Phighting Phire Blight with Phagewrir4.ucdavis.edu/events/2017_SLR_Meeting/Presentations... ·...
Transcript of Phighting Phire Blight with Phagewrir4.ucdavis.edu/events/2017_SLR_Meeting/Presentations... ·...
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