytoplasma making the host weak resulting in losses both in quantity and quality. The important foliar diseases of sesame are Alternaria blight (Alternaria sesami); Cercospora leaf spot (Cercospora sesami and C.sesamicola); Corynespora blight (Corynespora cassicola); Cylinderosporium leaf spot (Cylinderosporium sesami); anthracnose (Colletotrichum sp.); powdery mildew (Leveillula taurica, Oidium sp., Sphaerotheca fuligena); Phoma leaf blight, bacterial leaf spot (Pseudomonas syringae pv. sesami); bacterial blight (Xanthomonas campestris pv. sesami) Leaf curl (Nicotiana virus lo); Phyllody (Phytoplasma) etc.

The Studies on efficacy of foliar spray on incidence of Alternaria leaf spot disease conducted during two years 2008 and 2009 at ARS Mandor Jodhpur results revealed that among eleven fungicides tested minimum incidence of Alternaria leaf spot (4.4%) and highest seed yield ( 551 kg/ha) was observed in foliar spray of Carbendazim + Iprodione @0.1 % followed by Iprodione @ 0.1% (PDI 4.9 %, seed yield 544 kg/ha), Carbendazim+ Mancozeb (PDI 5.37% Yield 532 Kg/ha) and Propiconazole (PDI 6.1 %, seed yield 539 kg/ha).

In another experiment conducted during two years 2008 and 2009 on Management of sesame foliar diseases through botanicals revealed that among seven plant products tested minimum phyllody (1.71 %), leaf curl 1.0 % and highest seed yield (547 kg/ha) was observed in foliar spray of raw neem oil (0.2%) followed by Neem leaf extracts @ 5% (Phyllody 2.15 % and seed yield 536 Kg/ha).

The third field experiment was also conducted in randomized block design with five replications on sesame during kharif 2008 and 2009 at ARS, Mandor‐Jodhpur India to evaluate the efficacy of three integrated disease management module in comparison to farmers practice. Integrated disease management module M1 reduced Macrophomina stem & root rot from 16.2 to 4.7 %, Alternaria leaf spot from 15.6 to 3.9 %, Phyllody from 6.4 to 1.4% and increased seed yield from 410 to 642 kg/ha with B:C ratio 2.43.Organic modules M3 were also found effective in reducing foliar diseases and at par with chemical IDM module for management of foliar diseases.

In lab experiments A. sesami produced pectinolytic and cellulolytic enzymes. The Alternaria is an intercellular fungus the production of these enzymes appears to facilitate the dissolution of a part of the host cell wall and help entry and establishment of the pathogen. Current status of foliar diseases and their integrated management through cultural practices like crop rotation, suitable planting date, integrated fertilizer dose, proper plant population, phytosanitary measures etc. Sesame varieties and lines have been identified resistant to foliar diseases caused by Phytophthora blight, Alternaria blight, Cercospora leaf spot, powdery mildew, bacterial leaf spot, bacterial blight, phyllody singly and several varieties have been identified with resistance to two, three and four diseases, and chemical control measures will be discussed and presented in this paper.


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Studies on the efficacy of soil amendments with neem cake and with bio‐control agent on the incidence of Macrophomina stem and root rot of sesame

T.S. Rajpurohit

Agricultural Research Station , (S. K. Rajasthan Agricultural University), Mandor, Jodhpur‐ 342 304 (Rajasthan) India

Stem and root rot of sesame caused by Macrophomina phaseolina infects high percentage of plants and consequently leads to great yield losses in rainfed crop especially in Rajasthan. The continuous use of chemicals has deleterious effect on the beneficial microorganism in soil, in addition to the residual problem and development of resistance by the pathogen. An experiment was conducted in randomized block design with eight treatments and four replications with plot size of 4x2.4m. on sesame during kharif 2006 ‐ 2007 at Agricultural Research Station, Mandor ‐ Jodhpur (Rajasthan)‐ India to find out the efficacy of soil amendments with neem cakes and with bio‐control agent on the incidence of Macrophomina stem and root rot. The cakes were incorporated in soil and mixed thoroughly before sowing. Bio agent Trichoderma viride was added in FYM 15 days prior to its application and kept in shed and the incidence of Macrophomina stem and root rot was recorded before harvesting. Minimum incidence of Macrophomina stem & root rot (3.32%) and highest seed yield (924 Kg/ha) was recorded in soil application of Neem cake (250 kg/ha) + seed treatment with Trichoderma viride (0.4%) + soil application of T. viride @ 2.5 kg/ha, This treatment gave 82.27 % disease control and 43.92 % yield increase with B : C ratio of 2.88, this was followed seed treatment with T. viride (0.4%) + soil application of T. viride @ 2.5 kg/ha (PDI 6.08%, seed yield 816 kg/ha). Highest disease (13.03%) was recorded in control. Two years results revealed that soil application of Neem cake( 250 kg/ha) + seed treatment with Trichoderma viride (0.4%) + soil application of T. viride @ 2.5 kg/ha reduced Macrophomina stem & root rot from 18.17 to 3.32% and increased seed yield from 642 kg/ha to 924 kg/ha with B : C ratio of 2.88.

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Corresponding Author: T. S. Rajpurohit: E‐mail address: [email protected]


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Salmonella transcriptional signature in the Tetrahymena phagosome and role of acid tolerance in passage through the protist

Rehfuss, MY, Parker, CT and Brandl, MT.

Produce Safety and Microbiology Research Unit, USDA, ARS, Albany, CA.

Salmonella enterica Typhimurium remains undigested in the food vacuoles of the common protist, Tetrahymena. Contrary to its interaction with Acanthamoeba spp., S. Typhimurium is not cytotoxic to Tetrahymena and is egested as viable cells in its fecal pellets. We investigated the factors in S. Typhimurium that are involved in its resistance to digestion by Tetrahymena via microarray gene expression profiling. The transcriptome of S. Typhimurium in Tetrahymena phagosomes revealed that 989 and 1282 genes were altered in expression compared to in water and in LB culture medium, respectively. A great proportion of the upregulated genes have a role in anaerobic metabolism and the use of alternate electron acceptors. Many genes required for survival and replication within macrophages and human epithelial cells also had increased expression in Tetrahymena, including mgtC, one of the most highly induced genes in all three cells types. A ∆mgtC mutant of S. Typhimurium did not display decreased viability in Tetrahymena, but paradoxically, was egested at higher cell density than the wild‐type. The expression of adiA and adiY, which are involved in arginine‐dependent acid resistance, also was increased in the protozoan phagosome. A ∆adiAY mutant had lower viability after passage through Tetrahymena, and a higher proportion of S. Typhimurium wild‐type cells within pellets remained viable after exposure to pH 3.4 compared to uningested cells. Our results provide evidence that acid resistance has a role in the survival of Salmonella to digestion by Tetrahymena and that passage through the protist confers physiological advantages relevant to its contamination cycle.


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Trichomes do not influence bacterial community composition on Arabidopsis thaliana leaf surfaces

E. Reisberg, U. Hildebrandt, M. Riederer, U. Hentschel

University of Würzburg, Department of Botany II, Julius‐von‐Sachs Institute for Biosciences, Julius‐von‐Sachs‐Platz 3, D‐97082 Würzburg, Germany

Several studies have indicated that plant trichomes may affect microbial colonization of the phyllosphere. Various A. thaliana mutants exist that exhibit different trichome densities on their leaves. In this study the A. thaliana ecotype Col‐0 and the gl‐1 mutant, devoid of trichomes on the leaf surface, were compared with regards to natural bacterial community diversity on their leaf surfaces. The wildtype and mutant leaf waxes were analysed by GC/MS and GC/FID. The gl‐1 mutant showed a similar wax composition as the Col‐0 wildtype with slightly reduced amounts of C29, C31 and C33 alkanes. For bacterial diversity analyses, communities were harvested from leaves of ten week old plants grown under controlled climatic conditions. The cultivation of A. thaliana Col‐0 leaf surface associated bacteria on six different media resulted in 120 isolates that were classified into 39 bacterial genera based on partial 16S rDNA sequences. About 20% of the analysed sequences were found to be affiliated with the genus Pseudomonas. Eight sequences exhibited high homologies to the genus Microbacterium and Bacillus and Rhizobium were represented by 6 sequences each. Comparative analyses of the bacterial communities on A. thaliana Col‐0 versus gl‐1 leaf surfaces were performed using cultivation independent denaturing gradient gel electrophoresis (DGGE). Community patterns of the two plant lines showed only minor differences suggesting that the presence of trichomes does not affect bacterial diversity under the given conditions. Sequencing of representative DGGE‐bands is on‐going to reveal the identity of the major community members. Further studies will be performed in order to assess the effects of different plant surface characteristics on microbial abundance and community composition.


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Distribution of epiphytic bacteria on pistachio nut trees in Iran

Saeed Tarighi1, Behrooz Jafarpour1, and Heshmatollah Rahimian2

1Laboratory of Phytopathology, Department of Crop Protection, Ferdowsi University of Mashhad, Mashhad, Iran; 2Laboratory of Phytobacteriology, Department of Crop Protection, Mazandaran University, Sari, Iran

Cultivable Mesophilic heterotrophic, aerobic, anaerobic or facultatively anaerobic bacteria that grow on nutrient yeast glucose extract agar were isolated from the surface of young pistachio leaves in spring, summer and autum. Phenotypic classification of 395 representative strains put them in 44 phena. Of these, 9 were identified to genus or lower level. From the identity of the representative strains, the frequency of occurrence among the phylloplane bacteria over the 2‐year period was estimated at 30% for Flavobacterium spp., followed by Pantoea agglomerans (29.2%), Burkholderia cepacia (28.6%), Enterobacter cloacae (24.7%), Bacillus sp. (18.3%), Pseudomonas fluorescens (12.9%), Burkholderia plantarii (10.8%), Erwinia sp.(3.1%), Brenerria nigrifluens (2.8%), Curtobacterium plantarum (2.2%), Pantoea ananas (2.2%). In addition, the isolated bacteria were analyzed for N‐acyl‐homoserine lactone (AHL) production, which have been reported to associate with bacterial colonization, survival and pathogenesis. Bacterial communities on leaves at a given time during any one year displayed a very similar structure. Communities on the leaves at a given time of the year were variable. During spring and summer time the community of gram‐negative bacteria were higher than gram‐positive bacteria while in autumn the leaf surface of pistachio trees were occupied with gram‐positive bacteria. The results indicated that none of strains had antifungal activity against Aspergilus flavus among predominant bacterial groups. Analysis of AHL production revealed that most bacterial strains in the dominant groups exhibited no production of AHLs in growth medium except Pseudomonas strains. Interestingly, some Bacillus strains showed AHL quenching activities, which suppose one reason that why we don’t have any bacterial disease on pistachio nut trees in Iran. Thus, the differences of these physiological properties among dominant bacteria may be associated with the disease suppression ability on pistachio plants.

Keywords: Pistachio, Bacteria, Epiphyte


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Management of bacterial blight of lilac caused by Pseudomonas syringae by growing plants under plastic shelters

Stockwell, V. O.1, Shaffer, B. T.2, Henkels, M. D.2, Pscheidt, J. W.1 and Loper, J. E.2

1Dept. of Botany and Plant Pathology, Oregon State University, USA.; 2USDA/ARS, Horticultural Crops Research Lab, Corvallis, Oregon, USA.

Pseudomonas syringae pv. syringae causes some of the most economically‐important bacterial diseases affecting woody perennials grown by the nursery industry in the Pacific Northwest of the United States. Symptoms of these diseases include leaf spots, cankers on woody tissues, bud and blossom blights, and shoot tip diebacks. In the past, growers managed these diseases by applying copper‐based bactericides and/or streptomycin from fall through spring bloom to reduce populations of P. syringae on plant surfaces. Unfortunately, these chemicals are no longer as effective in managing diseases caused by P. syringae because many strains of the pathogen isolated from nurseries today are resistant to these compounds. Alternative methods are urgently needed for the management of these diseases. In some commercial nurseries, plants are grown in unheated plastic shelters during the cool and rainy winter months to manage these diseases. This study was initiated to document the efficacy of this cultural control practice against bacterial blight of lilac (Syringa vulgaris ‘Angel White’). Because plastic shelters are thought to manage disease by protecting plants from rain and/or frost, we monitored environmental conditions inside and outside of experimental shelters. To determine the contribution of frost protection to the efficacy of this cultural control practice, the experiment included four treatments: 1) plants grown with no shelters, 2) plants grown under plastic shelters, 3) plants grown with no shelters, but placed under shelters when frost was predicted, and 4) plants grown under shelters, which were removed when frost was predicted. The experiment was done twice in an experimental nursery yard in Corvallis, Oregon (13 February to 30 April 2008 and 11 February to 22 April 2009) with a randomized block design composed of four replicates, each with 12 plants in one‐gallon containers. Plastic shelters provided excellent management of bacterial blight of lilac. In 2008 and 2009, necrotic spots symptomatic of the disease were observed on 55% to 60% of the leaves of plants grown with no shelters vs. <5% of leaves of plants grown under shelters. At the end of the experiments, epiphytic populations of P. syringae exceeded 106 cfu/leaf for plants grown with no shelters vs. <102 cfu/leaf for plants grown under shelters. There were 29 freezing events in 2008 and 22 freezing events in 2009. Average low air temperatures did not differ significantly inside vs. outside of the experimental shelters. Disease severity was similar between treatments 1 and 3, and between treatments 2 and 4, indicating that cover during frost events alone was not a major factor influencing the efficacy of the experimental shelters. Over the course of the experiment, plants grown with no shelters were exposed to rain, with detectable precipitation on 48 days in 2008 and 46 days in 2009. In contrast, plants under shelters remained dry throughout the experiment. These results demonstrate that lilac plants grown under plastic shelters exhibit few symptoms of bacterial blight and support low epiphytic population sizes of P. syringae. Limiting free moisture on leaf surfaces appears to be an important component of the disease control provided by the experimental shelters evaluated in this study.


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Relationship between leaf stomata size and density of lettuce cultivars and severity of bacterial leaf spot.

Toussaint, V., S. Jenni, A. Brillaud and M. Ciotola

Horticulture Research and Development Centre, Agriculture and Agri‐Food Canada, 430 Gouin Blvd, St‐Jean‐sur‐Richelieu, J3B 3E6 Canada

Bacterial leaf spot (BLS) is a serious worldwide disease of lettuce crop caused by Xanthomonas campestris pv. vitians. Disease control recommendations are essentially based on standard prophylactic measures to avoid pathogen proliferation and dispersion. Copper‐based bactericides usually used on vegetable crops are known to be toxic to lettuce. Currently, the use of less susceptible cultivars is the most effective way to restrain the BLS development. Therefore, a breeding program is presently being pursued in our institution to develop resistant cultivars for this bacterial disease. Significant differences among lettuce cultivars for the resistance to BLS have been reported in recent studies and our results support these observations. In general, resistance to bacterial diseases in plants is mostly horizontal meaning that several plant traits play a role in the resistance. Factors inherent to the plant that mostly affect the bacterial leaf spot development are currently being investigated with the aim to better understand the mechanisms of resistance and possibly help the cultivar selection process. The first symptoms of the BLS can develop on the leaf blade, suggesting that the stomata are an important entrance for the pathogen and consequently, their size and density may affect the disease development. To validate this first hypothesis, the number and size of stomata have been determined on 24 cultivars of lettuce and these data were correlated to disease severity index obtained for each of these cultivars. Plants were grown in a 4 inches diameter pots filled with a peat moss medium until the five leaf stage. For each cultivar, fingerprints were made on the second leaf of three replicates. The fingerprints were prepared by applying a transparent polymer on abaxial (lower) and adaxial (upper) leaf surface in the upper third of the leaf and letting it dry for 5 minutes. After the drying period, the polymer layer was peeled off from the leaf surface using pliers and placed on a microscope slide. The density and size of stomata were determined by counting and measuring them over three different uniform fields of 0.80 mm2 for each sample using Infinity Analyze software v. 4.0 from Lumenera Corporation. The analysis of variance showed that the size and the number of stomata were significantly different among cultivars. While the number of stomata was significantly higher on the abaxial than on the adaxial side of the leaves, the size of stomata was not significantly different. Moreover, the size of the stomata was inversely correlated to the number of stomata (r=‐0.87). The size of stomata varied from 40 to 64 μm and their number from 8 to 39 per field. The principal component analysis showed that the disease severity was correlated to the number of stomata, with those on the abaxial side having the greater coefficient (r=0.71). Density of stomata on leaf surface explained 50% of the variation of the disease severity and consequently may be considered an important indicator to predict the cultivar susceptibility to the bacterial leaf spot of lettuce.


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Subsurface Examination of a Foliar Biofilm Using Scanning Electron‐ and Focused‐Ion‐Beam Microscopy

Patricia Kay Wallace1, Bruce Arey2, Walter F. Mahaffee1,3

1 Department of Botany and Plant Pathology, Oregon State University, Corvallis OR; 2Pacific Northwest National Laboratory‐Environmental Molecular Sciences Laboratory, Richland, WA; 3 United States Department of Agriculture – Agriculture Research Service, Horticultural Crops Research Laboratory, Corvallis, OR

The dual beam scanning electron microscope, equipped with both a focused ion‐ and scanning electron‐ beam (FIB SEM) is a novel tool for the exploration of the subsurface structure of biological tissues. The focused ion beam is capable of removing small cross sections to view the subsurface features of fixed and unfied tissue and may be suitable to examining the subsurface structure of bacterial biofilms on the leaf surface. The suitability of chemical and cryofixation was examined for use with the FIB SEM to examine bacterial biofilms on leaf surfaces. The biological control agent, Burkholderia pyroccinia FP62, that rapidly colonizes the leaf surface and forms biofilms, was inoculated onto geranium leaves and incubated in a greenhouse for 7 or 14 days. Cryofixation was not suitable for examination of leaf biofilms because it created a frozen layer over the leaf surface that cracked when exposed to the electron beam and prevented accurate deposition of the protective cap required to stabilize surface for filling. With chemically fixed samples, it was possible to precisely FIB mill a single cross section (5 µm) or sequential cross sections from a single site without any damage to the surrounding surface. Biofilms, 7 days post‐inoculation (DPI), were composed of 2 to 5 bacterial cell layers while biofilms 14 DPI ranged from 5 to greater than 30 cell layers. Empty spaces between bacteria cells in the subsurface structure were observed in biofilms 7‐ and 14‐DPI. Sequential cross sections inferred that the empty spaces were often continuous between FP62 cells and could possibly make up a network of channels throughout the biofilm. FIB SEM was a useful tool to observe the subsurface composition of a foliar biofilm.


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The relationship biofilm production to biocontrol activity of Burkholderia pyroccinia FP62

Patricia K. Wallace1, Walter F. Mahaffee2, Tara M. Neill2, Caroline M. Press2, Meredith M. Larsen2

1Dept. Botany and Plant Pathology, Oregon State University, Corvallis, OR; 2USDA‐ARS Horticulture Crops Research Lab, Corvallis, OR

The efficacy of foliar biocontrol agents is often inconsistent due to poor colonization and survival. Burkholderia pyroccinia FP62 a superior leaf colonist biocontrol agents of Botrytis cinerea that forms unsaturated biofilms on leaves. A mini Tn5 mutant library was screened for biofilm and biocontrol deficiency. The mutant 55B1 was found to have an insertion in a region homologous to TrmE GTPase which is involved in resistance to environmental stresses. 55B1 was completed by marker exchange (55B1C). Washed bacterial cell suspensions with and without polysaccharides were inoculated onto geraniums then incubated in a greenhouse with bottom watering for 0, 1, 3, or 7 days. Population dynamics were monitored by sonicating leaves in phosphate buffer and plating washates on 5% yeast extract broth agar. Leaf samples were taken pre and post sonication, chemically fixed and viewed with scanning electron microscopy (SEM). Inoculated plants were incubated for 1 or 7 days, then challenged with B. cinerea, and incubated at 20ºC with >85% RH for 5‐7 days before rating for percent diseased leaf area. Biocontrol activity was significantly reduced in 55B1 and restored in 55B1C. Culturable populations were similar, however; SEM revealed that significant numbers of FP62 and 55B1C cells remained attached to the leaf surface. The addition of polysaccharides to the 55B1 inoculum restored wildtype activity. These data suggests that biofilm production is vital to the biocontrol activity of FP62.


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Directory of

Participants


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Alamri, Dr Saad PO Box 10255 Abha, Asir 61321 Saudi Arabia P: 96‐655‐233‐9919 [email protected] Amato, Pierre CNRS ‐ Clermont université SEESIB Université Blaise Pascal Aubiere, France 63177 France P: 33 (0)4 73 40 53 20 pierre.amato@univ‐bpclermont.fr Arlat, Matthieu UNiversity Toulouse 3/INRA Chemin de Borde‐Rouge BP52627 Castanet Tolosan, Haute Garonne 31326 France P: (+33)561 28 50 47 [email protected] Beattie, Gwyn Iowa State University Department of Plant Pathology 207 Science I Ames, IA 50011 USA P: 515‐294‐5571 [email protected] Belkin, Shimshon Hebrew University of Jerusalem Institute of Life Sciences Jerusalem, Israel 91904 Israel P: 972‐2‐6584192 [email protected]

Brandl, Maria USDA, ARS, WRRC 800 Buchanan Street Albany, CA 94710 USA P: 510‐559‐5885 [email protected] Burch, Adrien 111 Koshland Hall University of California Berkeley, CA 94720 USA P: 510‐643‐6498 [email protected] Burkhardt, Juergen University of Bonn, INRES‐PE Karlrobert‐Kreiten‐Str. 13 Bonn, Germany 0 Germany P: 49‐228‐732186 j.burkhardt@uni‐bonn.de Cho, Juliana UC Berkeley‐ Lindow Lab 385 Jayne Avenue #306 Oakland, CA 94610 USA P: 510‐599‐5862 [email protected] Crowley, David Dept Environmental Sciences, University of California Riverside, CA 92521 United States P: 951‐827‐3785 [email protected] Davis, Chuck Salisbury University Department of Biology Salisbury, MD 21801 USA P: 443‐523‐5908 [email protected]


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Dulla, Glenn 1202 N 160th St. Shoreline, WA 98133 P: 415‐336‐8546 [email protected] Dunlap, Christopher USDA/ARS/NCAUR/CBP 1815 N University Street Peoria, IL 61604 USA P: 309‐681‐6339 [email protected] Finkel, Omri Hebrew University of Jerusalem Department of Plant and Environmental Sciences Edmond J Safra Campus, Givat Ram Jerusalem, Israel 91904 Israel P: 972‐2‐6584862 [email protected] Gravouil, Clement Scottish Crop Research Institute Errol Road Invergowrie Dundee, Scotland 0 United Kingdom P: +44 (0)1382 562731 [email protected] Hartney, Sierra Oregon State University USDA‐ARS 3420 NW Orchard Avenue Corvallis, OR 97330 P: 541‐738‐4108 [email protected]

Hildebrandt, Ulrich University of Wuerzburg Julius‐von‐Sachs‐Institute for Biosciences Julius‐von‐Sachs‐Platz 3 Wuerzburg, Bavaria 97082 Germany P: 49(0)931 3186206 [email protected]‐wuerzburg.de Hockett, Kevin UC Berkeley 1421 Walnut Street Unit 1 Berkeley, CA 94709 USA P: 503‐701‐9892 [email protected] Hogenhout, Saskia The John Innes Centre Norwich Research Park Colney Lane Norwich, Norfolk 0 United Kingdom P: 44 1603 450393 [email protected] Holland, Mark A Salisbury University Department of Biology Salisbury, MD 21801 USA P: 410‐548‐5590 [email protected] Hovinga (Lego), Sarah AgraQuest, Inc. 1540 Drew Avenue Davis, Ca 95618 USA P: 530‐304‐4197 [email protected], [email protected]


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Kisluk, Guy Zahal st. 12 Tirat Carmel, Tirat Carmel 30200 Israel P: 972‐4‐8294259 [email protected] Knief, Claudia Institute of Microbiology, ETH Zurich Wolfgang‐Pauli‐Strasse 10 Zurich, Switzerland 8093 Switzerland P: 41‐4463‐23‐830 [email protected] Lambais, Marcio Universidade de São Paulo Av. Pádua Dias 11 LSO, ESALQ Piracicaba, São Paulo 0 Brazil P: 55 19 3417‐2107 [email protected] Leveau, Johan University of California One Shields Avenue Davis, CA 95616 USA P: 530‐752‐5046 [email protected] Lindow, Steven University of California Dept. Plant and Microbial Biology 111 Koshland Hall Berkeley, CA 94720 USA P: 510‐642‐4174 [email protected]

Lonjidkhorloo, Erdenebulgan lecturar and researcher at mongolian national university Apartment 8 Building 1‐ 2 VII Khoro, Sukhbaatar District ulaanbaatar, ‐ 976 mongolia P: 976‐95648822 [email protected] Loper, Joyce USDA‐ARS 3420 NW Orchard Avenue Corvallis, OR 97330 USA P: 541‐738‐4057 [email protected] Mahaffee, Walter USDA‐ARS 3420 NW Orchard Avenue Corvallis, OR 97330 USA P: 541‐738‐4036 [email protected] Mannanov, Rustam Tashkent State Agro University Chilanzar 10‐9‐1 Tashkent, Tashkent 100123 Republic of Uzbekistan P: +998 97 1576516 [email protected] Marco, Maria University of California, Davis One Shields Avenue Davis, CA 95161 P: 530‐752‐1516 [email protected] Martin, Danielle 3420 NW Orchard Avenue Corvallis, OR 97330 USA P: 541‐738‐4085 [email protected]


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Meric, Guillaume Institute of Food Research Norwich Research Park Colney Ln, Colney Norwich, Norfolk 0 United Kingdom P: 44160325‐5246 [email protected] Millican, Michael Iowa State University Department of Plant Pathology 207 Science I Ames, IA 0 USA P: 515‐294‐3198 [email protected] Monteil, Caroline INRA Domaine St. Maurice BP 94 Montfavet Cedex, France 84143 France P: 04 32 72 28 40 [email protected] Morris, Cindy INRA Plant Pathology Research Unit BP 94, Domaine St. Maurice Montfavet, France 84140 France P: 33 432 72 28 41 [email protected] Mukhtar, Irum house # A‐1 Waris colony, wahdat road lahore pakistan lahore, punjab 54600 P: 092‐3024067818 [email protected]

Mundt, Christopher Oregon State University Dept. of Botany and Plant Pathology 2082 Cordley Hall Corvallis, OR 97331 USA P: 541‐737‐5256 [email protected] Nadiradze, Kakha Biotechnology Center 30 App 5 B 1 M.D. Vazisubani 1 A Str, Sarajishvili Tbilisi, GE 197 Georgia P: 99532654878 [email protected] Namjilsuren, Nandin‐Erdene Researcher at Institute of Biology, Mongolian Academy of Sceinces Apartment 27 Building 125 XIII Khoro, Enkhtaivan Avenue Bayanzurkh District Ulaanbaatar, none 976 Mongolia P: 976 98111396 [email protected] Neill, Tara 3420 NW Orchard Avenue Corvallis, OR 97330 USA P: 541‐738‐4028 [email protected] Nickel, Regina Iowa State University Department of Plant Pathology 207 Science I Ames, IA 0 USA P: 515‐294‐9792 [email protected]


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Nix, Shannon Clarion University Department of Biology 840 Wood Street Clarion, PA 16214 USA P: 814‐393‐1905 [email protected] Ottesen, Andrea fda 4118 wexford ct. kensington, md 20895 P: 240 277 4595 [email protected] Parihar, Dr. Pradeep Lovely Professional University Phagwara Punjab Phagwara, Punjab 144002 India P: 9197792‐10621 [email protected] Paul, Prabir Kumar Amity Institute of Biotechnology Amity University , Uttar Pradesh ,Sector 125 NOIDA, Uttar Pradesh 201303 India P: 011‐22379821, 9818789144 [email protected] Powell, Tracy University of California, Berkeley 111 Koshland Hall, PMB University of California, Berkeley Berkeley, CA 94720 USA P: 510‐643‐6498 [email protected]

Rajpurohit, T. S. S. K. Rajasthan Agricultural University Agricultural Research Station Mandor Jodhpur 34, Imratia Bera, Paota c road, Jodhpur Jodhpur, Rajasthan 342304 India P: 91 9414921262, 91 291 2549130 [email protected] Ramos, Eugenia UC‐Berkeley 111 Koshland Hall Berkeley, CA 94720 USA P: 510‐643‐6498 [email protected] Rangel, Lorena Oregon State University USDA‐ARS Horticultural Crops Research Lab 3420 NW Orchard Avenue Corvallis, OR 97330 USA P: 541‐738‐4071 [email protected] Rasheed, Adedeji CRIN Cocoa Research Institute of Nigeria Idi‐Ayure Ibadan, Oyo 0 Nigeria P: 234‐805‐560‐7861 [email protected] Reisberg, Eva University of Wuerzburg Julius‐von‐Sachs‐Institute for Biosciences Julius‐von‐Sachs‐Platz 3 Wuerzburg, Bavaria 97082 Germany P: 49 (0) 931 31 80866 eva.reisberg@stud‐mail.uni‐wuerzburg.de


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Remus‐Emsermann, Mitja Boterhoeksestraat 48 Heteren, Gelderland 0 Netherlands P: 31264791412 m.remus‐[email protected] Sammer, Ulrike Friedrich Schiller University of Jena Institute of Microbiology/ Microbial Phytopathology Neugasse 25 Jena, Thuringia 0 Germany P: 49 3641 949293 ulrike.sammer@uni‐jena.de Scheublin, Tanja Netherlands Institute of Ecology (NIOO‐KNAW) Boterhoeksestraat 48 Heteren, The Netherlands 0 The Netherlands P: +31‐(0)26 4791316 [email protected] Schreiber, Lukas Bonn University Department of Ecophysiology Kirschalle 1 Bonn, North Rhine‐Westphalia 53115 Germany P: 0049 228 73 4687 lukas.schreiber@uni‐bonn.de Scott, Russell U.C. Berkeley, Dept. of Plant and Microbial Biology, Lindow Lab 311 Koshland Hall Berkeley, CA 94720 USA P: 510‐643‐6498 [email protected]

Sela, Shlomo Department of Food Quality and Safety, Institute of Postharvest and Food Sciences, Agricultural Research Organization, The Volcani Center P.O.B 6 Beth‐Dagan, N/A 50250 ISRAEL P: 972‐3‐9683750 [email protected] Shaffer, Brenda USDA‐ARS Horticultural Crops Research Lab 3420 NW Orchard Avenue Corvallis, OR 97330 USA P: 541‐738‐4055 [email protected] Shipp, Les Agriculture and Agri‐Food Canada 2585 County Road 20 Harrow, Ontario 0 Canada P: 519‐738‐1235 [email protected] Shirron, Natali Prof.Sima Yaron, Biotechnology and Food Engineering Technion Haifa, Israel 32000 Israel P: 972‐545790388 [email protected] Stapleton, Ann University of North Carolina at Wilmington Department of Biology and Marine Biology UNCW 601 S. College Wilmington, NC 28403 USA P: 910‐962‐7267 [email protected]


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Steinkraus, Don University of Arkansas Department of Entomology 319 AGRI Fayetteville, AR 72701 USA P: 479‐575‐3187 [email protected] Stoll, Rob Department of Mechanical Engineering University of Utah 50 S. Central Campus Drive, RM 2110 Salt Lake City, UT 84112 USA P: 801‐581‐3405 [email protected] Suslow, Trevor Department of Vegetable Crops University California, Davis Davis, CA P: 530‐754‐8313 [email protected] Tecon, Robin NIOO‐KNAW Department of Plant Pathology, University of California Davis 465 Hutchison, One Shields Avenue Davis, CA 95616 USA P: 530‐752‐5191 [email protected] Teviotdale, Beth 1072 Rorden Avenue Selma, CA 93662 P: 559‐891‐1497 [email protected] Toussaint, Vicky Agriculture and Agri‐Food Canada 430 Boul. Gouin St‐Jean‐sur‐Richelieu, Quebec 0 Canada P: 450‐515‐2098 [email protected]

van der Wal, Annemieke Boterhoeksestraat 48 Heteren, Holland 0 Holland P: 0031‐26‐4791111 [email protected] Vasquez David, Luz Estella Street 84A # 52D‐90 Medellin, Antioquia Colombia P: 057‐4‐2854578 [email protected] Vorholt, Julia ETH Zurich Wolfgang‐Pauli‐Strasse 10 Hoenggerberg HCI F429 Zurich, CH 8093 Switzerland P: 41‐44‐632‐5524 [email protected] Ward, Nicole Louisiana State University 302 Life Sciences Baton Rouge, LA 70753 USA P: 225‐718‐2652 [email protected] Wiedinmyer, Christine National Center for Atmospheric Research P.O. Box 3000 Boulder, CO 80307 US P: 303‐497‐1414 [email protected] Williams, Thomas UC Davis Food Science One Shields Ave. Davis, California 95616 P: 530‐752‐7066 [email protected]


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Wu, Liang Iowa State University Department of Plant Pathology 207 Science I Ames, IA 50011 USA P: 515‐294‐9792 [email protected] Yaron, Sima Technion‐ Israel Institute of Technology Faculty of Biotechnology and Food Eng Technion Haifa, Israel 32000 Israel P: 972 4 8292940 [email protected] Yu, Xilan Iowa State University Department of Plant Pathology 207 Science I Ames, IA 50011 USA P: 515‐294‐9792 [email protected]

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The 9th International of the Microbial Ecology of Plant ...people.oregonstate.edu/~mahaffew/MeetingProgramandAbstracts.pdf · of the Microbial Ecology of ... Ninth International Symposium