OPTIMIZATION OF BACTERIOPHAGE FOR MANAGEMENT OF FIRE BLIGHT DISEASE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: OTHER GRANTS
• Reporting Frequency: Annual
• Accession No.: 1017086
• Grant No.: 2018-51106-28776
• Cumulative Award Amt.: $499,996.00
• Proposal No.: 2018-03548
• Project Start Date: Sep 1, 2018
• Project End Date: Aug 31, 2021
• Grant Year: 2018
• Program Code: [112.E]- Organic Transitions

Project Director
Sundin, G.

Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824

Performing Department
PLANT SOIL MICROBIAL

Non Technical Summary
Organic apple production east of the Mississippi river will not be sustainable without effective management of fire blight disease. The main goials of this project are to develop alternatives to antibiotics for organic fire blight management in humid regions, in particular in the midwestern and eastern United States. Bacteriophage represent a promising tool for the management of bacterial plant diseases; however, phage have not been developed commercially for reasons including reduced efficacy, and environmental issues such as sensitivity to solar UV radiation that impact phage survival. In this project, we will examine the efficacy of phage for the biological control of fire blight in organic orchard systems. Field testing will be conducted in Michigan and North Carolina with goals to optimize implementation. Additives including UV sunscreens and spray adjuvants will be evaluated for effects on increasing phage dispersal and survival. Integrated programs with phage and existing biological control agents will be examined to select the best combinations that maximize disease management efficacy. Larger-scale field testing will be targeted against natural populations of the fire blight pathogen Erwinia amylovora so that we can understand how phage deployment impacts natural pathogen genotypes and ultimately how this affects disease control potential throughout the apple bloom period. Extension activities will include a yearly on-farm field day with demonstration plots available to growers, production and dissemination of scripted videos outlining project research results, regular grower presentations, and site visits.

Animal Health Component

70%

Research Effort Categories

Basic

15%

Applied

70%

Developmental

15%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	1110	1100	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1110 - Apple;

Field Of Science
1100 - Bacteriology;

Keywords

erwinia amylovora

biocontrol

blossom blight

pome fruit

Goals / Objectives
The major goals of this project are to develop, evaluate, and optimize bacteriophage as biological control agents for management of the disease fire blight. While various bacteriophage have been tested previously for fire blight management, these materials have not been rigorously evaluated and optimized for ultimate use by commercial growers. For example, very little is known about bacteriophage survival on apple flowers following application. In addition, we plan to test optimized phage biocontrols in mixes with other registered biological control agents to maximize the level of efficacy against the blossom blight phase of fire blight. We will also perform detailed economic analyses of phage deployment for commercial organic growers. We have basic research goals in this project as well, and will study the effect of bacteriophage deployment on the apple flower microbiome. Finally, we will utilize our extension programs to educate growers about the use of bacteriophage in fire blight management, proper handling of phage etc to maximize the biocontrol ability of these agents.

Project Methods
In the first set of phage experiments, we will test combinations with phages φ21-4 and φ31-3 deployed either in equal mixtures or at 3:1 ratios in the field at sites in Michigan and North Carolina. The efficacy of these phages in controlling blossom blight will be examined in cocktails, as mixtures of phage have always performed better than single phage treatments in blossom blight tests and in tests conducted on other plant diseases (Schnabel et al. 1999; Nagy et al. 2012). The total phage concentration (overall plaque-forming units (pfu)/ml) will be 10^11 pfu/ml, as maximal phage densities are consistently associated with better disease efficacy. In addition to altering phage ratios, we will also examine the effect of addition of two UV sunscreen materials, carrot juice and peptone, on phage survival and efficacy, and will determine if utilization of the OMRI-approved adjuvant Kinetic (Helena Chemical Company; Collierville, TN) improves the efficacy of phage preparations by improving phage distribution among sprayed apple flowers. The experiment will be repeated in Year 2 of the project giving us two years of field data from two locations. Also in Year 2 and 3, we will add treatments with testing with the sunscreens and adjuvant added to the best phage ratio (if different from 50:50) to ensure testing of the best combinations for disease efficacy replicated over two years in the field.We will focus on Serenade Opti (Bayer) and Blossom Protect (Westbridge Biological) as biological control materials that we anticipate would work well in programs with phage for blossom blight control. Experiments will consist of using alternation strategies with phage and Serenade Opti or tank mixes of both materials. Since Blossom Protect is a living yeast biocontrol agent, earlier sprays are required to facilitate flower colonization. Because of that requirement, we will only run one treatment with two applications of phage integrated into a Blossom Protect program.We will scale up the field experiments to perform tests assessing the effect of bacteriophages on managing natural fire blight infections. Experiments will be conducted in the certified organic apple orchard located at the MSU Clarksville Research Station in Clarksville, MI and the transitioning to organic apple orchard at the NCSU MHCRC in Mills River, NC. Tests will be conducted on the highly fire blight susceptible cultivar Gala. We will use a randomized complete block design, 3-tree plots, 6 replicates per treatment. All treatments will be applied to trees using air blast sprayers. We will use the MaryBlyt fire blight disease prediction model to indicate when spray applications are needed targeting fire blight infection during the bloom period; all applications will be applied during evening hours to facilitate phage survival on flower surfaces. We hope to be making a minimum of 2-3 applications during bloom.Field experiments will be conducted in Michigan and North Carolina using the apple cultivar Gala. Phages φ21-4 and φ31-3 will be prepared for field application as described above. Individual flower clusters will be tagged for each treatment (25 flower clusters per tree on each of 2 trees). At two phenological timings (2-3 flowers open [40-60% bloom] and all flowers open [100% bloom]), phage preparations of φ21-4, φ31-3, or φ21-4 + φ31-3 (each at 10^11 pfu/ml) will be applied in the evening. In a second set of treatments, E. amylovora Ea110 will also be applied at a low rate (1 x 10^5 cfu/ml; 10X lower than normal rate) approximately 2 hr prior to phage application. Samples for microbiome analyses will consist of stigmas from 15 open flowers; microbial cells will be separated from flowers prior to DNA extraction. Extracted microbial DNA will be subjected to 16S rRNA tag-encoded amplicon pyrosequencing using a primer set that inhibits chloroplast amplification. Flowers will be sampled, immediately placed on ice, and processed for DNA extraction within 30 minutes. DNA extractions will be conducted in both Michigan and North Carolina; after extraction, DNA will be stored at -20°C. DNA samples obtained in North Carolina will be shipped overnight to Michigan on dry ice. All sequencing will be conducted at the MSU RTSF sequencing facility. Bioinformatics analysis, including sequence identification, diversity index analyses, and tracking of frequencies of individual species following phage application, will be conducted.We will utilize electronic and printed media outlets as well as presentations and grower field days to extend project results. We will produce a series of scripted videos for YouTube that will detail the concepts and findings of this research for post-antibiotic management of fire blight. Recommendations for organic fire blight management using phage will be incorporated into the MyIPM-NED app, which aids users in fruit disease diagnostics and management and includes organic management recommendations. Project team members will produce a series of one page bulletins that highlight fire blight management tactics using phage that will be updated and distributed online and at grower meetings.

Progress 09/01/18 to 08/31/21

Outputs
Target Audience:The target audience for this work is the apple grower community, organic growers, extension agents, representatives for chemical companies and disease scouting consultants, in states where fire blight is a serious problem, especially those east of the Mississippi river. Additional target audience is the research community of plant pathologists that work on bacterial diseases of tree fruit and bacterial pathogens as well as the microbiology research community that works on bacteriophages. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training opportunities include mentoring of one Ph.D. student and two M.S. graduate students. How have the results been disseminated to communities of interest?Results have been disseminated to grower audiences, especially organic apple growers, through a presentation at a special grower meeting on fire blight, held in Traverse City, MI in June 2019. Other presentations of this work include to the Great Lakes Fruit and Vegetable EXPO in Grand Rapids, MI in 2020 and to the Michigan Apple Committee in 2019, 2020, and 2021. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We examined the in vitro sensitivity of two bacteriophage of Erwinia amylovora, phi21-4 and phi31-3. Both of these phage are highly sensitive to UV-C and UV-B wavelengths; these wavelengths cause direct DNA damage. We found that both peptone and kaolinite clay were highly effective as UV-screening agents and conferred significantly-enhanced UVB survival to both phages phi21-4 and phi31-3. In 2019, we tested the efficacy of peptone at 5 mg / ml as a sunscreen additive to phages phi21-4 and phi31-3 and also to a recently-registered new commercial biocontrol product, AgriPhage (Certis Corp.). The addition of peptone resulted in significantly better blossom blight control by AgriPhage, but did not increase control with phi21-4 and phi31-3. In 2020 and 2021, we were forced to work with AgriPhage alone as our cooperator was unable to generate the phages phi21-4 and phi31-3 due to COVID impacts on their laboratory. In 2020, under lower disease pressure, the addition of peptone or of kaolinite clay did not increase the efficacy of AgriPhage in a field experiment. In 2021, the addition of either peptone or of kaolinite clay to AgriPhage resulted in numerically better blossom blight control, but the amount of control was not significantly better. Thus, the importance of adding sunscreen to phage preparations is not clear yet. We also conducted experiments aimed at detemrining the ffect of bacteriophage on the apple flower microbiome. Field experiments using AgriPhage were conducted, samples taken, DNA isolated and was sequenced at a commercial facility. These data are still being analyzed with analysis slated tobe completed by March 2022.

Publications

• Type: Journal Articles Status: Under Review Year Published: 2022 Citation: Dobbins, M.R., Outwater, C.A., Slack, S.M., Nesbitt, D., Svircev, A., Lauwers, E.M., Villani, S.M., and Sundin, G.W. 2022. Assessment of the role of ultraviolet radiation protection in the field efficacy of bacteriophage against fire blight. Plant Dis. (under review)

Progress 09/01/19 to 08/31/20

Outputs
Target Audience:The target audience for this work is the apple grower community, organic growers, extension agents, representatives for chemical companies and disease scouting consultants, in states where fire blight is a serious problem, especially those east of the Mississippi river. Additional target audience is the research community of plant pathologists that work on bacterial diseases of tree fruit and bacterial pathogens as well as the microbiology research community that works on bacteriophages. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training opportunities include mentoring of two M.S. graduate students. How have the results been disseminated to communities of interest?Results have been disseminated to grower audiences, especially organic apple growers, through a presentation at the Great Lakes Fruit and Vegetable EXPO, held in Grand Rapids, MI in December, 2020. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue field experiments in Michigan and in North Carolina working with sunscreen additives to either phages phi21-4 and phi31-3 or AgriPhage. We plan to complete the in vitro work we have done to identify the best UV sunscreens for phage protection in the environment. We will also conduct experiments assessing the impact of phage application on the apple flower microbiome.

Impacts
What was accomplished under these goals? We examined the field efficacy of a commercial AgriPhage product, because our ability to use the experimental bacteriophages phi21-4 and phi31-3 was impacted by COVID. In the North Carolina experiment, the AgriPhage treatment did not provide any reduction in the incidence of blossom blight compared to a non-treated control. Alternation of AgriPhage with other biological controls such as Serenade Opti or Blossom Protect did not increase the level of disease control of blossom blight. In the Michigan experiment, Agriphage use resulted in a significant reduction in blossom blight disease incidence compared to a non-treated control. However, the rate of control was approximately 39%, although this level of control was equivalent to other biological controls used in the experiment such as LifeGard and Stargus. The addition of the UV protectant peptone at 5 mg / ml to AgriPhage did result in significantly better disease control than AgriPhage alone, increasing the disease control to 65% (we had previously shown that the Erwinia bacteriophage are highly sensitive to UV radiation).

Publications

• Type: Other Status: Published Year Published: 2020 Citation: Kreis, R.A., S. Ellis, and S.M. Villani. 2020. Evaluation of biological bactericides for the management of fire blight on 'Gala' apple in NC, 2019. Plant Dis. Manage. Rep. 14:PF078.
• Type: Other Status: Published Year Published: 2020 Citation: Outwater, C.A., T.J. Proffer, S.M. Slack, and G.W. Sundin. 2020. Evaluation of new and existing biological control materials for the control of fire blight on Gala apples, 2019. Plant Dis. Manage. Rep. 14:PF071.

Progress 09/01/18 to 08/31/19

Outputs
Target Audience:The target audience for this work is the apple grower community, organic growers, extension agents, representatives for chemical companies and disease scouting consultants, in states where fire blight is a serious problem, especially those east of the Mississippi river. Additional target audience is the research community of plant pathologists that work on bacterial diseases of tree fruit and bacterial pathogens as well as the microbiology research community that works on bacteriophages. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Training opportunities include mentoring of one Ph.D. student, two M.S. graduate students, and one undergraduate student. How have the results been disseminated to communities of interest?Results have been disseminated to grower audiences, especially organic apple growers, through a presentation at a special grower meeting on fire blight, held in Traverse City, MI in June 2019. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue field experiments in Michigan and in North Carolina working with sunscreen additives to either phages phi21-4 and phi31-3 or AgriPhage. We plan to complete the in vitro work we have done to identify the best UV sunscreens for phage protection in the environment. We will also conduct experiments assessing the impact of phage application on the apple flower microbiome.

Impacts
What was accomplished under these goals? We examined the in vitro sensitivity of two bacteriophage of Erwinia amylovora, phi21-4 and phi31-3. Both of these phage are highly sensitive to UV-C and UV-B wavelengths; these wavelengths cause direct DNA damage. Since UV-B wavelengths are present in solar radiation, it can be surmised that these phage would be sensitive to inactivation by solar radiation. We tested a variety of materials to utilize as potential sunscreens including peptone (5-50 mg / ml), carrot juice, and kaolinite clay. We found that both peptone and kaolinite clay were highly effective and conferred significantly-enhanced UVB survival to both phages phi21-4 and phi31-3. In 2019, we tested the efficacy of peptone at 5 mg / ml as a sunscreen additive to phages phi21-4 and phi31-3 and also to a recently-registered new commercial biocontrol product, AgriPhage (Certis Corp.). The addition of peptone resulted in significantly better blossom blight control by AgriPhage, but did not increase control with phi21-4 and phi31-3. In a second field experiment using AgriPhage, we observed significantly better blossom blight control (compared to AgriPhage used alone without sunscreens) when Surround (kaolinite clay) was added, but not when peptone was added. In a third field experiment, we added the commercial biocontrol Serenade Opti to AgriPhage, but did not observe significantly better control compared to the untreated control treatment.

Publications