Biology of the Fire Blight Pathogen Erwinia amylovora Under Starvation Conditions: Survival Strategies and Virulence

Bilgy f the Fire Blight Pathgen Erwinia amylvra Under Starvatin Cnditins: Survival Strategies and Virulence Ricard Delgad Santander 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 1

Unknwn Aspects f the E. amylvra Life Cycle Hw des E. amylvra deal with nutrient scarcity? Are rts suitable sites f entry f E. amylvra int the hst? 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 2

Unknwn Aspects f the E. amylvra Life Cycle Hw des E. amylvra deal with nutrient scarcity in cankers, plant surfaces r, in general, utside the hst? Nutrient Limitatin Other stressing envirnmental cnditins: Lw/high Temperature, Relative Humidity, etc. 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 3

Unknwn Aspects f the E. amylvra Life Cycle Hw des E. amylvra deal with nutrient scarcity in cankers, plant surfaces r, in general, utside the hst? What happens under Nutrient Rich cnditins? Flagella Rd shaped cells Nn stressed cells Bacterial mtility. Surce: classes.midlandstech.edu 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 4

Unknwn Aspects f the E. amylvra Life Cycle Hw des E. amylvra deal with nutrient scarcity in cankers, plant surfaces r, in general, utside the hst? What happens under Nutrient Rich cnditins? Millins f cells becme visible t the human eye Culture medium Culture medium Bacterial Clny Surce: http://www.bruker.cm 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 5

Unknwn Aspects f the E. amylvra Life Cycle Hw des E. amylvra deal with nutrient scarcity in cankers, plant surfaces r, in general, utside the hst? What happens under Nutrient Rich cnditins? Culture medium Fruit blight picture by Sctt Nelsn Basis fr Plant Disease Diagnsis Surce: http://www.bruker.cm 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 6

Unknwn Aspects f the E. amylvra Life Cycle Hw des E. amylvra deal with Nutrient Scarcity in cankers, plant surfaces r, in general, utside the hst? Prgressive Lss f Flagella / Mtility Nutrient Limitatin Dwarfing + Acquisitin f runded shapes Nn stressed cells Develpment f Surface Vesicles Starved cells 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 7

Unknwn Aspects f the E. amylvra Life Cycle Hw des E. amylvra deal with nutrient scarcity in cankers, plant surfaces r, in general, utside the hst? Prgressive Lss f Flagella / Mtility Nutrient Limitatin Dwarfing + Acquisitin f runded shapes Nn stressed cells Develpment f Surface Vesicles 200 nm TEM images f E. amylvra starved cells, by Ricard D. SaNtander 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 8

Unknwn Aspects f the E. amylvra Life Cycle Hw des E. amylvra deal with nutrient scarcity in cankers, plant surfaces r, in general, utside the hst? But what happens with Bacterial Culturability? Prgressive Lss f Flagella / Mtility Nutrient Limitatin Dwarfing + Acquisitin f runded shapes Nn stressed cells Develpment f Surface Vesicles 200 nm 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 9

Unknwn Aspects f the E. amylvra Life Cycle Effects f Starvatin n E. amylvra culturability Nutrient rich cnditins Fruit blight picture by Sctt Nelsn E.g. Within the hst under favrable grwth temperature cnditins Early infectin Late Infectin Starvatin cnditins After days f starvatin After mnths f starvatin After years f starvatin e.g. Rainwater 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 10

Unknwn Aspects f the E. amylvra Life Cycle Effects f Starvatin n E. amylvra culturability Starvatin cnditins After days f starvatin After mnths f starvatin After years f starvatin e.g. Rainwater CONCLUSION Years Ag EXPOSURE OF BACTERIA TO STARVATION KILLS CELLS OVER TIME 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 11

Unknwn Aspects f the E. amylvra Life Cycle Effects f Starvatin n E. amylvra culturability Starvatin cnditins After days f starvatin After mnths f starvatin After years f starvatin e.g. Rainwater CONCLUSION Years Ag Nwadays EXPOSURE OF BACTERIA TO STARVATION KILLS CELLS OVER TIME EXPOSURE OF BACTERIA TO STARVATION INDUCES THE ENTRY INTO A DORMANT VIABLE BUT NONCULTURABLE (VBNC) STATE 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 12

Unknwn Aspects f the E. amylvra Life Cycle Effects f Starvatin n E. amylvra culturability Starvatin cnditins After days f starvatin After mnths f starvatin After years f starvatin e.g. Rainwater Drmant bacteria by David Parkins 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 13

Unknwn Aspects f the E. amylvra Life Cycle Effects f Starvatin n E. amylvra culturability Starvatin cnditins After days f starvatin After mnths f starvatin After years f starvatin e.g. Rainwater Plant Diagnstic Prtcls based n CULTURE DEPENDENT METHODS Apple seedling. Surce: https://www.dreamstime.cm Recvery f VBNC Cells after passage thrugh a susceptible hst 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 14

Unknwn Aspects f the E. amylvra Life Cycle Effects f Starvatin n bacterial culturability BACTERIAL DORMANCY The Viable But Nnculturable (VBNC) State Characteristics f VBNC cells Metablically active Increased tlerance t stress (including antibitics, high temperatures, cpper, etc) Gene expressin and ptential virulence. Ability t recver culturability after remving the stressing factr, passage thrugh the hst, etc Drmant bacteria by David Parkins 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 15

Unknwn Aspects f the E. amylvra Life Cycle Effects f Starvatin n bacterial culturability BACTERIAL DORMANCY The Viable But Nnculturable (VBNC) State Hw can we determine BACTERIAL VIABILITY in VBNC cells? VIABILITY STAININGS MOLECULAR METHODS Drmant bacteria by David Parkins 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 16

Unknwn Aspects f the E. amylvra Life Cycle Effects f Starvatin n bacterial culturability BACTERIAL DORMANCY The Viable But Nnculturable (VBNC) State VIABILITY STAININGS E.g. SYTO9 + PROPIDIUM IODIDE DNA Live Bacteria Intact membrane DNA Dead Bacteria Damaged membrane 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 17

Unknwn Aspects f the E. amylvra Life Cycle Effects f Starvatin n bacterial culturability BACTERIAL DORMANCY The Viable But Nnculturable (VBNC) State VIABILITY STAININGS E.g. SYTO9 + PROPIDIUM IODIDE Starved cells Live Bacteria Intact membrane Dead Bacteria Damaged membrane NO GROWTH On regular culture media DETECTION OF VIABLE CELLS By culture independent methds 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 18

Unknwn Aspects f the E. amylvra Life Cycle Effects f Starvatin n bacterial culturability BACTERIAL DORMANCY The Viable But Nnculturable (VBNC) State VIABILITY STAININGS Live Bacteria Intact membrane Stresses inducing the VBNC state in E. amylvra: Cpper Chlrine (in tap water) Starvatin in rainwater, distilled water, river water and mineral water Starvatin in sil Starvatin in/n insects Starvatin in the apple calyx Starvatin in artificial media Dead Bacteria Damaged membrane 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 19

Unknwn Aspects f the E. amylvra Life Cycle Effects f Starvatin n bacterial culturability BACTERIAL DORMANCY The Viable But Nnculturable (VBNC) State VIABILITY STAININGS Live Bacteria Intact membrane Stresses inducing the VBNC state in E. amylvra: Cpper Chlrine (in tap water) Starvatin in rainwater, distilled water, river water and mineral water Starvatin in sil Starvatin in/n insects Starvatin in the apple calyx Starvatin in artificial media Dead Bacteria Damaged membrane 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 20

Unknwn Aspects f the E. amylvra Life Cycle Effects f Starvatin n bacterial culturability BACTERIAL DORMANCY The Viable But Nnculturable (VBNC) State Effect f Temperature n starvatin respnses. Maintenance f viability 98.6ºF 82.4ºF 68ºF 57.2ºF 39.2ºF 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 21

Unknwn Aspects f the E. amylvra Life Cycle Effects f Starvatin n bacterial culturability BACTERIAL DORMANCY The Viable But Nnculturable (VBNC) State Effect f Temperature n starvatin respnses. Maintenance f viability Starvatin-survival state (CULTURABILTY) 98.6ºF 82.4ºF 68ºF 57.2ºF 39.2ºF 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 22

Unknwn Aspects f the E. amylvra Life Cycle Effects f Starvatin n bacterial culturability BACTERIAL DORMANCY The Viable But Nnculturable (VBNC) State Effect f Temperature n starvatin respnses. Maintenance f viability Starvatin-survival state VBNC respnse 98.6ºF 82.4ºF 68ºF 57.2ºF 39.2ºF 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 23

Unknwn Aspects f the E. amylvra Life Cycle Effects f Starvatin n bacterial culturability BACTERIAL DORMANCY The Viable But Nnculturable (VBNC) State Effect f Temperature n starvatin respnses. Is the VBNC a stage f the E. amylvra life cycle? Rle in Survival / Overwintering / Asymptmatic infectins? CURRENT PROJECT Determine the presence and quantify E. amylvra culturable and viable cell ppulatins in cankers f trees f different hsts (apple and pear) and cultivars 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 24

Unknwn Aspects f the E. amylvra Life Cycle Effects f Starvatin n bacterial culturability BACTERIAL DORMANCY The Viable But Nnculturable (VBNC) State Mlecular Methds E.g. digital PCR + Prpidium Mnazide Live Bacteria Intact membrane Dead Bacteria Damaged membrane E. amylvra specific DNA sequence E. amylvra specific DNA sequence PCR amplificatin If E. amylvra is present: POSITIVE DETECTION Hw can we differentiate DNA cming frm live r dead cells?? 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 25

Unknwn Aspects f the E. amylvra Life Cycle Effects f Starvatin n bacterial culturability BACTERIAL DORMANCY The Viable But Nnculturable (VBNC) State Mlecular Methds E.g. digital PCR + Prpidium Mnazide Live Bacteria Intact membrane E. amylvra specific DNA sequence Dead Bacteria Damaged membrane E. amylvra specific DNA sequence digital PCR Quantificatin f LIVE CELLS DNA UNSUITABLE fr PCR 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 26

Unknwn Aspects f the E. amylvra Life Cycle Effects f Starvatin n bacterial culturability BACTERIAL DORMANCY The Viable But Nnculturable (VBNC) State E. amylvra cells expsed t starvatin at 7ºC Plate cunts at time 0 10 6 CFU/mL Plate cunts after 3 mnths at 7ºC 10 4 CFU/mL digital PCR + Prpidium Mnazide after 3 mnths at 7ºC 10 6 CFU/mL 9.9 10 5 CFU/mL VBNC CELLS Drmant bacteria by David Parkins 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference

Unknwn Aspects f the E. amylvra Life Cycle Effects f Starvatin n bacterial culturability BACTERIAL DORMANCY The Viable But Nnculturable (VBNC) State Average 2.7 10 4 live cells/canker Radial decrease f E. amylvra cell ppulatins, utwards frm the canker edge. Canker n apple (Surce: https://fff.hrt.purdue.edu/) 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference

Unknwn Aspects f the E. amylvra Life Cycle Effects f Starvatin n bacterial culturability BACTERIAL DORMANCY The Viable But Nnculturable (VBNC) State Rt Blight Are rts suitable sites f entry int the hst? 360 sampled rtstcks at 7 farms in Champlain Valley (Nv 2016) # PCR+ Samples / Ttal # samples % Infected Rtstcks Orchard #1 19/54 35.2 Orchard #2 27/52 51.9 Orchard #3 19/54 35.2 Orchard #4 18/54 33.3 Orchard #5 27/63 42.9 Orchard #6 11/55 20.0 Orchard #7 3/28 10.7 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 29

Unknwn Aspects f the E. amylvra Life Cycle Effects f Starvatin n bacterial culturability BACTERIAL DORMANCY The Viable But Nnculturable (VBNC) State Rt Blight Are rts suitable sites f entry int the hst? Previus Data (assays in apple): E. amylvra cells inculated int the stem migrate t the rts Inculatin f rts des nt invlve stem invasin In pear? 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 30

Unknwn Aspects f the E. amylvra Life Cycle Effects f Starvatin n bacterial culturability BACTERIAL DORMANCY The Viable But Nnculturable (VBNC) State Rt Blight Are rts suitable sites f entry int the hst? 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 31

Unknwn Aspects f the E. amylvra Life Cycle * 100 % f rt inculated plants develped symptms in the aerial rgans 2-3 days pst-inculatin E. amylvra migrates frm the rts t the stem thrughut the vascular vessels Cells escape frm the vascular cylinder in petiles and the tip f the stem Fire Blight Symptms Migratin f cells thrugh the crtex ccurs upwards and dwnwards 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 32

Unknwn Aspects f the E. amylvra Life Cycle Effects f Starvatin n bacterial culturability BACTERIAL DORMANCY The Viable But Nnculturable (VBNC) State Rt Blight Are rts suitable sites f entry int the hst? Green Flurescent transfrmant 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 33

Unknwn Aspects f the E. amylvra Life Cycle 6 5 1 4 2 3 06/04/2018 2018 Eastern New Yrk Fruit and Vegetable Cnference 34

Acknwledgements This material is based upn wrk that is supprted by the USDA's Natinal Institute f Fd and Agriculture, U.S. Department f Agriculture, Multistate under 2017-18-267: W3185: Bilgical Cntrl in Pest Management fr the prject "Ppulatin Dynamics f Fire Blight Bacterium Erwinia amylvra in Cankers Under Drught Stress and Winter Cld.