CRITERIA FOR RANKING EVALUATIONS OF IR-4 EARLY …ir4.rutgers.edu/Biopesticides/Proposals/5E.pdf · CRITERIA FOR RANKING EVALUATIONS OF IR-4 EARLY STAGE BIOPESTICIDE PROPOSALS-2014

Reviewer’s Initials

CRITERIA FOR RANKING EVALUATIONS OF IR-4 EARLY STAGE BIOPESTICIDE PROPOSALS-2014

Proposal number/Title/PI: 5E, A Natural Treatment for Fire Blight: Pilot Tests in Apple Orchards, Grose

The following criteria were established to assist the reviewers in selecting biopesticide projects for funding that: (1) in an exploratory or early stage of development (2) have a high probability of being registered/marketed in a reasonable period of time; and (3) will be useful in meeting pest control needs involving minor crops (uses), including minor uses on major crops.

Criteria Score

(0 to 10 or 20)

1. Adequacy of investigators and facilities. of 10

2. Experimental design, work plan and preliminary research. of 10

3. Evaluation of budget. of 10

4. Time to completion and probability of attaining objectives in the proposed time frame. of 10

5. Relevance of the proposal toward the development of data for

registration or label expansion of the biopesticide. of 10

6. Evidence of Efficacy. of 20

7. Probability of biopesticide being used by growers (factors such as effectiveness and economics of use rates should be considered). of 10

8. Adverse environmental risks including crop safety, safety to

beneficials, safety to ecosystems, and stability. of 10

9. Other control measures currently available to control target pest. of 10

10. Probability of biopesticide being registered, time to registration, and if label expansion, time to market. of 10

11. Availability of a potential registrant. Likelihood of developing a

formulated commercial product. of 10 TOTAL* of 120

Funding Recommendation YES ____________ (Check appropriate line) NO ____________

MAYBE ____________

Note: Attach a comment page, should you have specific comments related to the proposal not covered in the above criteria. * There is a possibility of 10 points per criteria(except efficacy=20) for a total of up to 120 points. A rating of 0 means that the proposal does not meet the criteria at all, while a rating of 10 means it is ideal.

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IR-4 BIOPESTICIDE GRANTS COVER PAGE

2014

Proposal Number(For IR-4 Use): Principal Investigator: Julianne H. Grose Proposal Title: A Natural Treatment for Fire Blight: Pilot Tests in Apple Orchards Institution: Brigham Young University Total dollars Requested (Year 1 only): $25,000

Enter each biopesticide /crop/ pest combination

No. Biopesticide and/or Conventional Product TRADE Name

Active Ingredient

Crop Pest (Weeds, Diseases, Insects)

1 Fire Quencher Bacteriophages Apple Trees Fire Blight (Disease) 2 Streptomycin Streptomycin Apple Trees Fire Blight

3 Fire Quencher + Streptomycin 17%

Bacteriophages +Streptomycin Apple Trees Fire Blight 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

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Biopesticide Grants Contact Information Form

Proposal Title: A Natural Treatment for Fire Blight: Pilot Tests in Apple Orchards

Name

Address

Phone Number & Fax Number

E-mail Address

Street

City/ State

Zip+4

Project Director (Principal Investigator): Julianne H. Grose

685 East University Parkway

Provo, Utah 84602

Utah 84602 (801) 422-‐4940

(801) 422-‐0519

[email protected]

Administrative Contact: Gene Larson


Provo, Utah 84602

Utah 84602 (801) 422-‐3360

(801) 422-‐0620

gene_larson@byu. edu

Financial Grant Officer: Kathleen Rugg


Provo, Utah 84602

Utah 84602 (801) 422-‐8025

(801) 422-‐0620

[email protected]

Authorized Grant Official: Alan Harker


Provo, Utah 84602

Utah 84602 (801) 422-‐3582

(801) 422-‐0620

alan_harker@byu. edu

Individual Responsible for Invoicing: David Morris


Provo, Utah 84602

Utah 84602 (801) 422-‐7548

(801) 422-‐0620

david.morris@byu. edu

NOTE: THIS IS FOR INFORMATIONAL PURPOSES ONLY. THIS IS NOT MEANT TO BE SIGNED. DO NOT DELAY SUBMITTING YOUR PROPOSAL BY ATTEMPTING TO GET THIS SIGNED. THIS IS NOT MEANT AS A REPLACEMENT FOR ANY INSTITUTIONAL APPROVAL PAGES.

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I. Grant Stage What is the grant Stage to which you are applying? Early or Advanced (Check appropriate line)

✓ Early – Biopesticide not yet registered and has not completed the Tier I toxicology data requirements.

Advanced – the biopesticide is registered or at least has completed the Tier I toxicology data requirements.

If you are applying for any Advanced Stage Proposal, and the product is not currently registered with EPA, provide a list of the toxicology work that has been completed. Ask registrant or have company provide information to IR-4.

II. Introduction (Limit 1 page)

OBJECTIVE: Our objective is to provide a natural and effective treatment for fire blight through the use of bacteriophages. Bacteriophages are viruses that infect bacteria. They are the most abundant organism on the planet and are essential to maintaining ecological balance due to their ability to infect and kill their hosts. The term bacteriophage literally means “to devour bacteria” as they infect their host organisms by injecting their DNA, pirating cellular machinery to produce progeny, and finally lysing the host cells to release tens or hundreds of progeny that then infect more bacteria. These natural predators are highly specific for their host organism and thus do not affect humans, plants, animals, or the environment. Bacteriophages are biodegradable and considered “organic,” making them a valuable ally in the fight against agricultural diseases. Bacteriophages are commonly used to treat several agricultural diseases, including those that infect tomatoes and peppers, in the United States and abroad. We have isolated 41 bacteriophages that infect and kill the causative agent of fire blight. These bacteriophages have been isolated and amplified from local orchards in Utah. We have combined the best five bacteriophages into a natural bactericide named Fire Quencher. This proposal outlines our goals to 1) further characterize these bacteriophages, 2) conduct small-scale efficacy tests of Fire Quencher in controlled green houses, 3) produce characterize and compare liquid and solid Fire Quencher for large-scale distribution, and 4) test the efficacy of Fire Quencher in conjunction with apple orchards. DESCRIPTION OF DISEASE: Fire blight is a bacterial disease that affects a wide variety of plants including apple trees, pear trees, cherry trees, rose bushes and chrysanthemums. Reported apple produce losses due to fire blight are in excess of $100 million annually in the United States alone ((Norelli et al., 2003). Fire blight is caused by the gamma-proteobacterium Erwinia amylovora that invades wounds on trees and infects almost all tissue types, including flowers, stems, and leaves (Vanneste, 2000). Because the bacteria can overwinter within the bark of tree limbs 2-years-old and older, infected branches often serve as the reservoir of bacteria in subsequent seasons (Khan et al.).. Infected leaves bend inwards towards the stem, release infectious exudates, wilt, and die. As dead plant matter falls to the ground the bacteria can also subsist in the soil. Since the bacteria prefers to amplify in newly formed plant tissue, spring blossoms and new shoots of

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infected trees suffer the greatest damage. Death of blossoms, leaves and shoots, results in low fruit yields and smaller fruits.

The current primary treatment for fire blight is the antibiotic streptomycin sulfate. Other treatment options include heavy metals (such as copper), and Blossom Protect (a new yeast-based spray); however, both of these non-antibiotic options can cause russet. All of these options are considered preventative since none are very effective after symptoms of fire blight appear. Once symptoms appear, trees are generally remove from the orchard to prevent spread. Thus, treatments are currently used at blossom time.

JUSTIFICATION The need for an alternative form of fire blight control is imperative as bacterial resistance to antibiotics and heavy metals has increased (Martinez et al., 2009b). In addition, many farmers would prefer to decrease the use of antibiotics and heavy metals in agriculture (Lipsitch et al., 2002; Martinez, 2009; Martinez et al., 2009a; Stockwell and Duffy, 2012; Stockwell et al., 2011) and still maintain healthy, productive crops. The goal to eliminate antibiotic use is especially important since most Erwinia amylovora strains isolated from orchards have developed antibiotic resistance (Ngugi et al., 2012). For fire blight, antibiotic and heavy metal use, as well as the associated development of resistant bacteria is particularly alarming because Erwinia amylovora is closely related to E. coli and Salmonella and may thus be able to transfer antibiotic and heavy metal resistance gene cassettes. For organic farmers, an alternative option is imperative because antibiotics will no longer be allowed beginning fall of 2014. III. Experimental Plan (Please limit this section to 10 pages)

Proposed experiments: The numerical list of treatments below corresponds to experiments outline in this table.: All tests of Fire Quencher efficacy will be performed with Fire Quencher alone or in combination with streptomycin sulfate, the current standard treatment for fire blight prevention. Experiments are designed with streptomycin sulfate only controls when necessary.

Plants

# plants treated/ untreated Treatment timing Treatment form

1 Apple seedlings 25/5 Pre-‐ and post-‐ infection Fire Quencher (liquid) 2 Apple seedlings 25/5 Pre-‐ and post-‐ infection Streptomycin sulfate(17%) 3 Apple seedlings 25/5 Pre-‐ and post-‐ infection Fire Quencher (liquid)

Combined with Streptomycin sulfate(17%) 4 Apple seedlings 25/5 Post-‐infection Fire Quencher (liquid) 5 Apple seedlings 25/5 Post-‐infection Streptomycin sulfate(17%) 6 Apple seedlings 25/5 Post-‐infection Fire Quencher (liquid)

Combined with Streptomycin sulfate(17%) 7 Apple seedlings 25/5 Pre-‐ and post-‐ infection Fire Quencher (solid) 8 Apple seedlings 25/5 Pre-‐ and post-‐ infection Fire Quencher (solid)

Combined with Streptomycin sulfate(17%) 9 Apple seedlings 25/5 Post-‐infection Fire Quencher (solid) 10 Apple seedlings 25/5 Post-‐ infection Fire Quencher (solid)

Combined with Streptomycin sulfate(17%) 11 Apple trees 6/4 Pre-‐ and post-‐ infection Fire Quencher (liquid) 12 Apple trees 6/4 Pre-‐ and post-‐ infection Fire Quencher (liquid)

Combined with Streptomycin sulfate(17%)

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13 Apple trees 6/4 Pre-‐ and post-‐ infection Fire Quencher (solid) 14 Apple trees 6/4 Pre-‐ and post-‐ infection Fire Quencher (solid)

Combined with Streptomycin sulfate(17%) 15 Apple trees 16/4 Pre-‐ and post-‐ infection Fire Quencher (liquid) 16 Apple trees 16/4 Pre-‐ and post-‐ infection Streptomycin sulfate(17%) 17 Apple trees 16/4 Pre-‐ and post-‐ infection Combined with Streptomycin sulfate(17)

1. Treatment Title: Greenhouse pre- and post- infection treatment of apple seedlings

using Fire Quencher (liquid) Product Trade Name: Fire Quencher (liquid) Active Ingredient: A mixture of 5 bacteriophages that infect Ewinia amylovora Rate (units): Fire Quencher ( 0.1 mL of bacteriophages in nutrient broth at a final concentration of 1012 plaque forming units) will be diluted into water and distributed at a final concentration of 109 plaque forming units. A total final volume of 100 mL will be sprayed onto 25 apple seedlings. Water will be sprayed on a total of 5 apple seedlings as a control. Application Timing: All 30 apple seedlings will be sprayed with either water or Fire Quencher one hour before infection with Erwinia amylovora, then one hour after infection. Treatment will continue with one application once every other day for the following six days. Facility: Quarantined greenhouse owned by Brigham Young University.


using streptomycin sulfate (17%) Product Trade Name: streptomycin sulfate (17%) Active Ingredient: streptomycin sulfate (17%) Rate (units): Streptomycin sulfate (17%) will be sprayed over 25 apple seedlings at 200 ppm final concentration. Water will be sprayed on a total of 5 apple seedlings as a control. Application Timing: All 30 apple seedlings will be sprayed with either water or streptomycin for one hour before infection with Erwinia amylovora, then one hour after infection. Treatment will continue with one application once every other day for the following six days. Facility: Quarantined greenhouse owned by Brigham Young University.


using Fire Quencher (liquid) and streptomycin sulfate (17%) Product Trade Name: Fire Quencher (liquid) and Streptomycin sulfate(17%) Active Ingredient: Fire Quencher ( a mixture of 5 bacteriophages that infect Ewinia amylovora) and Streptomycin sulfate (17%) Rate (units): Fire Quencher ( 0.1 mL of bacteriophages in nutrient broth at a final concentration of 1012 plaque forming units) will be diluted into water and distributed at a final concentration of 109 plaque forming units. A total final volume of 100 mL will be sprayed onto 25 apple seedlings. Streptomycin sulfate (17%) will then be sprayed at 200 ppm. Water will be sprayed on a total of 5 apple seedlings as a control. Application Timing: All 30 apple seedlings will be sprayed with either water or Fire

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Quencher plus streptomycin one hour before infection with Erwinia amylovora, then one hour after infection. Treatment will continue with one application once every other day for the following six days. Facility: Quarantined greenhouse owned by Brigham Young University.

4. Treatment Title: Greenhouse post-infection treatment of apple seedlings using Fire

Quencher (liquid) Product Trade Name: Fire Quencher (liquid) and Streptomycin sulfate (17%) Active Ingredient: A mixture of 5 bacteriophages that infect Ewinia amylovora Rate (units): Fire Quencher (0.1 mL of bacteriophages in nutrient broth at a final concentration of 1012 plaque forming units) will be diluted into water and distributed at a final concentration of 109 plaque forming units. A total final volume of 100 mL will be sprayed onto 25 apple seedlings. Water will be sprayed on a total of 5 apple seedlings as a control. Application Timing: All 30 apple seedlings will be sprayed with either water or Fire Quencher one day following infection with Erwinia amylovora. Treatment will continue with one application once every other day for the following six days. Facility: Quarantined greenhouse owned by Brigham Young University.

5. Treatment Title: Greenhouse post-infection treatment of apple seedlings using streptomycin sulfate (17%) Product Trade Name: Streptomycin sulfate (17%) Active Ingredient: Streptomycin sulfate (17%) Rate (units): Streptomycin sulfate (17%) will then be sprayed at a final concentration of 200 ppm onto 25 apple seedlings. Water will be sprayed on a total of 5 apple seedlings as a control. Application Timing: All 30 apple seedlings will be sprayed with either water or Fire Quencher one day following infection with Erwinia amylovora. Treatment will continue with one application once every other day for the following six days. Facility: Quarantined greenhouse owned by Brigham Young University.

6. Treatment Title: Greenhouse post-infection treatment of apple seedlings using Fire Quencher (liquid) and Streptomycin (17%) Product Trade Name: Fire Quencher (solid) and Streptomycin sulfate (17%) Active Ingredient: Fire Quencher (a mixture of 5 bacteriophages that infect Ewinia amylovora) and Streptomycin sulfate (17%) Rate (units): Fire Quencher (0.1 mL of bacteriophages in nutrient broth at a final concentration of 1012 plaque forming units) will be diluted into water and distributed at a final concentration of 109 plaque forming units. A total final volume of 100 mL will be sprayed onto 25 apple seedlings. Streptomycin sulfate (17%) will then be sprayed at 200 ppm. Water will be sprayed on a total of 5 apple seedlings as a control. Application Timing: All 30 apple seedlings will be sprayed with either water or Fire Quencher and streptomycin sulfate one hour before infection with Erwinia amylovora, then one hour after infection. Treatment will continue with one application once every other day for the following six days. Facility: Quarantined greenhouse owned by Brigham Young University.

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7. Treatment Title: Greenhouse pre- and post-infection treatment of apple seedlings

using Fire Quencher (solid) Product Trade Name: Fire Quencher (solid) Active Ingredient: A mixture of 5 bacteriophages that infect Ewinia amylovora Rate (units): Solid Fire Quencher (lyophilized bacteriophages at a total concentration of 1012 plaque forming units) will be reconstituted in water and distributed at a final concentration of 109. A total final volume of 100 mL will be sprayed onto 25 apple seedlings. Water will be sprayed on a total of 5 apple seedlings as a control. Application Timing: All 30 apple seedlings will be sprayed with either water or Fire Quencher one hour before infection with Erwinia amylovora, then one hour after infection. Treatment will continue with one application once every other day for the following six days. Facility: Quarantined green house owned by Brigham Young University.

8. Treatment Title: Greenhouse pre- and post-infection treatment of apple seedlings using Fire Quencher (liquid) and streptomycin sulfate (17%) Product Trade Name: Fire Quencher (solid) and streptomycin sulfate (17%)

Active Ingredient: Fire Quencher (a mixture of 5 bacteriophages that infect Ewinia amylovora) and streptomycin sulfate (17%). Rate (units): Fire Quencher (lyophilized bacteriophages at a total concentration of 1012 plaque forming units) will be reconstituted in water and distributed at a final concentration of 109. A total final volume of 100 mL will be sprayed onto 25 apple seedlings. Streptomycin sulfate (17%) will then be sprayed at 200 ppm. Water will be sprayed on a total of 5 apple seedlings as a control. Application Timing: All 30 apple seedlings will be sprayed with either water or Fire Quencher and streptomycin sulfate one hour before infection with Erwinia amylovora, then one hour after infection. Treatment will continue with one application once every other day for the following six days. Facility: Quarantined green house owned by Brigham Young University

9. Treatment Title: Greenhouse post-infection treatment of apple seedlings using Fire

Quencher (solid) Product Trade Name: Fire Quencher (solid) Active Ingredient: A mixture of 5 bacteriophages that infect Ewinia amylovora Rate (units): Solid Fire Quencher (lyophilized bacteriophages at a total concentration of 1012 plaque forming units) will be reconstituted in water and distributed at a final concentration of 109. A total final volume of 100 mL will be sprayed onto 25 apple seedlings. Water will be sprayed on a total of 5 apple seedlings as a control. Application Timing: All 30 apple seedlings will be sprayed with either water or Fire Quencher one hour after infection with Erwinia amylovora. Treatment will continue with one application once every other day for the following six days. Facility: Quarantined green house owned by Brigham Young University.

10. Treatment Title: Greenhouse post-infection treatment of apple seedlings using Fire Quencher (liquid) and streptomycin sulfate (17%)

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Product Trade Name: Fire Quencher (solid) and streptomycin sulfate (17%) Active Ingredient: Fire Quencher (a mixture of 5 bacteriophages that infect Ewinia amylovora) and streptomycin sulfate (17%) Rate (units): Fire Quencher (lyophilized bacteriophages at a total concentration of 1012 plaque forming units) will be reconstituted in water and distributed at a final concentration of 109. A total final volume of 100 mL will be sprayed onto 25 apple seedlings. Streptomycin sulfate (17%) will then be sprayed at 200 ppm. Water will be sprayed on a total of 5 apple seedlings as a control. Application Timing: All 30 apple seedlings will be sprayed with either water or Fire Quencher and streptomycin sulfate one hour after infection with Erwinia amylovora. Treatment will continue with one application once every other day for the following six days. Facility: Quarantined green house owned by Brigham Young University.

11. Treatment Title: Greenhouse pre- and post- infection treatment of apple trees using Fire Quencher (liquid)

Product Trade Name: Fire Quencher (liquid) Active Ingredient: A mixture of 5 bacteriophages that infect Ewinia amylovora Rate (units): Fire Quencher ( 0.1 mL of bacteriophages in nutrient broth at a final concentration of 1012 plaque forming units) will be diluted into water and distributed at a final concentration of 109 plaque forming units. A total final volume of 100 mL will be sprayed onto 6 apple trees (3 years old). Water will be sprayed on a total of 4 apple seedlings as a control. Application Timing: All 10 apple trees will be sprayed with either water or Fire Quencher one hour before infection with Erwinia amylovora, then one hour after infection. Treatment will continue with one application once every other day for the following six days. Facility: Quarantined greenhouse owned by Brigham Young University.

12. Treatment Title: Greenhouse pre- and post- infection treatment of apple trees using

Fire Quencher (liquid) and streptomycin sulfate (17%) Product Trade Name: Fire Quencher (liquid) and Streptomycin sulfate(17%) Active Ingredient: Fire Quencher (a mixture of 5 bacteriophages that infect Ewinia amylovora) and Streptomycin sulfate (17%) Rate (units): Fire Quencher ( 0.1 mL of bacteriophages in nutrient broth at a final concentration of 1012 plaque forming units) will be diluted into water and distributed at a final concentration of 109 plaque forming units. A total final volume of 100 mL will be sprayed onto 6 apple trees (3 years old). Streptomycin sulfate (17%) will then be sprayed at 200 ppm. Water will be sprayed on a total of 4 apple trees as a control. Application Timing: All 10 apple trees will be sprayed with either water or Fire Quencher plus streptomycin one hour before infection with Erwinia amylovora, then one hour after infection. Treatment will continue with one application once every other day for the following six days. Facility: Quarantined greenhouse owned by Brigham Young University.

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13. Treatment Title: Greenhouse pre- and post-infection treatment of apple trees using Fire Quencher (solid)

Product Trade Name: Fire Quencher (solid) Active Ingredient: A mixture of 5 bacteriophages that infect Ewinia amylovora Rate (units): Solid Fire Quencher (lyophilized bacteriophages at a total concentration of 1012 plaque forming units) will be reconstituted in water and distributed at a final concentration of 109. A total final volume of 100 mL will be sprayed onto 6 apple trees (3 years old). Water will be sprayed on a total of 4 apple trees as a control. Application Timing: All 10 apple trees will be sprayed with either water or Fire Quencher one hour before infection with Erwinia amylovora, then one hour after infection. Treatment will continue with one application once every other day for the following six days. Facility: Quarantined green house owned by Brigham Young University.

14. Treatment Title: Greenhouse pre- and post-infection treatment of apple trees using Fire Quencher (liquid) and streptomycin sulfate (17%) Product Trade Name: Fire Quencher (solid) and streptomycin sulfate (17%)

Active Ingredient: Fire Quencher (a mixture of 5 bacteriophages that infect Ewinia amylovora) and streptomycin sulfate (17%) Rate (units): Fire Quencher (lyophilized bacteriophages at a total concentration of 1012 plaque forming units) will be reconstituted in water and distributed at a final concentration of 109. A total final volume of 100 mL will be sprayed onto 6 apple trees (3 years old). Streptomycin sulfate (17%) will then be sprayed at 200 ppm. Water will be sprayed on a total of 4 apple trees as a control. Application Timing: All 10 apple trees will be sprayed with either water or Fire Quencher and streptomycin sulfate one hour before infection with Erwinia amylovora, then one hour after infection. Treatment will continue with one application once every other day for the following six days. Facility: Quarantined green house owned by Brigham Young University

15. Treatment Title: Pre-infection treatment of apple orchards at Oregon State University and Washington State University using Fire Quencher (liquid)

Product Trade Name: Fire Quencher (liquid) Active Ingredient: A mixture of 5 bacteriophages that infect Ewinia amylovora Rate (units): Fire Quencher (bacteriophages in 3.9 mL of nutrient broth at a final concentration of 1012 plaque forming units) will be diluted into one gallon of water and distributed at a final concentration of 109. The total final volume will be sprayed onto 4 apple trees. This will be repeated 3 times for a total of 16 trees. Application Timing: Trees will be sprayed one hour before infection with Erwinia amylovora, then one hour after infection. Treatment will continue with one application once every other day for the following six days. Three different application times will be tested this spring, on approximately 4 trees each for a total of 16 treated and infected trees. In addition, 4 trees will also be sprayed with water and infected as control. Application times will be determined by weather and blossoming of the trees. Facility: Sacrificial orchards USDA approved and funded for testing located at both Oregon State University and Washington State University.

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16. Treatment Title: Pre-infection treatment of apple orchards at Oregon State University and Washington State University (liquid) using streptomycin sulfate

Product Trade Name: Streptomycin sulfate (17%) Active Ingredient: Streptomycin sulfate (17%) Rate (units): Streptomycin sulfate (17%) will be sprayed onto 4 apple trees for a final concentration of 200 ppm. This will be repeated 3 times for a total of 16 trees. Application Timing: Trees will be sprayed one hour before infection with Erwinia amylovora, then one hour after infection. Treatment will continue with one application once every other day for the following six days. Three different application times will be tested this spring, on approximately 4 trees each for a total of 16 treated and infected trees. In addition, 4 trees will also be sprayed with water and infected as control. Application times will be determined by weather and blossoming of the trees. Facility: Sacrificial orchards USDA approved and funded for testing located at both Oregon State University and Washington State University.

17. Treatment Title: Pre-infection treatment of apple orchards at Oregon State University and Washington State University using Fire Quencher (liquid) and streptomycin sulfate Product Trade Name: Fire Quencher (liquid) and streptomycin sulfate (17%) Active Ingredient: A mixture of 5 bacteriophages that infect Ewinia amylovora and streptomycin sulfate (17%) Rate (units): Fire Quencher (bacteriophages in 3.9 mL of nutrient broth at a final concentration of 1012 plaque forming units) will be diluted into one gallon of water and distributed at a final concentration of 109. The total final volume will be sprayed onto 4 apple trees. The trees will then be sprayed with streptomycin sulfate (17%) to a final concentration of 200 ppm. This will be repeated 3 times for a total of 16 trees. Application Timing: Trees will be sprayed one hour before infection with Erwinia amylovora, then one hour after infection. Treatment will continue with one application once every other day for the following six days. Three different application times will be tested this spring, on approximately 4 trees each for a total of 16 treated and infected trees. In addition, 4 trees will also be sprayed with water and infected as control. Application times will be determined by weather and blossoming of the trees but are anticipated to be one week apart. Facility: Sacrificial orchards USDA approved and funded for testing located at both Oregon State University and Washington State University.

2. What crops or sites will this study be conducted on?

Treatments #1-14 will be performed in a quarantined green house owned by Brigham Young University. We currently are growing hundreds of apple seedlings and 40 young trees (3 years old) for these studies. Treatments #15 -17 will be performed at both the Washington State University and the Oregon State University on test orchards that have been designed and USDA approved for the testing of fire blight treatment options. These facilities have tested antibiotic, heavy metal, and various natural product treatments (including combined treatments) the past five years. Please see their letters of support. Old red delicious trees are used which are resistant to fire blight.

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3. What experimental design will be utilized?

Treatments #1-14 will be performed in a quarantined green house owned by Brigham Young University. In addition to the Fire Quencher treatment, water will be used as a negative control. The studied will be blinded in that each plant will be given a number, plants receiving the control or Fire Quencher will be chosen randomly, and different students will be used to infect versus score efficacy. Plants will be scored based on degree of disease, scores will be averaged and reported as percent infected relative to wild type. A students T test will be applied to determine validity of the efficacy. In trial tests (see Appendix 3- Supporting Preliminary Data) 100% of plants infected with Erwinia amylovora died. Thus we are confident that our numbers will provide statistical significance if our treatment provides 10% efficacy for treatments #1-10 and 20% efficacy for treatment #11-14. Scales for scoring disease are reported below. Treatments #15-17. These experiments are performed by experts Ken Johnson (Oregon State University) and Tim Morris (Washington State University). Four trees will be treated at each time point for a total of 16 per treatments. Only 50 blossoms are inoculated per tree. Evaluation is based on the number of blossoms displaying fire blight our of each of the 50 flower clusters. The diseases does not spread further into the tree since old red delicious trees are used which are resistant to fire blight spreading from the blossom. Their experimental set-up has proven to detect efficacies as low as 20% (Stockwell, 2011). All data is reported as percent trees rescued and is compared to the untreated controls and is analyzed by a Bayesian approach (Mila, 2011).

4. How many locations (field or greenhouse)? How many replications? Treatments #1-14 will all be performed at the Brigham Young University Life Science Greenhouses. Treatments #1-10 include 30 plants each (300 total), treatments #11-14 include 10 trees each (40 trees total). Treatments #1-8 will be replicated once several months apart to ensure reproducible. This replication will increase the total number of apple seedlings (2-4 inch plants) to 540 plants. Treatments #15-18 will be performed once at two separate locations, Washington State University and Oregon State University. These treatments include a total of 20 trees each, or 1/5 of the acre devoted to each study at each location for a total of 120 trees or 1.2 acres.

5. Describe how this proposal is designed to provide information on how it fits into an integrated pest management program.

Fire Quencher is designed to be an early season treatment. Optimally, it will be applied prior to plant blooming to reduce the occurrence of fire blight symptoms. All experiments (Treatments #1-18) are designed to test phage treatment prior to the appearance of fire blight symptoms, which is generally 4-10 days post infection. Efficacy will be determined by the occurrence of fire blight symptoms in treated versus untreated samples. Every type of experiments includes treatment alone and in combination with streptomycin sulfate. Streptomycin sulfate alone generally has an efficacy rate of ~87% in controlled environments (Tim Smith, unpublished data. Please see his letter of support). Note that this efficacy rate is dramatically lower in most orchards due to antibiotic resistant bacteria in the environment.

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6. Data collection – Treatments #1-14: Data will be collected based on visual efficacy. A rating scale of 1-7 will be used for each sample in treatments #1-10. For treatments #1-10, samples will be evaluated each day following inoculation up to 14 days. For treatments #11-14, samples will be evaluated for 4 months to determine if Fire Quencher delays onset of the disease in trees. Samples will be compared to a control sample that was sprayed with water or streptomycin sulfate only. Standard statistical analysis will be performed (students t-test). In addition, we will attempt to isolate the pest (Erwinia amylovora) from each sample including the controls each day of the experiment by swabbing the infected area of the plant and plating to CCT plates that are semi-selective for Erwinia amylovora. Arising colonies will be verified as Erwinia amylovora isolates by amplifying regions of DNA specific to Erwinia amylovora via colony Polymerase Chain Reaction (PCR). Rating Scale:

1- No appearance of disease. 2- Leaves appear dry and shriveled but maintain normal coloring. 3- Leaves are dry, shriveled and are turning brown/black. 4- Leaves are dry, shriveled, are turning towards the stem and are brown/black 5- Leaves are dry, shriveled, are turning towards the stem and are brown/black, stem of plant is discolored 6- Leaves are dry, shriveled, are turning towards the stem and are brown/black, stem of plant is discolored and plant has ooze that exudes from cankers. 7- Plant is dead (entire plant is discolored and dry, all leaves and stem and brown/black).

Treatments #15-17: Data will be collected throughout spring/summer and times of disease appearance will be recorded to determine in Fire Quencher delays onset (suggesting multiple treatments may be necessary). For each tree, 50 blossoms will be infected with the Erwinia amylovora and each of the 50 scored for blight (color/withering) of the blossom (times 4 replicates) Old delicious trees are used since they are resistant to fire blight. The disease doesn’t travel very far into the wood before it stops.

7. Describe the pests to be controlled, the degree to which they are a problem in your state or region and the frequency that they occur (season long problem, every year, every few years).

The pest to be controlled is the gram-negative bacterium Erwinia amylovora, the causative agent of fire blight. Reported apple produce losses due to fire blight are in excess of $100 million annually in the United States alone (Norelli et al., 2003). The development of alternative treatment strategies for fire blight is imperative to protect the 5,710 acres of fruit bearing trees valued at 16.7 million in utilized production in Utah. There are no reported statistics for the frequency of fire blight in Utah, however when we called 18 local orchards along the Wasatch front all but one orchard was experiencing an infection of at least one tree in their orchard this year. Fire blight generally originates in the spring when the plant is blossoming. Insects carry it from plant to plant. It may also originate later in the season due to natural damage to the tree and/or pruning. It is a problem every year, however, the degree of the problem varies with weather conditions. Surveys of the fire blight pathogen in Utah County have demonstrated variable rates of antibiotic resistance from 10% to 90% (DG

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Aston, Utah State University). Washington State University reports a majority of Washington State Orchards infected with antibiotic resistant bacteria (as much as 70%)(Ngugi, 2011).

8. Will the crop be inoculated with the target pest or otherwise be brought into the test system to ensure that it will be available for evaluation? If not, describe the frequency of occurrence.

Yes, in each treatment #1-17 the plants will be inoculated with Erwinia amylovora. For treatments #1-14 the plants will be inoculate by scoring the underside of a leaf and rubbing a 108 culture of Erwinia amylovora over the wound. For treatments #15-17 trees will be inoculated by applying directly to the 50 blossoms per tree.

9. What is the proposed start date and completion date? Also describe this in chronological order in the context of the experimental plan.

In all, are goals are to: 1) further characterize the 41 Erwinia bacteriophages we have isolated through DNA sequencing and phage host specificity, 2) conduct small-scale efficacy tests in controlled greenhouses, 3) produce, characterize and compare liquid and solid Fire Quencher for large-scale distribution, and 4) test the efficacy of these solutions in conjunction with apple orchards. All tests of Fire Quencher efficacy will be performed with Fire Quencher alone or in combination with streptomycin sulfate, the current standard treatment for fire blight prevention. February - April 2014 Goal #1) Further characterize the 41 Erwinia bacteriophages. DNA sequencing and host range specificity of Erwinia phages will be completed. DNA sequencing results and analysis will verify our current data on whether the phages are lytic or temperate. Lytic phages simply infect their host and produce progeny through lysis and destruction of the host cell. These are desired for agricultural treatment. Temperate phages are able to infect a host and integrate their DNA into the host genome. They can lie ‘dormant’ until awakened by an environmental cue such as stress. Temperate phages are not desired. Temperate phages can be identified in the lab (see appendix) and verified by DNA sequencing because phage that are lytic should lack the DNA encoding for proteins necessary for integration into the host genome. DNA sequencing of entire phage genomes is relatively expensive so we have requested support for this analysis. Our research team has a combined total of 14 years of experience with phage isolation and characterization including DNA sequencing and analysis (see Appendix 3, Supplementary Preliminary Data). Host specificity tests are important for testing the safety of our phage. Host specificity tests have been performed for 13 of our 41 phage. These phage were tested for their ability to infect close relatives of Erwinia amylovora, the bacteria E. coli and Salmonella. None of the 13 phages were able to infect either of these hosts. We are in the process of testing the remaining 28 phages. In addition, we have tested for deleterious affects of Fire Quencher on healthy apple seedlings and have seen no negative affects.

Goal 3) Produce, characterize and compare liquid and solid Fire Quencher for large-scale distribution. Studies will be conducted on the stability of bacteriophage solutions for long-term use. Specifically, bacteriophage will be stored at room temperature and in the fridge in various solutions (nutrient broth versus a saline buffer) and analyzed each week for plaque

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forming units (viable bacteriophage). In addition, bacteriophages will be lyophilized using standard procedures (Paupermpoonsiri et al, 2010), and resuspended at one month intervals to determine phage viability. These tests will be initiated February and will continue through April. High quantities of bacteriophages are currently being produced successful to begin these tests on our target date. February 2014- December 2014 Goal 2) Conduct small-scale efficacy tests in controlled greenhouses. Treatments #1-14 will be performed to test efficacy of Fire Quencher Treatment of apple trees. Efficacy will be determined alone and in combination with streptomycin sulfate, the current standard treatment for fire blight prevention. Experiments #1-8 will be replicated, initiated in February and again in July to test reproducibility several months apart.

March 2014- August 2014 Goal 4) To test the efficacy of these solutions in conjunction with apple orchards. Treatments #15-17 will be performed. Efficacy will be determined alone and in combination with streptomycin sulfate, the current standard treatment for fire blight prevention.

10. Describe the test facilities where these studies will be conducted.

Treatments #1-14 will all be performed at the Brigham Young University Life Science Greenhouses. This facility includes 12 wings, each 30 x 125 feet, including one quarantined facility that we will use. For a full description of this facility please visit: http://lsmagazine.byu.edu/Issues/Spring2012/LifeSciencesGreenhouses.aspx Treatments #15-17 will be performed once at two separate locations, Washington State University and Oregon State University. Each location is a one acre test orchard run by qualified fire blight investigators funded by the USDA. Letters of support and contact information for each of our collaborators is provided. Washington State University Oregon State University Tim Smith Kenneth B. Johnson WSU Integrate Pest Management Professor Dept. Botany and Plant Pathology Wenatchee WA 98801 Corvallis, OR 97330 97331-2902 [email protected] [email protected]

11. Budget: Provide an itemized budget, with categories such as labor, supplies, travel, etc. Provided below (see Appendix 7).

12. Describe why this product is needed and why growers are likely to use this product. (Also list alternative conventional and alternative biopesticide treatments)

The need for an alternative form of fire blight control is imperative, as bacterial resistance to antibiotics and heavy metals has increased dramatically in recent years with 10- 90% of antibiotic treatments proving ineffective at preventing disease. Recent data have proven antibiotic and bacteriophage combination therapy effective in the treatment of a variety of diseases (Knezevic, P., 2013, Chhibber, S., 2012; Kirby, AE,2012; Zang, Q.G, 2012). In addition, for organic farmers, an alternative option is imperative because antibiotics will not

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longer be allowed beginning fall of 2014. Fire Quencher is a natural, bacteriophage-based biopesticide amplified from the environment. It is safe, biodegradable, easy to apply by conventional methods and proven to be extremely effective at destroying the causative agent of fire blight (Erwinia amylovora) in the lab. If we are able to show efficacy in orchards through this study, alone or in combination with antibiotics, the product will be widely received.

List of Appendix (pages 16-‐24 and others attached separately):

Appendix 1: PCR Forms. Embedded below Appendix 2: Fire Quencher Label –Attached separately Appendix 3: Supporting preliminary data – Embedded below Appendix 4: Description of Principal Investigator and Co-PI’s is embedded below. Resumes are attached separately. Appendix 5- Not applicable Appendix 6- Submitted separately by registrant. Appendix 7- Budget Appendix 8- References. Embedded below

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FOR OFFICE USE ONLY Date: Cat: PR#:

IR-4 Minor Use Biopesticide (*Required Fields) Project Clearance Request (PCR) Form

1. *Requestor: Julianne H. Grose Affiliation: Brigham Young University

*Address 751 WIDB, 685 East University Parkway *City: Provo *State/Territory: UT *Zip: 84602 *Telephone: (801) 422-4940 FAX: (801) 422-0519 *E-mail address: [email protected]

2. *Pest Control Product (Active Ingredient {a.i.}): Fire Quencher (Bacteriophages)

*Trade Name/Formulation: Fire Quencher / 5 Bacteriophages that infect Erwinia Registrant (manufacturer): Ben Miller and Eric Peery Method of Production (Fermentation, in vivo, extraction from plants): Amplification in Erwinia amylovora in nutrient broth (NB) followed by centrifugation, filtration (0.45 uM filter) and chloroform treatment to ensure no bacteria remain.

3. *Commodity (one crop or crop group per form): Apple trees and produce

*Use Site (e.g., field, greenhouse, post-harvest): greenhouse and field/orchard Parts Consumed: 40 trees; produce from 60 Animal Feed By-Products: Yes No x Planting Season: Harvest Season: State/Territory Acreage: <.01 % National: <.01 Average Field Size: 1 acre

4. Insect/Disease/Weed: Fire blight caused by the bacterium Erwinia amylovora

Damage caused by pest: destruction of fruit and tree 5. *Why is this use needed?: antibacterial/heavy metal resistance and pollution

6. *Proposed Label Instructions

*Rate per Application (lbs a.i. per acre or 1000 linear ft): 100 gallons (108 pfu) per acre Type of sprayers that may be used (e.g., fixed wing, ground boom sprayer, chemigation, air blast, ULV, granular spreader): fixed wing, ground broom, chemigation, ULV, may all be used. Range of Spray Volume (if applicable): up to 1 galloon. Maximum Acreage Treated per Day: 1 acre

*Crop Stage during Application(s): early season (just prior to blossom or right at blossom) *Maximum no. of applications: 4 Minimum interval betw. applications: 2 days

Maximum lbs active ingredient per acre per year/season: (4 x108 pfu)*PHI: 7. *Availability of Supporting Data1: *Phytotoxicity(P) P _ *Efficacy(E) E *Yield(Y)

1Supporting data may be required before a residue study will be initiated.

8. *Submitted By (print name): Julianne H. Grose *Signature: *Date: Dec. 7, 2013

Send this completed form to: IR-4 Project Headquarters, 500 College Road East; Suite 201 W; Princeton, NJ 08540-6635;

Telephone (732)932-9575 ext 4610 (Michael Braverman) FAX (609) 514-2612 or e-mail: [email protected]

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Appendix 2-‐ Fire Quencher Label

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DIRECTIONS FOR USE It is a viola4on 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 driG. Only protected handlers may be in the area during applica4on. For any requirement specific to your State and Tribe, consult the agency responsible for pes4cide regula4on. USER SAFETY RECOMMENDATIONS Wash hands before ea4ng, 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 Protec4on Standard, 40 CFR part 170. This standard contains requirements for the protec4on of agricultural workers on farms, forests, nurseries, and green houses, and handlers of agricultural pes4cides. It contains requirements for training, decontamina4on, no4fica4on and emergency assistance. It also contains specific instruc4ons and excep4ons pertaining to the statements on this label about personal protec4ve equipment (PPE), no4fica4on to workers, and restricted-‐entry interval. The requirements in this box apply to uses of this product that are covered by the Worker Protec4on Standard. No restricted entry interval (REI) – 0 hours No Pre-‐Harvest interval (PHI) – 0 hours No PPE requirements. For preven4on 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 solu4on should be applied by conven4onal 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 condi4ons. More frequent applica4on may be necessary during periods of heavy or persistent rains. To prepare diluted solu4on, fill the mix tank with the desired amount of water and add Fire Quencher with agita4on to mix thoroughly. The volume of diluted solu4on necessary for adequate coverage will depend on spray equipment weather, and local condi4ons. Apply enough solu4on 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, aGer the first 5 minutes , then con4nue 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 inter4dal 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. Pes4cide Storage: Store this product in a cool dry area, away from direct sunlight and heat to avoid deteriora4on. Pes4cide Disposal: Wastes resul4ng 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 recondi4oning, or puncture and dispose of in a sanitary landfill, or by incinera4on, or if allowed by state and local authori4es, by burning. If burned, stay out of smoke. WARRANTY STATEMENT XXXXXXX, Inc. warrants that this material conforms to the descrip4on on the label and is reasonably fit for the purposes referred to in the Direc4ons 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 consequen4al, special, or indirect damages resul4ng from the use or handling of this product including, but not limited to, loss of profits, business reputa4ons, or customers; labor cost, or any other expenses incurred in plan4ng, cul4va4ng, or harves4ng.

Fire Quencher !

Bactericide!for!Use!on!Apples!and!Pears!!

Biological!Control!for!Fire!Blight!!

Active!Ingredient! Bacteriophages*! 0.0002%!Other!Ingredients! ! 99.998%!Total! ! 100.000%!

!*For!(Erwinia'amylovora)!

!Contains!at!least!1012!phages!per!pint!!

!Keep!Out!Of!Reach!of!Children!

CAUTION!See!side!panels!for!precautionary!statements.!

!!

!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!EPA!Reg!No.!XXXXXNX! ! ! ! !EPA!Est.!No.!XXXXXXNUTN001!

!Manufactured!by:!

!!!!!!

Net!contents:!!!1!pint!!

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APPENDIX 3 - Supporting preliminary data

Phage Isolation Between Fall 2009-Spring 2013 the BYU Phage Hunters program has captured and isolated over 55 phages, sequenced 52 of the phages and published 20 of the sequences. Figure 1 is an example of a control plate with bacteria and a test plate of bacteria with exposure to a phage sample. Clear ‘plaques’ indicate an area where a phage has landed on the plate and devoured the bacteria. As the phage produce more progeny this clearing spreads radially through the plate.

Figure 1. Bacteria are grown on the surface of agar in a dish to generate a ‘lawn” of bacteria (A). when phage is added to the plate, clear plaques indicate where phage have infected and killed the bacteria (B).

Prior to this spring, only a single phage was isolated that infects Erwinia amylovora . As phage isolation conditions were optimized, the BYU Phage Hunters Program has isolated 40 more this spring, summer and fall bringing our library total to 41 phages that infect Erwinia amylovora. Electron micrographs of each of these phages have been taken, one DNA genome has been sequenced, and all 41 bacteriophages have been grown to a concentration of at least 1010.

Figure 2. Electron micrographs of two bacteriophages that infect Erwinia amylovora. (A) bacteriophage Hapsheptsut and (B) bacteriophage Phobos.

A B

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Phage Characterization

Lytic vs. Temperate Phage. There are two basic types of phage lifestyles, those of lytic phage and those of temperate phage. Lytic phages simply infect their host and produce progeny through lysis and destruction of the host cell. These are desired for agricultural treatment. Temperate phages are able to infect a host and integrate their DNA into the host genome. They can lie ‘dormant’ until awakened by an environmental cue such as stress. Temperate phages are not desired. Temperate phages can be identified in the lab through close inspection of the plaques produced by phage. Lytic phage produce a clear plaque of killing whereas temperate phage produce a turbid plaque since they can integrate into the host genome instead of lysing the host (Figure 3). Only one of the 41 bacteriophages that infect Erwinia amylovora appears to be a temperate phage. These results will be validated by whole genome DNA sequencing of all 41 bacteriophages. The bacteriophages that are temperate should contain DNA that codes for proteins necessary for the temperate lifestyle (proteins for integrating DNA into the host genome, etc.). Characterization of all 41 bacteriophages is desired so that we can use the more lytic and stabile in our fight against fire blight.

Figure 3. The plaque appearance is indicative of the phage’s ability to enter lysogeny. Lytic phage produce clear plaques (A) and lysogenic phage produce turbid plaques (B)

Bacteriophage Stability. Of the 41 bacteriophages, 12 we isolated by June of 2013. These 12 phages have been stored at a concentration of 1010 plaque forming units/mL (pfu/mL) for six months and have not lost titer. We will be conducting stability tests of all 41 phages in this study, but our initial results are encouraging.

DNA isolation. Of the 41 bacteriophages, we have successfully isolated DNA for 28. This DNA is of sufficient quantity and purity to ensure high quality whole genome sequencing as assessed by DNA gel electrophoresis (Figure 4).

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Figure 4. Purified whole genome DNA isolated from six bacteriophages that infect Erwinia amylovora. The DNA appears as a solid, bright, single band of appropriate size (lane 1 is a DNA size standard ladder, lanes 2-7 is genomic DNA isolated from six different bacteriophages).

Phage Safety Studies

Bacteriophage Specificity Tests. In order to ensure the safety of our bacteriophages, we have tested their ability to infect closely related bacteria, E. coli and Salmonella. No bacteriophage has ever been isolated that infects bacteria that are very distantly related, for example, no bacteriophage can infect gram-negative and gram-positive bacteria, however some have a broader host range than others. We desire bacteriophages that have a fairly narrow host specificity, broad enough to infect different Erwinia amylovora hosts but narrow enough to be unable to infect closely-related bacteria of a different genus such as E. coli and Salmonella. To perform these studies we have collected over 30 strains of Erwinia amylovora from Utah and obtained a strain from Canada. In addition, our collaborators Tim Smith and Ken Johnson are sending us strains from Washington and Oregon to expand our library. A variety of E. coli and Salmonella strains were obtained form Professor John Roth (UC Davis). We conducted host specificity test for 13 of the 41 phages using a three Erwinia amylovora strains, and two E. coli and two Salmonella strains. All 13 of our phages were unable to infect E. coli and Salmonella. Thus our bacteriophage have a high specificity for Erwinia amylovora and should not harm any other organism whether it be bacteria, plant or animal.

Fire Quencher Safety Tests. We have tested Fire Quencher for safety by spraying 100 mL at a concentration of 1010 onto ten apple seedlings. Apple seedlings appeared unaffected and remained healthy after treatment for the duration of the study (10 days) post treatment.

Figure 5. Healthy apple seedlings treated with Fire Quencher appear identical to healthy untreated apple seedlings for the duration of the study. Pictures were taken at day five. Treated (left tray), untreated (right tray).

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Production of Fire Quencher

Fire Quencher was produced by combining five of our most potent and lytic phage at equal concentrations to a final concentration of 1012 in spent nutrient broth (Difco). Because these phages were isolated and amplified from the environment by culturing in Erwinia amylovora, special precautions are taken to ensure the final product does not contain the pest, Erwinia amylovora. Specifically, cultures are centrifuged at high speed (10,000 x g) for 20 minutes, the supernate is treated with chloroform to eliminate any remaining bacteria, and the sample is then filtered through a 0.45 uM filter as a third precaution. Lysates are then streaked to NB to confirm the absence of bacterial growth.

Preparing for Trials in Apple Seedlings

We have tested our ability to infect apple seedlings with Erwinia amylovora. Thus far we have infected 12 apple seedlings with the bacterium by scoring the underside of a leaf at the vein and rubbing an overnight culture of Erwinia amylovora into the wound. All (100%) of the 12 infected apple seedlings were dead within five days. This novel method for testing fire blight treatments allows for testing large numbers in controlled/quarantined green houses without the lose of valuable trees.

Conclusions of Preliminary Data

Our preliminary data includes the isolation and partial characterization of 41 bacteriophages that infect Erwinia amylovora, the causative agent of fire blight. Our characterization of these bacteriophages indicates that they are highly lytic, reaching concentrations of 1012 pfu easily and forming clear plaques. In addition, we have performed tests on the safety of 13 of these phages by determining if they infect closely related bacteria, which they do not. They also do not affect the health of apple seedlings after four treatments at high concentration. Five of these phages were used to produce our product “Fire Quencher”, a mixture of Furthermore, we have demonstrated the ability to infect apple seedlings with Erwinia amylovora. These infections resulted in 100% of the plants showing a level seven infection (the highest rating on our scale). Together, our preliminary data suggests that we have produced a highly potent, safe, natural biopesticide that is ready for early stage testing on apple trees.

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APPENDIX 4 -Attach resume for Principal Investigator and Co-PI’s.

Principal Investigator: Julianne H. Grose Assistant Professor of Microbiology and Molecular Biology 751 WIDB Brigham Young University Provo, Utah 84602 Co-PI’s: Sandra H. Burnett, Ph. D., and Donald P. Breakwell, Ph. D. Dr. Julianne Grose obtained her Ph.D. in 2003 under the direction of Dr. John Roth, a respected Salmonella geneticist who studies many aspects of Salmonella and bacteriophages that infect it. Erwinia is a close relative to Salmonella making many of the experimental approaches and scientific designs similar. Dr. Sandra Burnett and Don Breakwell have been conducting research on bacteriophage since 2009 when they joined the national research project established by the Howard Hughes Medical Institute (HHMI). In 2010 Dr. Julianne Grose became actively involved with this research team and initiated the project to isolate bacteriophages that infect Erwinia amylovora in 2012. Dr. Grose has a USDA permit (PPQ 526) for the transportation of and use of Erwinia amylovora (Application number P526-‐120612-‐015). None of the PI’s have experience with EPA approval or bringing a product to market. Thus, we have joined with experienced researchers Tim Smith (Washing State University) and Ken Johnson (Oregon State University) to obtain the necessary efficacy data. Letters of support from these collaborators are attached. In addition, we are working with Ben Miller and Eric Peery, MBA’s with experience in entrepreneurship. No funding is currently available for these studies. IR4 support would help to bridge this gap between these PI’s and persons experienced with efficacy testing as well as bringing products to market.

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Appendix 4 Continued: Principal Investigator and Co-‐PI Resumes.

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Julianne H. Grose Curriculum vitae

Julianne H. Grose, Ph.D.

Brigham Young University Email: [email protected] Microbiology and Molecular Biology Webpage: http://groselab.byu.edu 751 WIDB Office Phone: (801) 422-4940 Provo, UT 84602 Fax: (801) 422-0519 I am a tenure track professor in the Department of Microbiology and Molecular Biology. My university position consists of 45% effort for teaching, 45% effort for mentoring/research and 10% effort for citizenship. I teach approximately 12 credit hours of undergraduate courses per year and currently mentor 7 graduate students and 18 undergraduates in my lab. My teaching is dedicated to bringing novel research experiences into the classroom through our phage hunters program. My long term goal is to mentor students while contributing novel scientific findings to our field of study, the isolation and characterization of bacteriophages that infect the causative agent of fire blight, Erwinia amylovora. EDUCATION AND TRAINING Education 2003 Ph.D. Biology, University of Utah 1996 B.S. Chemistry, University of Utah Research Positions 2008 – Present Assistant Professor, Brigham Young University, Department of Microbiology and Molecular Biology. Includes research on Erwinia amylovora bacteria

and bacteriophages as part of the BYU Phage Hunters program. 2006 – 2008 Postdoctoral Research Associate for Bioenergenics, Department of Biochemistry, University of Utah. Development of compounds that inhibit PAS kinase. 2004 – 2008 Postdoctoral Scholar, Lab of Dr. Jared Rutter, Department of Biochemistry,

University of Utah. Molecular characterization of the regulation and function of yeast PAS kinase.

1996-2003 Ph.D. Student ,Lab of Dr. John Roth, Department of Biochemistry, University of Utah. Regulation of NAD(P) metabolism in Salmonella typhimurium.

1994-1995 Undergraduate Research Assistant, Lab or Dr. Marion Woods, MD, Department of Infectious Disease, University of Utah School of Medicine 1992-1993 ACCESS Program for Women in Mathematics and Science, instruction and laboratory work in Mathematics and Science, University of Utah PROFESSIONAL ACTIVITIES AND AWARDS: Member of the American Society for Microbiology Member of the Genetics Society of America ASM Early-career Travel Award (2009) Reviewer for Pilot Research Projects Southwest Environmental Health Sciences Center (2012) Ad Hoc Reviewer for the following journals: Acta Biochimica et Biophysica Sinica, Trends in Microbiology, FEMS Microbiology Letters EXTRAMURAL RESEARCH SUPPORT Principal Investigator: Julianne H. Grose National Institutes of Health R15 . Grant number R15 GM100376-01. Amount: $346,949 End date: 5/31/2015 Subaward Principal Investigator: Julianne H. Grose (I. Benjamin PI) National Institutes of Health Subaward. Grant number R10011765-01. Amount: $30,000 End date: 9/31/2013

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Julianne H. Grose Curriculum vitae

RELEVANT RESEARCH GRANTS A Phage-Based Treatment for Fire Blight and American Foulbrood Principal Investigator: Julianne H. Grose, Sandra H. Burnett, Donald P. Breakwell BYU Technology Transfer Bridging Fund Amount: $15,000 End date: 12/13/2012 RELEVANT PUBLICATIONS 1. Jordan D. Jensen, Bryan D. Merrill, Sandra H. Burnett, Julianne H. Grose, and Donald P. Breakwell# The Genomes of Three Novel Bacillus cereus Bacteriophages. Genome Announcements in December 2013. 3. Donald P. Breakwell, E. Zane Barrus, Alex B. Benedict, Alicia K. Brighton, Joshua N. B. Fisher, Adam V. Gardner, Brittany J. Kartchner, Kara C. Ladle, Bryce L. Lunt, Bryan D. Merrill, John D. Morrell, Sandra H. Burnett, and Julianne H. Grose. (2013) Genome Sequences of Five Cluster B1 Mycobacteriophages. Genome Announcements 1(6). 4. Michael A. Sheflo, Adam V. Gardner, Bryan D. Merrill, Joshua N. B. Fisher, Bryce L. Lunt, Donald P. Breakwell, Julianne H. Grose, Sandra H. Burnett. (2013) Complete Genome Sequences of Five Paenibacillus larvae Bacteriophages. Genome Announcements 1(6). 5. Smith, KC, Castro- Nallor, E, Breakwell, DP, Grose, JH.,Burnett, S. (2013) Phage Cluster Relationships Identified Through Single Gene Analysis. BMC Genomics 19;14:410 6. Hatfull, G. et al., (2012) Complete genome sequences of 138 mycobacteriophages. Journal of Virology 86(4): 2382-2384. 7. Grose, JH. (2010), Ch. 15, The Lure of Bacterial Genetics: a Tribute to John Roth. Eds. Maloy, S., Hughes, K.T., and Casadesus, J, ASM Press, Washington, DC, 9-22. *In addition, I am a coauthor on over 20 fully sequenced and annotated whole bacteriophage genomes published in GenBank RELATED RESEARCH PRESENTATIONS NATIONAL/INTERNATIONAL MEETINGS 1. Grose, JH. Isolation and Characterization of Bacteriophages that Infect Erwinia amylovora. (2013) Podium presentation. Analytical Genetic Meeting. 2. Brown, A, Christopher, A, Harrison, C, Kiser, K, Lasko, D, Li, X, Mrrill, B, Peck, K, Perry, LJ, Sabin, N, Schellhous M, Smith, K, Koooyman, D, Price, P, Grose, JH. Phage Pharming. (2013) Podium and Poster presentations iGEM Worldchampionship Jamboree, MIT. 3. Brown, A, Christopher, A, Harrison, C, Kiser, K, Lasko, D, Li, X, Mrrill, B, Peck, K, Perry, LJ, Sabin, N, Schellhous M, Smith, K, Koooyman, D, Grose, JH. Phage Pharming. (2013) Podium and Poster presentations iGEM Regional Jamboree, Toronto, Canada. 4. Gardner,AV, Adawi, EC, Christiansen, MR, Ferguson, NC, Irons, DL, Jensen, J, Kennedy, A, Lloyd, JS, Marlow, S, Mason, S, McCord, TM, Merrill, BD, Nelson, EP, Norton, CS, Pettersson, SM, Poe, DE, , RC, Smith, TC, Sullivan, S, Williams, KR, Morrell, JD, Brighton, AK, Fisher, JNB, Sheflo, MA, Breakwell, DP, Burnett, SH, Grose, JH (2012) Proposal for A1 Subcluster Division and Evidence of Evolutionary Events in B1 and B4 Subcluster Phage. Howard Hughes Medical Institute Fourth Annual Phage Symposium, Ashburn, VA. 5. Brighton, AK, Joshua N. B. Fisher, JNB, Lunt, BL, Taylor, MA, Smith, KC, Baker, B, Barrus, EZ, Chapman, KM, Drake, EA, Jackson, KR, Kartchner, BJ, Kiser, CD, Kiser, JT, Kitchen, JCB, McDaniel, SW, Ormsby, WR, Parker, M, Sheide, MG, Steck, RP, Vance, KS, Breakwell, DP, Burnett, SH, and Grose, JH. (2011) Additional Evidence for Frameshifts in A2 and Gene Mosaicism in F Mycobacteriophage. Howard Hughes Medical Institute Third Annual Phage Symposium, Ashburn, VA. 6. Grose, JH, Breakwell, DP, and Burnett, SH. (2011) Out of the SEA: Getting Students to Crawl on Land. Howard Hughes Medical Institute Third Annual Phage Symposium, Ashburn, VA.

REGIONAL/LOCAL MEETINGS 1. Ferguson, NC, Irons, DL, Marlow, SC, McCord, TM, Brighton, AK, Fisher, JNB, Sheflo, MA, Breakwell, DP, Grose, JH, Burnett, SH (2012) Division of the Mycobacteriophage A1 Subcluster Based on Phylogenetic Comparison. ASM Intermountain Branch Meeting, Idaho State University. 2. Mason, SJ, Gardner, AV, Nelson, EP, Christiansen, MR, Brighton, AK. Fisher, JNB, Sheflo, MA, Breakwell, DP, Grose, JH, Burnett, SH (2012) Mislabeling of the Second Tape Measure Protein. ASM Intermountain Branch Meeting, Idaho State University.

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Julianne H. Grose Curriculum vitae 3. Jensen, JD, Merrill, BD, Russell, RC, Smith, TC, Brighton, AK, Fisher, JNB, Sheflo, MA, Breakwell, DP, Burnett, SH, Grose, JH. (2012) Phylogenetic Origin of Glutaredoxin Gene Shared by Mycobacteriophage A1 Sub-cluster, Distantly Related Bacteria, and other bacteriophages. ASM Intermountain Branch Meeting, Idaho State University. 4. Joshua S. Lloyd, Christian S. Norton, Shea Sullivan, Shanette M. Pettersson, Brighton, AK, Fisher, JNB, Sheflo, MA, Breakwell, DP, Erickson, D, Burnett, SH, Grose, JH. (2012) Lack of Correlation between Phage Clusters and Ecoregions in the United States. ASM Intermountain Branch Meeting, Idaho State University. 5. Williams, KR, Adawi, EC, Kennedy, AK, Poe, DE, Brighton, AK, Fisher, JNB, Sheflo, MA, Breakwell, DP, Burnett, SH, Grose, JH. (2012) Divergent evolution of a RuvC holliday junction resolvase in the B1 subcluster. ASM Intermountain Branch Meeting, Idaho State University. 6. Gardner, AV, Brighton, AK, Fisher, JNB, Sheflo, MA, Breakwell, DP, Grose, JH, Burnett, SH. (2012) Environmental Effect on Phage Genomes: Analysis of the B4 Subcluster. ASM Intermountain Branch Meeting, Idaho State University. 7. Brighton, AK, Kaitlyn, SV, Parker, M, Jackson, KL, Steck, RP, Ormsby, WR, Taylor, MA, Fisher, J, and Lunt, B, Burnett, S.H., Grose, JH. and Breakwell, DP. (2011) Gene Mosaicism Demonstrated in Mycobacteriophage Shauna1. ASM Intermountain Branch Meeting, Weber State University. 8. Barrus, EZ, Sheide, MG, Taylor, MA, Fisher, J, and Lunt, B, Burnett, SH, Grose, JH. and Breakwell, DP. (2011) Shauna1 Mycobacteriaphage holin gene confirms common ancestry of all F cluster phage. ASM Intermountain Branch Meeting, Weber State University. 9. Kartchner, BJ, Kiser, JT, Kiser, CD, McDaniel, SW, Taylor, MA, Fisher, J, Lunt, B, Burnett, SH, Grose, JH, and Breakwell, DP. (2011) Clustering of Mycobacteriophage in the Utah Landscape. ASM Intermountain Branch Meeting, Weber State University. 10. Smith, KC, Burnett, SH, Grose, JH, and Breakwell, DP. (2011) Degenerate PCR Primers to Identify Mycobacteriophage Clusters and Sub-Clusters. ASM Intermountain Branch Meeting, Weber State University. 11. Chapman, KM, Baker, B, Drake, EA, Kitchen, JCB, Taylor, MA, Fisher, J, and Lunt, B, Burnett, SH, Grose, JH, and Breakwell, DP. (2011) TA17A: A Unique Member of the Mycobacteriophage Sub-Cluster A2. ASM Intermountain Branch Meeting, Weber State University. 12. Kitchen, JCB, Brighton, AK, Chapman, KM, Baker, B, Taylor, MA, Fisher, J, and Lunt, B, Burnett, SH, Grose, JH, and Breakwell, DP. (2011) Morphological Traits of Mycobacteriophage Clusters and Sub-Clusters. ASM Intermountain Branch Meeting, Weber State University.

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Donald Philip Breakwell, Ph.D.

Professor Department of Microbiology and Molecular Biology Brigham Young University 799 WIDB Provo, UT 84602 Telephone (801) 378-‐2378 Email [email protected] Education Doctor of Philosophy, August 1992, Purdue University Master of Science, August 1988, Purdue University Bachelor of Science, August 1986, Brigham Young University Honors and Awards College of Life Sciences Teaching Excellence Award, 2008, Brigham Young University Alcuin Fellowship in General Education, 2003-‐2006, Brigham Young University Professional Service American Society for Microbiology 2009-‐2012, Editorial Board, Journal of Microbiology and Biology Education. American Society for Microbiology 2008-‐2009, Chair of Steering Committee, American Society for Microbiology Conference for Undergraduate Educators. American Society for Microbiology 2008-‐2010, Member, Committee on Undergraduate Education American Society for Microbiology Co-‐Chair, 13th Annual American Society for Microbiology Conference for Undergraduate Educators. Orlando, FL. May 19-‐21, 2006. American Society for Microbiology Session Chair, 2010, Intermountain Branch Meeting, Provo, UT. Publications (since 2011) 1. D. P. Breakwell, E. Z. Barrus , A. B. Benedict , A. K. Brighton , J. N.B. Fisher , A. V. Gardner , B. J. Kartchner ,

K. C. Ladle , B. Lunt , B. D. Merrill , J. D Morrell , S. H Burnett , J. H. Grose. Genome Sequences of Five B1 Subcluster Mycobacteriophages. 2013. Genome Announcements. Genome Announc. November/December 2013 1:e00968-‐13;doi:10.1128/genomeA.00968-‐13

2. M. A. Sheflo, A. V. Gardner, B. D. Merrill, J. N. B. Fisher, B. L. Lunt, D. P. Breakwell, J H. Grose, and S. H. Burnett. 2013. Complete Genome Sequences of Five Paenibacillus larvae Bacteriophages. Genome Announc. November/December 2013 1:e00668-‐13;

3. K. C Smith, E. Castro-‐Nallar, J. N. B Fisher, D. P. Breakwell, J. H. Grose, S. H Burnett. 2013. Phage Cluster Relationships Determined by Single Gene Analysis. BMC Genomics 2013, 14:410 (19 June 2013)

4. P.S. Shen, M. J. Domek, E. Sanz-‐García, A. Makaju, R. M. Taylor, R. Hoggan,M. D. Culumber, C. J. Oberg, D. P. Breakwell, J. T. Prince, and D. M. Belnap. 2012. Sequence and Structural Characterization of Great Salt Lake Bacteriophage CW02, a Member of the T7-‐Like Supergroup. J. Virol. 86:15 7907-‐7917

5. Graham F. Hatfull, the Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science Program, the KwaZulu-‐Natal Research Institute for Tuberculosis and HIV Mycobacterial Genetics Course Students and the Phage Hunters Integrating Research and Education Program. 2012. Complete Genome Sequences of 138 Mycobacteriophages. J. Virol. 86:2382-‐2384

6. Jordon K. March, Kyle C. Jensen, Nathan T. Porter, and Donald P. Breakwell. 2011. Authentic Active Learning Activities Demonstrating the Use of Serial Dilutions and Plate Counts. Journal of Microbiology and Biology Education 12: 152-‐156.

Presentations (since 2010) 1. Jensen, JD, (2013), J.N.B. Fisher, J.H. Grose, S.H. Burnett, and D.P. Breakwell. Isolation and

Characterization of Three Novel Bacteriophages of Bacillus cereus. American Society for Microbiology General Meeting, Denver, CO.

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2. Ferguson, NC, Irons, DL, Marlow, SC, McCord, TM, Brighton, AK, Fisher, JNB, Sheflo, MA, Breakwell, DP, Grose, JH, Burnett, SH (2012) Division of the Mycobacteriophage A1 Subcluster Based on Phylogenetic Comparison. ASM Intermountain Branch Meeting, Idaho State University.

3. Mason, SJ, Gardner, AV, Nelson, EP, Christiansen, MR, Brighton, AK. Fisher, JNB, Sheflo, MA, Breakwell, DP, Grose, JH, Burnett, SH (2012) Mislabeling of the Second Tape Measure Protein. ASM Intermountain Branch Meeting, Idaho State University.

4. Jensen, JD, Merrill, BD, Russell, RC, Smith, TC, Brighton, AK, Fisher, JNB, Sheflo, MA, Breakwell, DP, Burnett, SH, Grose, JH. (2012) Phylogenetic Origin of Glutaredoxin Gene Shared by Mycobacteriophage A1 Sub-‐cluster, Distantly Related Bacteria, and other bacteriophages. ASM Intermountain Branch Meeting, Idaho State University.

5. Joshua S. Lloyd, Christian S. Norton, Shea Sullivan, Shanette M. Pettersson, Brighton, AK, Fisher, JNB, Sheflo, MA, Breakwell, DP, Erickson, D, Burnett, SH, Grose, JH. (2012) Lack of Correlation between Phage Clusters and Ecoregions in the United States. ASM Intermountain Branch Meeting, Idaho State University.

6. Williams, KR, Adawi, EC, Kennedy, AK, Poe, DE, Brighton, AK, Fisher, JNB, Sheflo, MA, Breakwell, DP, Burnett, SH, Grose, JH. (2012) Divergent evolution of a RuvC holliday junction resolvase in the B1 subcluster. ASM Intermountain Branch Meeting, Idaho State University.

7. Gardner, AV, Brighton, AK, Fisher, JNB, Sheflo, MA, Breakwell, DP, Grose, JH, Burnett, SH. (2012) Environmental Effect on Phage Genomes: Analysis of the B4 Subcluster. ASM Intermountain Branch Meeting, Idaho State University.

8. JH Grose, DP Breakwell, SH Burnett. 2011. Out of the SEA: Getting Students to Crawl on Land. Howard Hughes Medical Institute NGRI Third Annual Symposium, Ashburn, VA.

9. Brighton, AK, Fisher, JNB, Lunt, BL, Taylor, MA, Smith, KC, Baker, B, Barrus, EZ, Chapman, KM, Drake, EA, Jackson, KR, Kartchner, BJ, Kiser, CD, Kiser, JT, Kitchen, JCB, McDaniel, SW, Ormsby, WR, Parker, M, Sheide, MG, Steck, RP, Vance, KS, Breakwell, DP, Burnett, SH, and Grose, JH. (2011) Additional Evidence for Frameshifts in A2 and Gene Mosaicism in F Mycobacteriophage. Howard Hughes Medical Institute Third Annual Phage Symposium, Ashburn, VA.

10. Kartchner BK , JT Kiser, CD Kiser et al. 2011. Clustering of Mycobacteriophage in the Utah Landscape. American Society for Microbiology Intermountain Branch Meeting, Ogden, UT.

11. KC Smith, SH Burnett, JH Grose, DP Breakwell. 2011. Degenerate PCR Primers to Identify Mycobacteriophage Clusters and Sub-‐Clusters. American Society for Microbiology Intermountain Branch Meeting, Ogden, UT.

12. AK Brighton, KS Vance, KR Jackson et al. 2011. Gene Mosaicism Demonstrated in Mycobacteriophage Shauna1. American Society for Microbiology Intermountain Branch Meeting, Ogden, UT.

13. JCB Kitchen, AK Brighton, KM Chapman, et al. 2011. Morphological Traits of Mycobacteriophage Clusters and Sub-‐Clusters. American Society for Microbiology Intermountain Branch Meeting, Ogden, UT.

14. EZ Barrus, MG Sheide, MA Taylor, et al. 2011. Shauna1 Mycobacteriaphage holin gene confirms common ancestry of all F cluster phage. American Society for Microbiology Intermountain Branch Meeting, Ogden, UT.

15. KM Chapman, B Baker, EA Drake, et al. 2011. TA17A: A Unique Member of the Mycobacteriophage Sub-‐Cluster A2. American Society for Microbiology Intermountain Branch Meeting, Ogden, UT.

16. DP Breakwell and SH Burnett. (2010) Data Overload: Letting Freshmen Students “Have At” Mycobacteriophage Lab Work and Comparative Genomics. Howard Hughes Medical Institute NGRI Second Annual Symposium, Ashburn, VA.

17. CJ Sargent, DE Payne, II, BL Lunt, LB Argueta, PB, AB Benedict, LA Bull, ME Daetwyler, BJ Earley, JM Engle, JNB Fisher, I Giri, E Greenhalgh, AW Hansen, KJ Haskell, TF Issac, KL, ZS Liechty, SK Petersen, DS Sabin, MC Severson, KC Smith, MAR Taylor, TJ Woodward, BA Wright, SH Burnett, DP Breakwell. (2010) Genomic Analysis of the Newly-‐ Isolated Subcluster B1 Mycobacteriophage KLucky39 Reveals a Novel Putative Peptidase and a Primase, the Lack of Five Anticipated Genes, and the Relationship of KLucky39 to Other Phage. Howard Hughes Medical Institute NGRI Second Annual Symposium, Ashburn, VA.

18. Grose, J. and D.P. Breakwell. 2010. Using Bakers Yeast to Teach Life Cycles of Eukaryotic Microbes. American Society for Microbiology Conference for Undergraduate Educators, San Diego, CA.

19. KC Smith, ME Daetwyler, ZS Liechty, MC Severson, B Wright, BL Lunt, DE Payne II, DP Breakwell, and SH Burnett (2010) Tape Measure Protein in Mycobacteriophage KLucky39 Shows Evolution of Phage Clusters. American Society for Microbiology Intermountain Branch Meeting, Provo, UT.

20. L Argueta, S Petersen, D Sabin, C Sargent, MA Taylor, BL Lunt, DE Payne II, DP Breakwell, and SH Burnett

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(2010) Addition of Novel Mycobacteriophage to Pre-‐existing Subclusters of the B Cluster. American Society for Microbiology Intermountain Branch Meeting, Provo, UT.

21. A Hansen, K Ladle, A Benedict, BL Lunt, DE Payne II, SH Burnett, and DP Breakwell (2010) Mycobacteriophage Exhibit Discrepancies in the Distance of Shine-‐Dalgarno Sequences from the Start Codon. American Society for Microbiology Intermountain Branch Meeting, Provo, UT.

22. J Fisher, I Giri, T Issac, S Burnett, D Breakwell (2010) The Mycobacteriophage KLucky39 Genome Lacks Five Genes Commonly Found in Other Mycobacteriophage Subcluster B1 Genomes. American Society for Microbiology Intermountain Branch Meeting, Provo, UT.

23. J Engle, T Woodward, E Greenhalgh, K Haskell, BL Lunt, DE Payne II, DP Breakwell and SH Burnett (2010) The Genome of Mycobacteriophage KLucky39 Reveals a Putative M23 Peptidase Gene. American Society for Microbiology Intermountain Branch Meeting, Provo, UT.

24. K Haskell, B Earley, P Bajgain, D Breakwell, and S Burnett (2010) Comparison of KLucky39 Mycobacteriophage With Bacterium E. Coli. Microbiology and Molecular Biology Brigham Young University. American Society for Microbiology Intermountain Branch Meeting, Provo, UT.

GENBANK PUBLICATIONS The following are GenBank publications of complete phage genomes. All genomes include full genomes (not genome fragments) with complete annotation of all genes and identification of any present tRNAs. Genomes were peer reviewed by GenBank prior to acceptance and publication.

Year Phage Accession # Authors

2013 Alex

(Mycobacterio-‐phage)

JX649100 Benedict,A.B., Fisher,J.N.B., Gardner,A.V., Lunt,B.L., Payne,D.E., Burnett,S.H., Breakwell,D.P. and Grose,J.H.

2013 Gyarad


JX649099 Ladle,K.C., Fisher,J.N.B., Gardner,A.V., Lunt,B.L., Breakwell,D.P., Grose,J.H. and Burnett,S.H.

2013 Nacho


JX649098 Kartchner,B.J., Fisher,J.N.B., Gardner,A.V., Lunt,B.L., Grose,J.H., Burnett,S.H. and Breakwell,D.P.

2013 Piglet


JX649097 Barrus,E.Z., Adawi,E.C., Kennedy,A.K., Poe,D.E., Williams,K.R., Fisher,J.N.B., Gardner,A.V., Merrill,B.D., Grose,J.H., Burnett,S.H. and Breakwell,D.P.

2013

Serpentine (Mycobacterio-‐

phage) JX649096 Brighton,A.K., Fisher,J.N.B., Gardner,A.V., Lunt,B.L., Breakwell,D.P.,

Burnett,S.H. and Grose,J.H.

2013 TA17A


Lunt,B.L., Payne,D.E., Fisher,J.N.B., Smith,K.C.B., Taylor,M.R., Baker,B., Barrus,E.Z., Brighton,A.K., Chapman,K.M., Drake,E.A., Jackson,K.R., Kartchner,B.J., Kiser,C.D., Kiser,J.T., Kitchen,J.C.B.,McDaniel,S.W., Ormsby,W.R., Parker,M., Sheide,M.G., Steck,R.P., Vance,K.S., Breakwell,D.P., Burnett,S.H. and Grose,J.H.

2013 Basilisk (B. cereus phage) KC595511.1

Jensen,J.D., Fisher,J.N.B., Gardner,A.V., Irons,D.L., Lloyd,J., Pettersson,S.M., Smith,C., Sullivan,S., Brighton,A.K., Sheflo,M.A., Burnett,S.H., Breakwell,D.P. and Grose,J.H

2013 JL (B. cereus phage) KC595512.1

Lloyd,J., Fisher,J.N.B., Gardner,A.V., Hallam,S.J., Jensen,J.D., Pettersson,S.M., Smith,C., Sullivan,S., Brighton,A.K., Sheflo,M.A., Burnett,S.H., Breakwell,D.P. and Grose,J.H.

2013 Shanette (B. cereus phage) KC595513

Pettersson,S.M., Fisher,J.N.B., Gardner,A.V., Hallam,S.J., Jensen,J.D., Lloyd,J., Smith,C., Sullivan,S., Brighton,A.K., Sheflo,M.A., Burnett,S.H., Breakwell,D.P. and Grose,J.H.

2013 Jimmer1 (P. larvae phage) KC595515 Merrill,B.D., Sheflo,M.A., Gardner,A.V., Merrill,C.A., Williams,K.R.,

Lunt,B.L., Ayer,P.A., Grose,J.H., Breakwell,D.P. and Burnett,S.H 2013 Jimmer2

(P. larvae phage) KC595514 Sheflo,M.A., Gardner,A.V., Kennedy,A.K., Beckstead,A.P., Russell,R.C., Merrill,B.D., Merrill,C.M., Zimmerman,L.J., Lunt,B.L., Grose,J.H., Breakwell,D.P. and Burnett,S.H.

2013 Abuou (P. larvae phage) KC595517

Sheflo,M.A., Gardner,A.V., Kennedy,A.K., Beckstead,A.P., Russell,R.C., Merrill,B.D., Merrill,C.M., Zimmerman,L.J., Lunt,B.L., Grose,J.H., Breakwell,D.P. and Burnett,S.H.

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Sandra Hale Burnett CURRICULUM VITAE 791 WIDB, Provo, UT 94602, (801) 422-1310, Fax: (801) 422-0519, [email protected] EMPLOYMENT AND POST-DOCTORAL EXPERIENCE 2010- Associate Professor

Microbiology and Molecular Biology Department Brigham Young University

2004- Director, Research Instrumentation Core Facility College of Life Sciences Brigham Young University

2004-2010 Assistant Professor Microbiology and Molecular Biology Department Brigham Young University

2003-2004 Research Associate Microbiology, Immunology and Molecular Genetics Department University of Kentucky Medical School

2000-2003 Post-Doctoral Fellow Microbiology, Immunology and Molecular Genetics Department University of Kentucky Medical School

EDUCATION 1994-2000 Ph.D., Veterinary Science, Focus: Virology and Immunology.

Gluck Equine Research Center, Veterinary Science Department University of Kentucky College of Agriculture

Dissertation: Development of a competitive reverse transcription PCR assay to quantify equine IFN-γ, IL-2, IL-4 and G3PDH. Preliminary study of equine arteritis virus (EAV) and cytokine responses in horses. 1992-1993 M.S., BioVeterinary Science, Focus: Virology and Biochemistry.

Animal, Dairy and Veterinary Science Department Utah State University

Thesis: Inhibitors of equine arteritis virus replication. 1989-1992 B.A., German with Chemistry minor, Focus: Pre-veterinary medicine.

Utah State University PERSONAL STATEMENT The main focus of my research is to identify new bacteriophage, including isolation, whole genome sequencing, and genome comparison work. I am one of three faculty members to run the Phage Hunters program established through a grant from Howard Hughes Medical Institute in 2009. Our program includes approximately 20 undergraduate students per year. Students isolate and purify phage, and prepare DNA extractions for sequencing. Samples are sequenced and the students verify assembly and perform genome annotation for submission to GenBank. Since 2009, our students have isolated and sequenced the genomes of over 55 unique phages, published 20 whole phage genomes with more to come. The Phage Hunter program provides an opportunity to pursue phage isolation and characterization for future phage therapy against pathogenic bacteria.

LEADERSHIP ACCOMPLISHMENTS AND SERVICE 2012- 2008-2012 2008-2012 2007-2011 2007- 2005- 2004-

Faculty Development Committee Member, Undergraduate Curriculum Committee Chair, Microbiology & Molecular Biology Dept., BYU College Curriculum Committee Member, College of Life Sciences, BYU Computer Resources Committee Member, College of Life Sciences, BYU Executive Board Member, Autumn Immunology Conference Specific Pathogen-Free Animal Facility Advisory Committee Member, College of Life Sci., BYU Core Facility Directors Committee Member, College of Life Sciences, BYU

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PROFESSIONAL AFFILIATIONS 2010- 2009- 2007- 2003- 1997- 1993-2006

Member, International Society for Transgenic Technology Science Education Alliance “Phage Hunters,” Howard Hughes Medical Institute Executive Committee Member, Autumn Immunology Conference, Chicago Member, American Association of Immunologists Member, American Society for Microbiology Associate Member, American Society for Virology

TRANSGENIC MOUSE DEVELOPMENT 2003 Submission of the new transgenic mouse strain to Jackson Laboratories: Macrophage

Fas-Induced Apoptosis (Mafia) mice. Jackson stock number 005070. Strain: C57BL/6J-Tg(Csf1r-EGFP-NGFR/FKBP1A/TNFRSF6)2Bck/J

INTELLECTUAL PROPERTIES

U.S. Patents Pending (as well as 6 provisional patents pending and 2 invention disclosures) “Methods)and)Devices)for)Charged)Molecule)Manipulation,”)Aten,)Q.T.,)Burnett,)S.H.,)Howell,)L.L.,)Jensen,)B.D.,)assigned)to)Brigham)Young)University.)

Intellectual Property Agreements

“Methods)and)Devices)for)Charged)Molecule)Manipulation,”)Aten,)Q.T.,)Burnett,)S.H.,)Howell,)L.L.,)Jensen,)B.D.,)licensed)to)NanoInjection)Technologies,)LLC.))

SCHOLARSHIPS, HONORS AND AWARDS: 2002-2003 2001-2002 2000-2001 1997 1995 1994

NIH Research Training Grant in Microbial Pathogenesis, Univ. of Kentucky Medical School NIH Research Training Grant in Microbial Pathogenesis, Univ. of Kentucky Medical School NIH Research Training Grant in Cancer Etiology and Treatment, Univ. of KY Medical School Travel Grant, Graduate School, Univ. of KY, to American Society for Microbiology, Miami, FL Travel Grant, Society for Values in Higher Education, New Teacher Workshop, Claremont, CA Gamma Sigma Delta Honor Society of Agricultural, Univ. of KY

RESEARCH SUPPORT 2012-2013 2012 2011-2012 2011 2010-2011 2010 2009-2010

$ 16,000 $ 1,500 $ 49,950 $132,668 $ 97,500 $116,214 $127,386

Co-PI with J. Grose, D. Breakwell. A phage-based treatment for Fireblight and American Foulbrood. BYU Technology Transfer. PI. Transgenic mouse rederivation project. NIH-University of Utah. Co-PI with L.Howell, B.Jensen (PI). Cytoplasm-to-Pronucleus and YAC Nanoinjection. Nanoinjection and Cell Restraint Technologies. NanoInjection Technologies, LLC. Co-PI with L.Howell, B.Jensen (PI). Development of Nanoinjection Prototypes and Protocols, Phases I, II, and III. NanoInjection Technologies, LLC.

2009-2012 2008-2009 2006-2007 2005-2006

Co-PI with D. Breakwell to join Science Education Alliance, National Research Genomics Initiative to setup and run “Phage Hunters” research program, Howard Hughes Medical Institute. $15,000 Office of Research and Creative Activity Mentoring Grant, Brigham Young Univ. $30,000 Office of Research and Creative Activity Mentoring Grant, Brigham Young Univ. $19,900 College of Biology and Agriculture Mentoring Grant, Brigham Young Univ.

REPRESENTATIVE JOURNAL PUBLICATIONS (7 of 23 papers & 20 phage whole genomes) Breakwell DP, Barrus EZ, Benedict AB, Brighton AK, Fisher JNB, Gardner AV, Kartchner BJ, Ladle KC, Lunt BL, Merrill BD, Morrell JD, Burnett SH, Grose JH (20130) Genome Sequences of Five B1 Subcluster Mycobacteriophages Genome Announc. Nov/Dec 2013 1:e00968-13; doi:10.1128/genomeA.00968-13. Sheflo MA, Gardner AV, Merrill BD, Fisher JNB, Lunt BL, Breakwell DP, Grose JH, Burnett SH (2013)

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Complete Genome Sequences of Five Paenibacillus larvae Bacteriophages. Genome Announc. Nov/Dec 2013 1:e00668-13; doi:10.1128/genomeA.00668-13. Smith KC, Castro-Nallar E, Fisher JNB, Breakwell DP, Grose JH, Burnett SH (2013) Phage cluster relationships identified through single gene analysis. BMC Genomics 14:410. Wilson AM, Aten QT, Toone NC, Black JL, Jensen BD, Tamowski S, Howell LL, Burnett SH (2013) Transgene delivery via intracellular electroporetic nanoinjection. Transgenic Res. 22:993-1002. Teichert, G.H., Aten, Q.T., Easter, M., Burnett, S.H., Jensen, B.D., Howell, L.L. (2012) A Metamorphic Erectable Cell Restraint (MECR). Proceedings of the ASME 2012 International Mechanical Engineering Congress & Exposition, Chicago, IL. Aten, QT, Jensen, BD, Tamowski, S, Wilson, AM, Howell, LL, and Burnett, SH (2012) Nanoinjection: Pronuclear DNA Delivery using a Charged Lance. Transgenic Res., 21(6), 1279-1290.

Hatfull, et al. (2012) Complete genome sequences of 138 mycobacteriophages. J. Virology, 86(4) 2382. REPRESENTATIVE RESEARCH PRESENTATIONS (7 of 48 since 2004) Merrill BD, Sheflo MA, Ayer PA, Beckstead AP, Fajardo CP, Ferguson NC, Fisher JNB, Gardner AV, Graves KA, Hartmann KA, Kennedy AK, Liu JE, Lunt BL, Merrill CA, Russell RC, Wake BN, WilliamsKR, Zimmerman LJ, Grose JH, Breakwell DP, Burnett SH. (2013) Discovery and Characterization of Novel Paenibacillus larvae Bacteriophages. 5th Annual SEA-Phages Symposium, Ashburn, VA. Herring JA, Deus LM, Manci AM, Meadows HN, Heiner ME, Willyerd HJ, Gardner AV, Fisher JNB, Smith K, Grose JH, Breakwell DP, Burnett SH (2013) Phage cluster and subcluster identification using Tape Measure Protein primers in a PCR reaction. 5th Annual SEA-Phages Symposium, Ashburn, VA. Merrill BD, Sheflo MA, Ayer PA, Beckstead AP, Fajardo CP, Ferguson NC, Fisher JNB, Gardner AV, Graves KA, Hartmann KA, Kennedy AK, Liu JE, Lunt BL, Merrill CA, Russell RC, Wake BN, WilliamsKR, Zimmerman LJ, Grose JH, Breakwell DP, Burnett SH. (2013) Discovery and Characterization of Novel Paenibacillus larvae Bacteriophages. ASM Intermountain Branch Meeting, Idaho State University, Pocatello, ID. Ferguson NC, Irons DL, Marlow SC, McCord TM, Brighton AK, Fisher JNB, Sheflo MA, Breakwell DP, Grose JH, Burnett SH (2012) Division of the Mycobacteriophage A1 Subcluster Based on Phylogenetic Comparison. ASM Intermountain Branch Meeting, Idaho State University, Pocatello, ID. Grose JH, Breakwell DP, Burnett SH (2011) Out of the SEA: Getting Students to Crawl on Land. Howard Hughes Medical Institute Third Annual Phage Symposium, Ashburn, VA. A Wilson, Q Aten, B Jensen, L Howell, S Tamowski and S Burnett. (2011) Next Generation pronuclear injection: MEMS replaces the pump. Transgenic Research 20(5), p. 1159. Presented at the Tenth Transgenic Technology Meeting, St. Pete Beach, FL. Brighton AK, Vance KS, Parker M, Jackson KR, Steck RP, Ormsby WR, Taylor MA, Fisher JNB, and Lunt BA, Burnett SH, Grose JH, Breakwell DP. (2011) Gene Mosaicism Demonstrated in Mycobacteriophage Shauna1. ASM Intermountain Branch Meeting, Weber State University, Ogden, UT.

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APPENDIX 7-‐ Budget and Justification

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$ 1

$ $ $ $ $ $ NOT ALLOWED $ $ $

Project Period: 2014 BIOPESTICIDE PROJECT BUDGET To:

Funds Requested Matching Funds Totals ($)

A. Senior/Key Person $ $ B. Other Personnel $ 6,000 $ $ $ Total Number, Other Personnel C. Fringe Benefits $ $ Total Salary, Wages and Fringe Benefits $ $

D. Equipment NOT ALLOWED $

E. Travel $ $ 1. Domestic $1,000 $1,000 2. Foreign NOT ALLOWED $

F. Participant Support Costs $ $ 1. Travel $ $ 2. Other $ $

G. All Other Direct Costs 1. Materials and Supplies 11,000 $11,000 2. Publication Costs $ 3. Consultant Services $ 4. Computer Services $ 5. Subawards/Consortium/Contractual Costs $ 6. Equipment or Facility Rental/User Fees 7,000 $3,000 7. Alterations and Renovations $ 8. Other 1 $ 9. Other 2 $ 10. Other 3 $

Total Direct Costs $25,000 $15,000

**Each budget item requires documentation** **IMPORTANT**

On a separate sheet provide the following information: Project title, PI name and one paragraph statement of work Identify each budget item individually - provide cost and a written description and/or purpose for the cost. For rentals and fees: identify type of rental or fee and provide rental rate & purpose for the cost Any contractual work will require a separate budget and statement of work including rate and purpose

The Other category MAY NOT include construction or indirect overhead. These costs are not permitted, under any circumstances, under this grant. 1Indicate in a footnote if the matching funds are monetary or in kind and their source

Please enter all values to the nearest hundred dollars.

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APPENDIX 7- Budget and Justification.

Budget Justification A Natural Treatment for Fire Blight: Pilot Tests

in Apple Orchards Julianne H. Grose

Personnel wages: $6,000 This will cover the stipend for a graduate student to be dedicated to this

project from January – August 2014. This graduate student will oversee all treatments and preparation of materials directly. Other undergraduate research assistants will also assist as either volunteers or for course credit.

Travel: $1,000 ($2,000 total with match from BYU) This will cover the costs of travel for Julianne H. Grose to meet directly

with Tim Smith (Washington State University) and Ken Johnson (Oregon State University) to discuss experimental designs, etc.

Supplies: $11,000 ($22,000 total with match from BYU) This will cover basic research supplies to grow and mass produce phages

(including culture containers, nutrient broth, filters for bacterial removal, storage and transportation containers, pipet tips, gloves, etc.), reagents for PCR analysis of Erwinia strains and as well as phages (TAQ polymerase, dNTP’s and DNA ladder, New England Biolabs), kits to prepare phages for DNA sequencing (Norgen Biotek catalog #46850 ) DNA sequencing reagents, and phage shipping costs. In addition, it will cover greenhouse reagents including soil, plants, and pots.

Facility user fees: $7,000 ($10,00 total with match from BYU) $3,000 for DNA sequencing. This will cover the BYU sequencing

facility costs for all 41 phages to be sequenced (with $3,000 matched funds from the MMBIO department at BYU).

$4,000 for studies done at Washington State University and Oregon State University

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24

APPENDIX 8- REFERENCES

Chhibber, S., Kaur, T., and Sandeep, K. (2013). Co-‐therapy using lytic bacteriophage and linezolid: effective treatment in eliminating methicillin resistant Staphylococcus aureus (MRSA) from diabetic foot infections. PloS one 8, e56022. Escobar-‐Paramo, P., Gougat-‐Barbera, C., and Hochberg, M.E. (2012). Evolutionary dynamics of separate and combined exposure of Pseudomonas fluorescens SBW25 to antibiotics and bacteriophage. Evolutionary applications 5, 583-‐592. Kirby, A.E. (2012). Synergistic action of gentamicin and bacteriophage in a continuous culture population of Staphylococcus aureus. PloS one 7, e51017. Khan, M., Zhao, Y., Korban, S. (2012) Moleculr Mechanisms of Pathogenesis and Resistance of the Bacterial Pathogen Erwinia amylovora, Causal Agent of Fire Blight Diseases in Rosaceae. Plant Mol BIol Rep: 1-‐14. Knezevic, P., Curcin, S., Aleksic, V., Petrusic, M., and Vlaski, L. (2013). Phage-‐antibiotic synergism: a possible approach to combatting Pseudomonas aeruginosa. Research in microbiology 164, 55-‐60. Lipsitch, M., Singer, R.S., and Levin, B.R. (2002). Antibiotics in agriculture: when is it time to close the barn door? Proceedings of the National Academy of Sciences of the United States of America 99, 5752-‐5754. Martinez, J.L. (2009). Environmental pollution by antibiotics and by antibiotic resistance determinants. Environmental pollution 157, 2893-‐2902. Martinez, J.L., Fajardo, A., Garmendia, L., Hernandez, A., Linares, J.F., Martinez-‐Solano, L., and Sanchez, M.B. (2009). A global view of antibiotic resistance. FEMS microbiology reviews 33, 44-‐65. Mila, A.L., and Ngugi, H.K. (2011). A Bayesian approach to meta-‐analysis of plant pathology studies. Phytopathology 101, 42-‐51. Ngugi, H.K., Lehman, B.L., and Madden, L.V. (2011). Multiple treatment meta-‐analysis of products evaluated for control of fire blight in the eastern United States. Phytopathology 101, 512-‐522. Stockwell, V.O., and Duffy, B. (2012). Use of antibiotics in plant agriculture. Revue scientifique et technique 31, 199-‐210. Stockwell, V.O., Johnson, K.B., Sugar, D., and Loper, J.E. (2011). Mechanistically compatible mixtures of bacterial antagonists improve biological control of fire blight of pear. Phytopathology 101, 113-‐123. Vanneste, J.L. (2000) What is Erwinia amylovora? How to control it? CABI, New York.

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Supplemental Data to Accompany Timothy Smith’s Letter of Support.

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A Summary of Recent Results in Fire Blight Control Product Efficacy Trials

Timothy J. Smith, Washington State University, September 14, 2011

Overview: Fire blight is a bacterial disease that may attack apples and pears under certain weather conditions when flowers are present on the trees. For the disease to occur, a series of unusual events must occur in proper order:

The bacteria Erwinia amylovora must be present nearby, usually oozing from an active canker carried over from last season’s infections.

These bacteria must be transported from the oozing canker to the stigma surfaces of open flowers, usually by flies or pollinating insects.

Warm temperatures must occur, with sufficient warmth to allow the bacteria colony to grow rapidly to large numbers on the stigma.

The blossom then must be gently wetted, which allows the bacteria to be washed, or to swim from the tip of the stigma where they were growing, to the nectaries, which provide them the necessary open entry into the tree.

The bacteria that have gained entry into the highly susceptible young fruitlet must then find growing conditions to their benefit, allowing them to grow rapidly and overwhelm the flower tissues, leading to infection. Once the infection of the flower is successful, the bacteria move out of the flower into the other uninfected flowers of the cluster, into the fruit spur, and then beyond into the young wood of the tree. Within 10 to 30 days of initial infection, the damage appears on the tree as a “strike.” By this time, the bacteria are moving symptomlessly throughout the host tree, and may cause further damage to distant structures, such as shoot tips or sensitive rootstocks.

There are key points of this infection process that can be managed in the effort to prevent infection, or reduce damage to the host tree or orchard. First point, the presence of blight bacteria: Sanitation of the neighborhood is the most important step in control. Fire blight is very difficult to control if there are active cankers nearby, providing a constant source of high numbers of blight bacteria. The higher the number of live active cankers, and the closer they are, the higher the difficulty of control. Control of blight depends on the identification and removal of as many of these active cankers as possible. This is not easy, but a careful inspection and sanitation effort should be carried out prior to the normal pruning of the orchard. Blight cuttings can then be removed from the orchard prior to being intermingled with the non-‐infected pruning wood. Second point, the transport of bacteria to the blossoms: Efforts to reduce infection by controlling insects that visit blossoms have never been successful. Third point, warm temperatures and bacterial multiplication: Erwinia amylovora bacteria multiply best on the flowers’ stigma surfaces, using the same food and moisture resources that the pollen needs for germination and growth. The multiplication rate of the bacteria is dependent on moment by moment temperatures. The average temperature of the day is an outdated rough way to estimate bacterial growth rate. Totaling hourly temperature related to bacterial growth rate values over the time that the flowers are open is more precise. Fire blight infection risk models should serve as a method to

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determine when an infection is likely in the near future, or if infection has occurred during the current day. (See “CougarBlight 2010” for the recommended blight risk model, or WSU DAS.) If the infection risk forecast indicates infection conditions are possible in about three or four days, the flowers may be protected during the days leading up to that infection with products that retard the development of the blight bacteria that may be trying to grow on the blossoms. Most non-‐antibiotic products must be used this way. The “biological” fire blight control products are often living microorganisms, and are most effective if they are placed in the newly opened flowers soon after they open, in time to allow them to establish prior to the introduction of blight bacteria. Antibiotic or copper sprays used during this same time can hinder the development and effect of the “biologicals.” The fourth point, wetting of flowers: The actual infection event occurs when the blossoms are wetted by rain, dew, or light irrigation. If the infection risk model you trust indicates that infection conditions have occurred within the past several hours, and, especially if you believe that active cankers may be near your flowering orchard, antibiotics are the only effective treatment to reduce the degree of infection when sprayed at this timing. The antibiotic must be applied within 24 hours before or after the infection event to be most effective. The antibiotics act directly against the blight bacteria in the nectary by stopping their growth and multiplication. It is possible that the yeast product applied in the days prior to the infection event acts against the blight bacteria by changing the pH and sugar content in the nectary, and when the blight bacteria arrive, they don’t have the conditions or resources necessary to grow. The fifth point, infection occurs and the strikes appear: Nothing good happens after this point. Once blight appears, the degree of damage depends on the age and cultivar of the orchard host, and the relative number of blight strikes per tree or acre. In a young apple orchard, or a pear orchard younger than 15 – 20 years, immediate cutting of blight strikes usually leads to a reduction of total bearing surface removal. However, the blight often continues to occur during the season, and the blight manager often believes that the blight cutting is ineffective. It is best to put extra management resources into prevention, rather than reaction to this disease. Products used for prevention of blossom infection: Over the past decade, the author has tested numerous products and mixtures to assess their effect on fire blight infection. The results are summarized below. The data was developed using standardized evaluation methods that assure high levels of infection in the untreated check, and the “percent control” comparison should not be used as a direct indication of field results under natural infection conditions. To overcome the variability of natural infections, the test trees were all inoculated with high numbers of blight bacteria at full bloom to assure an even dose of bacteria per flower. In practice, products that control the infection of inoculated flowers at about 65% and above, if properly applied, will perform very well in the orchard under natural conditions. Most of the products have specific application requirements, and if correct rate and timing are not followed, are not likely to perform as in the trials. You may notice that results from the same product vary from year to year. This is likely due to variations in weather and degree of successful inoculation, rather than variation in dependability of the tested product. Generally, the various antibiotics have performed better than the other substances, and have been much easier to apply, as most other products require multiple applications prior to the infection period. However, there have been recent results with products that compete with the antibiotics for level of control.

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NOTE: Many of these products may not be registered for use on your crop. Results are provided here only to report research results, and are not intended as recommendations or endorsements. Read the label carefully before using any product to be certain that the product is registered on your crop. *Note also that the bacteria used to inoculate these plots were selected for comparative research purposes, and are susceptible to streptomycin. Many wild blight bacteria in the western USA are resistant to this substance. Streptomycin will not be as effective in most orchards as in these trials. Antibiotics:

Year Product Rate Timing Percent Control*

2011 Streptomycin 17%* 1 lb/A, 200 ppm 100% bloom @ inoculation 87.6

2011 Oxytet. (FireLine) 1 lb/A, 200 ppm 100% bloom @ inoculation 80.7

2011 Kasumin 8L pint/A, 100 ppm 100% bloom @ inoculation 74.4

2010 Streptomycin 17% 1 lb/A, 200 ppm 100% bloom @ inoculation 80.0

2010 Kasumin 2L (1x) 2 qt./A, 100 ppm 100% bloom @ inoculation 79.5

2010 Oxytet. (FireLine) 1 lb/A, 200 ppm 100% bloom @ inoculation 77.7


2009 Kasumin 2L 200 ppm 100% bloom @ inoculation 79.5

2009 Oxytet. 17% 1 lb/A, 200 ppm 100% bloom @ inoculation 70.5

2008 Gentamycin 10% (three treatments)

2.5, 3, & 3.5 lb/100/A

100% bloom @ inoculation 90.2, 87.0 & 86.8

2008 Oxytet. 17% (two treatments)

1 lb/A, 200 ppm 100% bloom @ inoculation 91.8 & 96.0

2008 Streptomycin 17% (two treatments)

1 lb/A, 200 ppm 100% bloom @ inoculation 90.1 & 87.0

2006 Oxytet. 17% 1 lb/A, 200 ppm 100% bloom @ inoculation 91.7


2006 Kasumin 2L 200 ppm 100% bloom @ inoculation 89.1

2005 Oxytet. FireLine 1 lb/A, 200 ppm 100% bloom @ inoculation 93.0

2005 Oxytet. Mycoshield 1 lb/A, 200 ppm 100% bloom @ inoculation 92.0


2004 Oxytet. FireLine 1 lb/A, 200 ppm 100% bloom @ inoculation 92.5


2004 Gentamycin 10% 3 lb/A 100% bloom @ inoculation + 1 day after

88.3


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Antibiotic Mixes:


2011 Actigard Pre-‐bloom, Sprayed on tree. + Strep, 100% bloom + Act. 1 – 2” shoots *

Actigard 2 oz./A Strep. 200 ppm Actigard 2 oz./A

Actigard 50% bloom Strep. 100% Bloom Act. @ 1 -‐ 2” shoot

98.5

2011 Actigard Pre-‐bloom, Sprayed on tree. + Strep 100% bloom*

Actigard 2 oz./A Strep. 200 ppm

Actigard 50% bloom Strep. 100% Bloom

95.5

2010

Actigard Pre-‐bloom, Sprayed on. + Strep 100% bloom

Actigard 1 oz./A, Strep. 200 ppm

Actigard 20 and 50% bloom Strep. 100% Bloom

98.2

2010

Kasumin 2L + oxytetracycline

2 qt./A, 100 ppm 1 lb/A, 200 ppm

100% bloom 82.4

2009

“Blossom Protect” + Buffer A, Then oxytet. 17%

1.34 lb/100gal/A 9.35 lb/100/A 1lb/100/A

BP + buffer @ 20 & 50% bloom, Oxytet. @ 100% bloom

70.4

2006 Oxytet. + Cal/Phos fertilizer

1 lb./A 200 ppm 2 qt./A

100% bloom 72.5

Copper and other fungicides:


2011

Copper Product GWN-‐9979

64 fl.oz./A 50% and 100% bloom

81.5

2011



81.5

2011 Cueva (copper soap) 1 gallon/100/A 20 & 50% 100% bloom 79.8

2011



76.7

2010 Kocide 3000 0.5 lb/100/A 80& 100% bloom 52.3

2010


4 qt./A 80, 100% bloom & 1 day post inoculation

90.0

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2009



98.4

2009



87.6

2009



53.3

2009 Kocide 3000 0.5 lb/100/A 80& 100% bloom 61.1

2006 Manzate 3.2 lb./A 80& 100% bloom 41.1

2005 Kocide 3000 0.5 lb/100/A 80& 100% bloom 56.1

2005 Dithane (manzate) 3.2 lb. / A 80& 100% bloom 44.0

2004 Dithane + Champ (Cu hydrox.)

3.2 lb. / A +0.67 pint

Pink + 100% bloom, then 3, 6 and 9 days later

71.0

2004 Champ (copper hydroxide 37.5%)

0.67 pint/A Pink + 100% bloom, then 3, 6 and 9 days later

27.4

Yeasts (Blossom Protect) and Bacteria (Serenade):


2011 “Blossom Protect” + Buffer A full rate

1.34 lb/100gal/A 9.35 lb. /100/A

20 & 50% 100% bloom

85.7

2011 “Blossom Protect” + Buffer A ½ rate

1.0 lb/100gal/A 5.0 lb. /100/A

20 & 50% 100% bloom 85.4

2011 Bacillus subtilis QRD146

1.5 lb/100gal./A 30 & 50% 100% bloom 70.0

2011 Serenade MAX 3.0 lb/100gal./A 30 & 50% 100% bloom 63.1

2010

“Blossom Protect” + Buffer A ½ rate

1.34 lb/100gal/A 4.7 lb/100/A

20, 50 & 100% bloom

82.4

2010

“Blossom Protect” + Buffer-‐A ½ rates

0.68 lb/100gal/A 4.7 l b/100 A

20, 50 & 100% bloom

82.4

2010

“Blossom Protect” + Buffer A

1.34 lb/100gal/A 9.35 lb. /100/A

20, 50 & 100% bloom

81.1

2010

Blossom Protect + alternative buffer

1.34 lb/100gal/A to pH 5

20, 50 & 100% bloom

62.8

2009


1.34 lb/100gal/A 9.35 lb/100/A

20, 40, 70 & 100% bloom

80.8

2009


1.34 lb/100gal/A 9.35 lb/100/A

40 & 80% bloom (fewer applications than above)

73.0

2009

“Blossom Protect” full rate No Buffer

1.34 lb/100gal/A

20, 40, 70 & 100% bloom

69.5

2009 “Blossom Protect” NO Acid Buffer Then Oxytet. 17%

1.34 lb/100gal/A 1 lb/100/A

BP @ 20 & 50% bloom Oxytet. @ 100% bloom

69.0

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2009 Serenade Max 1 lb/100/A 20, 50 & 100% bloom 66.0

2009 Serenade QRD 146 0.5 lb/100/A 20, 50 & 100% bloom 44.2

2008 “Blossom Protect” (full rate)+ Buffer A

1.34 lb/100gal/A 9.35 lb/100/A

10, 40, 70 & 90% bloom

90.0

2005 Serenade ASO 6 qt. /100 gal/ A 20, 50 & 100% bloom 84

2005 Serenade Max 2 & 3.5 lb/100/A 20, 50 & 100% bloom 63 & 71

2004

Serenade AS 6 qt. /100 gal/ A 90% Bloom, day before inoc.

14

2003 Serenade 6 lb./A @ 100% bloom, pre-‐inocul. 26.7

2003 Serenade 6 lb./A @ 100% bloom, pre-‐inocul. + 3 days after

42.1

Other:


2011 Actigard Pre-‐bloom, Sprayed 3 times prior to inoculation

Actigard 1 oz./A each spray

20 & 50% 100% bloom 56.5

2011 Actigard, Sprayed twice after 1st symptoms seen

1.34 oz / A Sprayed twice, three day interval, after 1st symptoms seen

37.8

2011 Actigard (SAR) soil treatment

1 oz./A Tight Cluster & 50% Bloom 40.5

2010 Acid Buffer (pH 5) 2 qt./A 20, 50 & 100% bloom 38.5

2010 Buffer A 9.35 lb. /100/A 20, 50 & 100% bloom 37.9

2008 Organic nutrient spray series

Various, N, P, K Calcium, micro

Pre-‐bloom 32.0

2006 Cal/Phos Fertilizer Series of various Pre-‐bloom 21.9

2006 Physpe (SAR) Series of various 4 times Pre-‐bloom 18.0

2006 Physpe (SAR) Series of various 2 times Pre-‐bloom 0

2005 Cal/Phos Fertilizer Series of various Pre-‐bloom 5.0

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Treatment Number of

Valid Treatments

Highest Percent Control

Lowest Percent Control

Average Percent Control

Strep + ASM* 6 98.4 90.6 95.1 Copper (new) 9 98 76.7 85.8 Streptomycin 9 90 75 85.3 BCYP + Buffer A 12 90 72 82.6 Oxytetracycline 15 93 53 78.9 Kasugamycin 8 89 62 77.5 Gentamycin 6 88 51 74.5 Serenade 12 84 38 63.5 Copper (old) 7 80 26 49.5 Fungicides 6 57 33 48.6 Acid Buffers 4 39 19 30.5 SAR (Claims) 10 46 0 30.2 Nutrient minerals 3 32 5 18.8 Summary of author’s current and past fire blight control efficacy trial results. Plots all inoculated. *ASM = Actigard, BCYP = Aureobasidium pullulans, “Blossom Protect.”

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DEPARTMENT OF MICROBIOLOGY & MOLECULAR BIOLOGY

BRIGHAM YOUNG UNIVERSITY 775 WIDB PROVO, UTAH 84602 (801) 422-2889/ FAX: (801) 422-0519

December 9, 2013 Dr. Michael Braverman IR-4 Project Headquarters 5000 College Road EastSuite 201 W Princeton, NJ 08540-6635 Dear Dr. Braverman, Please accept this letter of support for the IR-4 grant proposal submitted by Dr. Julianne Grose of Brigham Young University entitled, “A Natural Treatment for Fire Blight: Pilot Tests in Apple Orchards.” This project has received financial support from our department for the past four years, and we are enthusiastic about its progress and potential. We are glad to learn about the IR-4 funding mechanism because this project is rapidly approaching the point at which it will require EPA approval, and Brigham Young University has no institutional experience with or support system in place for navigating the EPA approval process. The IR-4 grant would be most helpful in that regard. The phage-based treatment for fruit tree Fire Blight being developed by Dr. Grose and her collaborators, Dr. Don Breakwell and Dr. Sandra Burnett, is potentially very valuable for combating diseases in fruit tree orchards while also decreasing the use of chemical toxins that pollute the environment. Our departmental commits to continue supporting this project, as we have done for the past four years, by supplementing the IR-4 funding as follows: we commit a $3,000 match toward DNA sequencing costs, a $1,000 match toward travel, and a $11,000 match toward supplies, for a total of $15,000. This proposal addresses a widespread problem in a novel and creative way, and it fits well within the scope of the IR-4 funding mechanism. I support it without reservation. Respectfully yours,

Laura C. Bridgewater, Ph.D. Chair, Department of Microbiology & Molecular Biology

Documents

CRITERIA FOR RANKING EVALUATIONS OF IR-4 EARLY …ir4.rutgers.edu/Biopesticides/Proposals/5E.pdf · CRITERIA FOR RANKING EVALUATIONS OF IR-4 EARLY STAGE BIOPESTICIDE PROPOSALS-2014