Phighting Phire Blight with Phage

Julianne H. Grose Brigham Young University

The Natural World Has Boundless Untapped Potential Natural Resources are Boundless

Bacteriophages are the Most Abundant Biological Entity on the Planet

Mass is about equal to the entire world population, but only if we are all sumo wrestlers. ~Costa Georgeopoulous

Introducing Bacteriophage As A Disease Therapy

Bacteria: Phage:

+ = +

head

tail

Viruses Turn a Host Cell Factory into a Viral Factory

Lytic Cycle of

Phages

Adapted from “Microbiology, and introduction”, Totora, Funk and Case, 2013

Tail fibers recognize the host cell

Advantages to Phage Therapy

• Typically Harmless to Host Organism and Other Beneficial Bacteria • High Therapeutic Index (few side effects) • Self replicating but also self limiting • Considered organic

www.education.ecspasy.org

Fredrick Twort

Phage Therapies

Felix d’Herelle

1940’s – Gangreen and

Dysentary

Staph & Step i.e

Lactococcus & Vibrio

Phage Therapies in the US

2006-Campylobacter, Listeria & Shigella (Listix and Intralytics)

Salmonella, E.coli & Campylobacter (Intralytics – E. coli and Salmonella)

Xanthamonas – Agriphage 2005 (Omnilytics)

Currently in clinical trials - Staph & Step (Intralytics)

PRODUCT COMPANY CROP EPA

Agriphage Omnilytics Tomatoes/peppers # 67986-1

Phage Therapies: Possible Problems

1. Narrow specificity toward bacterial strains and bacterial evolution/resistance

2. Phages can also contribute to pathogenicity of bacteria ooppacificassets.s3.amazonaws.com www.aarogya.com

The Two Lifecycles of Phages

E. amylovora ATCC

E. amylovora (Washington)

E. amylovora (Michigan)

E. coli Salmonella Yersinia Bacillus sp.

YES YES YES NO NO NO NO

Good Practices in Phage Therapy

• Use strictly lytic phage • Use a cocktail of unrelated phages

• Test host range

Good Practices in Phage Therapy

• Use strictly lytic phage • Use a cocktail of unrelated phages

ooppacificassets.s3.amazonaws.com

• Fully sequence phages

• Test host range

Erwinia amylovora is the Causative Agent of Fire Blight

Primary US Apple Production

Washington (64%) $1,142,105

New York $172,860

Fire blight leads to 100-500 million dollars worth of apple agricultural loss in the US/year

Current Fire Blight Prevention

Treatment of choice Agrimycin (streptomycin sulfate)

4 applications spring, 4 application fall

$30/acre/application = $240/year/acre Many non-responsive infections (20-70% efficacy reported)

Our Solution: Fire Quencher DIRECTIONS FOR USE It is a violation of Federal Law to use this product in a manner inconsistent with its labeling. Do not apply this product in a way that will contact workers or other persons, either directly or through drift. Only protected handlers may be in the area during application. For any requirement specific to your State and Tribe, consult the agency responsible for pesticide regulation. USER SAFETY RECOMMENDATIONS Wash hands before eating, drinking, chewing gum, using tobacco, or using the toilet. AGRICULTURAL USE REQUIREMENTS Use this product only in accordance with its labeling and with the Worker Protection Standard, 40 CFR part 170. This standard contains requirements for the protection of agricultural workers on farms, forests, nurseries, and green houses, and handlers of agricultural pesticides. It contains requirements for training, decontamination, notification and emergency assistance. It also contains specific instructions and exceptions pertaining to the statements on this label about personal protective equipment (PPE), notification to workers, and restricted-entry interval. The requirements in this box apply to uses of this product that are covered by the Worker Protection Standard. No restricted entry interval (REI) – 0 hours No Pre-Harvest interval (PHI) – 0 hours No PPE requirements. For prevention and treatment of Fire Blight (Erwinia amylovora) in apple trees, pear trees, cherry trees, rose bushes and chrysanthemums. Dilute Fire Quencher at a rate of 1 to 2 pints per 100 gallons of water to cover approximately 1 acre. The diluted solution should be applied by conventional aerial spray equipment prior to budding and/or flowering and every other day for the next six days (for a total of 4 treatments) depending on weather conditions. More frequent application may be necessary during periods of heavy or persistent rains. To prepare diluted solution, fill the mix tank with the desired amount of water and add Fire Quencher with agitation to mix thoroughly. The volume of diluted solution necessary for adequate coverage will depend on spray equipment weather, and local conditions. Apply enough solution for thorough coverage without runoff.

FIRST AID Call a poison control center or doctor for treatment advice. If in Eyes: Hold eye open and rinse slowly and gently with water for 15-20 minutes. Remove contact lenses, if present, after the first 5 minutes , then continue rinsing eye. If on Skin or Clothing: Take off contaminated clothing. Rinse skin immediately with plenty of water for 15-20 minutes. Have the product container or label with you when calling a poison control center or doctor, or going for treatment. PRECAUTIONARY STATEMENTS ENVIRONMENTAL HAZARDS Do not apply directly to water, or to areas where surface water is present or to intertidal areas below the mean high water mark, Do not contaminate water when cleaning equipment or disposing of equipment washes. STORAGE AND DISPOSAL Do not contaminate water, food, or feed by storage and disposal. Pesticide Storage: Store this product in a cool dry area, away from direct sunlight and heat to avoid deterioration. Pesticide Disposal: Wastes resulting from the use of this product may be disposed of on site or at approved waste disposal facility. Container Disposal: Non-refillable container. Do not reuse or refill this container. Triple rinse empty containers. Then offer for recycling or reconditioning, or puncture and dispose of in a sanitary landfill, or by incineration, or if allowed by state and local authorities, by burning. If burned, stay out of smoke. WARRANTY STATEMENT XXXXXXX, Inc. warrants that this material conforms to the description on the label and is reasonably fit for the purposes referred to in the Directions for Use. XXXXXXX, Inc. makes no other express or implied warranty or fitness or merchantability, or any other express or implied warranty. In no case or circumstance shall XXXXXXX, Inc. or seller be liable for consequential, special, or indirect damages resulting from the use or handling of this product including, but not limited to, loss of profits, business reputations, or customers; labor cost, or any other expenses incurred in planting, cultivating, or harvesting.

53 new phages that infect E. amylovora 31 have been fully sequenced – 8 unique families of phages

Frozen Phobos Stratton Huxley

Machina Parshik Quack Deimos-minion

Isolation of phages that infect E. amylovora

Fire Quencher - Good Practices in Phage Therapy

• Test host range • Use strictly lytic phage • Use a cocktail of phage • Fully sequence phage

ATP-Dependent DNA helicase

ATP-Dependent DNA helicase

phosphohydrolase

phosphohydrolase

D-alanyl-D-alanin carboxypeptidase

D-alanyl-D-alanin carboxypeptidase

RNA polymerase beta subunit

RNA polymerase beta subunit

chitinase and glycoside hydrolase

chitinase and glycoside hydrolase

thymidine kinase

thymidine kinase

DNA adenine

methylase

DNA adenine

methylase

nucleotide pyrophosphohydrolas

e

nucleotide pyrophosphohydrolas

e

integral membrane protein

PhoH

GDSL_hydrolase/lipase and tail sheath protein

HNH endonuclease

ribonuclease RNAseHI family lytic transglycosylase

major capsid protein

helicase

dihydrofolate reductase

NAD-dependent DNA ligase

phiKZ-like phage internal head proteins

cell divison protein ZipA

ATP-dependent DNA helicase

Bifunctional DNA-directed RNA polymerase beta subunit Calcineuring-like

phosphoesterase RNA pol I subunit A

thymidylate kinase

Tail fiber

RNA polymerase beta subunit

DNA-directed RNA polymerase beta subunit

DNA directed RNA polymerase beta subunit

dTMP kinase

ribonuclease RNAseHI family

NAD-dependent DNA ligase Tail fiber

RNA polymerase beta subunit

major capsid protein

helicase

phiKZ-like phage internal head proteins

cell divison protein ZipA

DNA-directed RNA polymerase beta subunit

ATP-dependent DNA helicase

Bifunctional DNA-directed RNA polyermase beta subunit

DNA directed RNA polymerase beta subunit Calcineuring-like

phosphoesterase

RNA pol I subunit A

thymidylate kinase

dTMP kinase

DNA-direct RNA polymerase beta

subunit

GroEL

GroEL

homing endonuclease GIU-YIG with DNA

binding motif

homing endonuclease GIU-YIG with DNA

binding motif

integral membrane protein

Serine/theronine phosphotase

Serine/theronine phosphotase

PhoH

GDSL_hydrolase/lipase and tail sheath protein

mycothiosynthase

actyltransferase

Exonuclease

Exonuclease: micothio synthase

dihydrofolate reductase

ribonuclease HI

ATP-dependent helicase

discoidin ribonuclease HI

ATP-dependent helicase

UvsX protein

UvsX protein discoidin

Domain of unknown function found in bacteria and phages DNA-direct RNA

polymerase beta subunit

Domain of unknown function found in bacteria and phages

Joad

RisingSun

Joad cont.

RisingSun cont.

Joad cont.

RisingSun cont.

Joad cont.

RisingSun cont.

Joad cont.

RisingSun cont.

Joad cont.

RisingSun cont.

Joad cont.

RisingSun cont.

Joad cont.

RisingSun cont.

Fire Quencher

Field Trials

Inoculate

Sample

Sample

Sample

Three Years of Promising Fire Quencher Field Trials

05

101520253035404550

untreated Oxy-tet FQ

Perc

ent o

f Blo

ssom

s Inf

ecte

d

Tim Smith Washington State 2014

Trials where Fire Quencher was Effective on Its Own

untreated FQ

George Sundin Michigan State 2015

Blossom Protect

+FQ

Blossom Protect

Cueva +FQ

Cueva

10

30

40

10

30

40

Three Years of Promising Fire Quencher Field Trials

0

10

20

30

40

50

60

70

Ken Johnson Oregon State 2015

Seranade FQ + Sernade

Oxy-tet untreated FQ

Trials where Fire Quencher + Seranade was effective

George Sundin Michigan State 2016

Seranade untreated FQ-A FQ-B FQ-C FQ-A + Seranade

40

30

20

10

40

20

10

30

Fire Quencher is Stable for > 8 months

Phage Concentration

July

September

October

November

December

January

February

March

108

25ºC

4ºC

New and Improved 2017 Fire Quencher • Best 4 of 8 phage families used • UV protection/sunblock • Stabilizing buffer • Bacterial species isolated from healthy apple

trees. • Licensed to Esplin Organic Solutions

Best use practices Timing: - Early bloom. Interval: -3-5 applications spaced 2-3 days apart Coverage: - conventional spray Tank: - Mix in phage last, just before spraying - check pH (between 5-8) -Do not mix with copper

Graduate Students Ian Esplin Ruchira Sharma Undergraduates Jordan Berg, Garrett Jensen, Hunter Smith, Mallorie Stratton, Kala Harbaugh, Brayden Foy, Daniella Anieves, Hannah Paz, Nathan Sabin, Chris Shurtleff, Jared Kruger, Malia Peck, Braden Brundage, Justin Crockett, Marlee Evans, Jonathan Hyde, Morgan McBride, Austin Simister, Trever Thurgood Faculty Sherwood Casjens (University of Utah) Donald P. Breakwell (BYU) Sandra H. Hope (BYU) Funding • HHMI SEA Phages program • Western Region IR-4 • USDA • Private donor- Kimo Esplin • Department of Molecular Biology and Microbiology

Acknowledgements