Source: VIRGINIA POLYTECHNIC INSTITUTE submitted to
AN ALL-STAGE FIRE BLIGHT CONTROL: REMOTE SENSING, DNA, ENZYME AND PLANT ACTIVATOR TECHNOLOGIES FOR CANKERS, BLOSSOM BLIGHT AND SHOOT BLIGHT
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1031507
Grant No.
2023-51181-41319
Cumulative Award Amt.
$5,717,639.00
Proposal No.
2023-05675
Multistate No.
(N/A)
Project Start Date
Sep 15, 2023
Project End Date
Sep 14, 2027
Grant Year
2023
Program Code
[SCRI]- Specialty Crop Research Initiative
Project Director
Acimovic, S.
Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
(N/A)
Non Technical Summary
In the last 20 years, epidemics caused by fire blight pathogen Erwinia amylovora caused losses of over $22 million per year in pear and apple production. If flowers aren't protected by spray applied antibiotics, blossom blight infections allow shoot blight infections with both leading to development of destructive fire blight cankers. Cankers are infected zones on wood bark which can kill more than 50% of trees in high density orchards by causing tree stem girdling. To control fire blight, growers apply copper in spring that is not always effective, antibiotics which can lead to resistance, biorational materials with inconsistent efficacy, and labor-intensive pruning removal of symptoms. This project's goals are to develop camera-robot system for canker mapping to speed up their manual pruning removal, determine biology and microbiome of cankers, screen for new biocontrol yeasts, determine efficacy of improved dormant copper and plant activator materials targeting canker viability, develop new antibiofilm enzymes and yeasts for blossom and shoot blight control, and conduct economic analyses of new control options resulting from project research objectives and research-driven results. Our cross-disciplinary team will use the latest technologies in robot engineering, genomics, transcriptomics, biochemistry, phytobiome, plant pathology and economics to provide growers with critically needed effective control options for all fire blight stages: cankers, blossom blight and shoot blight. To disseminate and implement new knowledge on E. amylovora and comprehensive fire blight management deliverables, our project team will use modern bilingual extension programming and in-depth impact analyses encompassing English and Spanish speaking agricultural communities.
Animal Health Component
50%
Research Effort Categories
Basic
25%
Applied
50%
Developmental
25%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2124010110015%
2124010106015%
2127210117020%
2051110116020%
6011110310010%
2011110104010%
2121110310010%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 601 - Economics of Agricultural Production and Farm Management; 205 - Plant Management Systems; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1110 - Apple; 4010 - Bacteria; 7210 - Remote sensing equipment and technology;

Field Of Science
1040 - Molecular biology; 1060 - Biology (whole systems); 1160 - Pathology; 1100 - Bacteriology; 1170 - Epidemiology; 3100 - Management;
Goals / Objectives
The long-term research goals of this project are to develop multipronged options targeting all fire blight disease stages. This work includes using computer vision and an autonomous mapping vehicle to guide the efficient removal of cankers, implementing tools for fire blight management with new modes of action against Ea, and increasing the number of spray-applicable options for pre- and post-infection fire blight management that are economical. Utilizing these products, we aim to prevent crop loss, tree death, and reduce Ea inoculum, while at the same time educating growers with new knowledge on biocontrol discovery and Ea biology, epidemiology, and management in cankers. This endeavor involves a series of multi-disciplinary team coordinated efforts that will result in reducing the incidence of fire blight and primary sources of inoculum. Within this framework we will pursue the following objectives:Objective 1. Develop a highly efficient, automated canker identification system on dormant trees by using an unmanned ground vehicle (UGV) equipped with a stereo RGB camera for geo-mapping.Objective 2. Understanding canker formation, reactivation, and ooze formation in the epidemiology of fire blight.Objective 3. Determine microbiome of fire blight cankers for discovery of novel biocontrol agents.Objective 4. Develop novel management solutions by exploring biofilm degrading enzymes, plant immunity inducing yeasts, and chemicals that reduce canker viability.Objective 5. Conduct economic analyses and cost modelling to identify high profit but low risk tools and practices for fire blight management.Objective 6. Develop and deliver impactful extension that combines research objectives with their results to improve stakeholder knowledge and adoption of effective control practices.
Project Methods
1.1. Camera hardware: data collection and development of stereo camera system: Current camera system will be modified to account for the appearance of the trees imaged and the canker infection on wood to be detected. 1.2. Detection and scanning software: development of the deep learning architecture for detection. We will train Faster and Mask Region Based Convolutional Neural Networks (FRCNNs) to perform pixelwise segmentation of cankers in images. 1.3. Spatial Mapping. This aspect will be addressed using processing at two domains: image and three-dimensional. The image processing will first determine what part of the image corresponds to a tree or parts of it (i.e., branches). 1.4. Autonomous navigation and mapping: GPS waypoint following. We will use the approach employed in our previous work on grapevines (SCRI led by Terry Bates) as the baseline to autonomously drive the robot in the field by following GPS coordinates. A model predictive controller (MPC), along with inertial (measures acceleration and velocity), lidar, and real time kinematics (RTK), GPS sensor measurements will be used for the control and perception system to drive between rows without collisions.2.1. - 2.3. We will combine approach of transposon sequencing (Tn-seq), dual RNA sequencing (dual-RNAseq, for both the pathogen and the host), and fluorescent activated cell sorting-RNAseq to decipher the mechanism of canker formation and reactivation. By comparing the abundance of each randomly barcoded transposon mutant at different stages of infection to those prior to infection, genes required for infection of a given stage will be identified. Stages will be 1. canker formation, 2. reactivation, 3. ooze formation. Dual-RNAseq will be performed to compare the transcriptome of both E. amylovora (Ea) and host plants at canker formation or reactivation to identify genes for a given stage of lifestyle transition. Finally, Ea cells isolated from cankers will be stained by BONCAT to differentiate metabolically active and dormant populations. The two populations will be sorted and sequenced for the identification and functional characterization of differentially expressed genes. 2.4 Experimental validation of selected genes using dual fluorescent reporters, mutagenesis, and complementation. Functions of genes related to stress tolerance and lifestyle transition will be validated through mutagenesis and complementation assay.3.1. We will determine the microbiome composition, and succession and function within fire blight cankers at different stages. 'Gala' apple trees will be used in this study DNA and RNA extracted from cankers to generate metagenomic and metatranscriptomic libraries and sequence them using the Illumina NovaSeq. Metagenomes will also be sequenced using the Oxford Nanopore PromethION. Annotation and mapping will be performed (Kyoto Encyclopedia of Genes, Genomes KEGG protein database). For microbial composition and diversity analyses, 16S rRNA and ITS sequences will be extracted. Taxonomic classification will be performed (Ribosomal Database Project classifier against the SILVA v132 reference alignment in mothur). We will use non-metric multidimensional scaling (NMDS), descriptive diversity statistics, MEGAN V.5, BLAT database within MG-RAST, and our published pipeline for metagenomic whole genome assembly to get to point to determine which microbes are carrying out the expressed functions. 3.2. a culture collection of yeast-like fungi from fire blight cankers will be formed and isolates identified that would induce apple defense response without causing significant fruit russeting.4.1. Antibacterial enzymes (ABEs) for Ea lab and field tests. We will determine minimum inhibitory concentration (MIC) for ABE vs. Ea and expand testing to a range of Ea field isolates. We will use electron microscopy to image Ea cell wall structure after ABE treatment to identify mechanisms of action on Ea cell wall structure as well as biofilm synthesis. We will combine ABEs and other enzymes with copper, streptomycin, oxytetracycline and kasugamycin to determine if there is synergism. We will test ABE on potted apple trees and mature apple and pear trees in orchard for blossom blight control in orchard trials. Optimal ABE formulations will be produced via fermentation at field-trial scale and then spray applied before inoculation of flowers with Ea110 strain.4.2. Blossom blight control experiments with resistance inducing yeasts from Objective 3. A 10 to 20 most promising yeast-like fungi resulting from the screening in Objective 3 will be evaluated for blossom blight control and compared to Blossom Protect, untreated control, and antibiotic streptomycin by 3 to 4 preventive yeast applications anywhere from 10% to 90% bloom, which is similar application schedule recommended for Blossom Protect biocontrol. We will use our previous published methods for this work. 4.3. Enhanced dormant copper spray programs against Ea in cankers. We will inoculate pome fruit trees to form cankers on mature apple and pear trees. We will spray-apply 5 treatments of copper hydroxide in mix with one bark penetrating oil to determine the most efficacious treatments to control Ea in cankers. We will collect cankers over time on each apple and pear tree and quantify Ea in them by v-ddPCR as per our previous work in Santander et al. We will collect healthy bark samples to determine copper ion abundance by ICP/MS or atomic absorbance instrument or similar method. 4.4. Post-infection use of prohexadione-calcium (PCA), giant knotweed extract (GKE) and resistance inducing yeasts (RIY) for canker viability reduction. Fire blight cankers will be developed on apples. 8 treatments of PCA,GKE and RIYs with bark penetrating oil for improved active material absorption into the bark. We will sample cankers at 10, 20 and 30 days after the treatment. We will determine Ea live populations in cankers with v-ddPCR as per our previous protocol. We will extract RNA from bark near cankers and perform RNA sequencing and transcriptome analysis to determine defense responses in bark as per our previous protocol.5. We will conduct economic analyses and cost modelling to identify high profit but low risk tools and practices for fire blight management. We will analyze the economic viability of deliverables developed in Objectives 1, 3 and 4 with comparison to alternative risk management practices and of likelihood of farmer adoption in modern HD orchards: 5.1. Fire blight canker mapping economics, 5.2. Economics of new Ea control practices, and 5.3. Investigation of cost of fire blight control practices and fire blight insurance rider.6. Develop and deliver impactful extension that combines research objectives with their results to improve stakeholder knowledge and adoption of effective control practices. We will disseminate project results and educate producers about the project's new fire blight management tools. We will coordinate with the Collaborators using central extension clearinghouse, project management website on monday.com, to share findings, develop, review, and edit extension resources (factsheets, newsletter articles, videos, blog posts) for extension to growers across all the partner states. We will develop and disseminate English and Spanish extension programs for new fire blight management systems, deliverables, and materials for all fire blight stages. To adopt new fire blight control strategies, we will educate and encourage stakeholders but also keep them engaged with yearly meetings, field days throughout the project, and one-on-one spray program crafting sessions. We will provide critical time-sensitive fire blight control recommendations from project results and disease prediction models by regularly delivering blog posts, extension presentations with built-in panel discussions at the annual grower meetings in major apple and pear producing states. 5 in-depth workshops on project results will be held.

Progress 09/15/23 to 09/14/24

Outputs
Target Audience:We presented the project's experiments to Virginia tree fruit growers at the Fruit School meetings at 4 locations in Virginia: Winchester, Syria, Roanoke and Lambsburg lasting 4 days from 1 - 4 Feb 2024. The results were then presented to the U.S. and Canadian extension specialists, tree fruit scientists and students (plant pathology, horticulture, entomology) at the 86th Annual Northeast Tree Fruit IPM Workshop in Northampton, MA, from 22-23 Oct 2024 and at the invited seminar presentations at the Clemson University on 6 Sep 2024, at Michigan State University on 9 Sep 2024, at Connecticut Agricultural Experiment Station, Invited Lockwood Lecture, in New Haven, CT, 18 Sep 2024, invited seminar at the Carnegie Mellon University, Robotics Program, in Pittsburg, PA on 8 Oct 2024. Results ere presented at the American Phytopathological Society annual meeting in Memphis, TN, 27-30 July 2024. The results have been presented at the 99th Annual Cumberland-Shenandoah Fruit Workers Conference in Winchester, VA, 30 Nov - 1 Dec, 2023. These meetings allowed a platform for direct delivery of the results from this project which reached over 120 growers, ~19 crop consultants, ~70 scientists and ~15 regional apple extension specialists in East Coast and Midwestern U.S. Results directly impacted by fire blight outbreaks in recent past, so this project's results are directly applicable to their farms. They are the following, including but not limited to: regional fruit tree growers, farm managers, extension specialists, applied scientists, Ag industry company representatives, plant health company representatives, apple packing businesses, nursery managers, Ag machinery and pesticide distributors and others. We presented the results at the 2024 Mid-Atlantic Fruit and Vegetable Convention in Hershey, PA, 1-2 Feb, 2024 in the General/Pome Fruit session, where we reached reach 112 Mid-Atlantic apple and pear growers, as well as plant health industry representatives and attending scientists and extension specialists. We plan to present project's results to Virginia tree fruit growers at the Fruit School meetings at 5 locations in Virginia (Winchester, Syria, Lovingston & Roanoke, Lambsburg) lasting 4 days in Feb 2025. Changes/Problems:We inoculated 59 trees of Gingergold and Jonagold apple cultivars in Winchester VA to generate enough cankers to conduct experiments with lower rates of copper for control of fire blight in cankers. Insufficient number of cankers have developed in per tree basis. We plan to conduct more inoculations on younger trees to develop cankers for these experiments. We also plan to search for naturally infected apple orchards to find enough cankers in per tree basis and ask the gorwers to allow us to use them in Jan 2025 or 2026 to conduct evaluating chemicals that reduce canker viability. What opportunities for training and professional development has the project provided?Hands on training, mock-up training, and actual canker sample runs for one technician (Research Specialist II) and two graduate students on how to process fire blight cankers with Geno/Grinder and use and run viability digital droplet PCR to process and analyze fire blight cankers treated with copper (Table 1). Training of one technician (RA II) how to prepare inoculum of Erwinia amylovora and how to inoculate apple and pear shoots to develop fire blight cankers and how to inoculate apple flowers to test control of blossom blight with Anti-biofilm enzyme. Training of RA II how to rate the fire blight incidnece on flowers and shoots. Training of one technician (RA II) how to drive a tractor, operate an experimental sprayer, and spray apply the project test materials using an experimental 4 x 25 gal tank experimental sprayer. How have the results been disseminated to communities of interest?We presented the results of apple and pear field trials to apple and pear growers attending two project-specific bilingual workshops - one in Central Point, Oregon at the 2024 Field Day, on 18 July 2024, and then in Winchester, VA on 25 July 2024 (Acimovic, KC, Berger). We reached the growers, crop consultants, farm managers, and local extension agents and chemical representatives at the annual Fruit School meetings in Feb 2024 and presented the results in person and at the AHS Jr AREC field day in northern Virginia on September 12, 2024. In Feb 2025 project results will be disseminated more to stakeholders. The results were then presented to the U.S. and Canadian extension specialists, tree fruit scientists and students (plant pathology, horticulture, entomology) at the 86th Annual Northeast Tree Fruit IPM Workshop in Northampton, MA, from 22-23 Oct 2024 and at the invited seminar presentations at the Clemson University on 6 Sep 2024, at Michigan State University on 9 Sep 2024, at Connecticut Agricultural Experiment Station, Invited Lockwood Lecture, in New Haven, CT, 18 Sep 2024, invited seminar at the Carnegie Mellon University, Robotics Program, in Pittsburg, PA on 8 Oct 2024. Results were presented at the American Phytopathological Society annual meeting in Memphis, TN, 27-30 July 2024. The results have been presented at the 99th Annual Cumberland-Shenandoah Fruit Workers Conference in Winchester, VA, 30 Nov - 1 Dec, 2023. What do you plan to do during the next reporting period to accomplish the goals?Continue specific activities in Objectives 1-6 outlined in the project description: 1. Most important is inoculation of shoots to develop fire blight cankers for evaluating chemicals that reduce canker viability. 2. Continue blossom blight control experiments in following years on multiple locaitons (ABE, yeasts, etc.) 3. Inoculate young trees in CT and VA to investigate canker formation, reactivation, and ooze formation in the epidemiology of fire blight. 4. Collect more fire blight cankers nationally and determine microbiome using ITS, 16SrDNA and virome sequencing and then use microbiome of fire blight cankers for discovery of novel biocontrol agents. 5. Continue extension using newly generated results.

Impacts
What was accomplished under these goals? For Objective 1. Preliminary data on visual appearance of fire blight cankers collected with a stereo RGB camera on 6 March 2024 by F. Yandun from Carnegie Mellon University. Objective 2. Supplies i.e. apple trees and pesticides were ordered to start this objective's experiments in early 2025. Postdoctoral Associate is in the process of getting the visa to be hired by the funding from this project. Objective 3. First fire blight cankers were collected, DNA from then extracted and 16SrDNA was sequenced. The gained data on microbiome in and on fire blight cankers is currently written into a scientific manucsript by Acimovic and Li at Virginia Tech. Objective 4. We evaluated biofilm degrading enzymes (ABE) in 2024 for control of bloccom blights and we got excellent results: one to two spray applicaitons of 1 g of ABE per liter of water gave 54.3% - 65.4% control of blossom blight incidence. The same treatments gave 61% - 69.7% control of shoot blight incidence. Objective 6. We presented ABE blossom blight control results to the growers at the organized, advertised and held twobilingual Workshops workshop in Central Point Oregon at the 2024 Field Day, on 18 July 2024, and then in Winchester, VA: CPPM Project Workshop - Winchester, VA, 25 July 2024 (Acimovic, KC, Berger):USDA CPPM Project's Fire Blight Workshop on 25 July 2024// Por favor Confirme su asistencia para unirse al taller del proyecto CPPM de la USCA sobre de fire blight, 25 de julio de 2024: https://www.arec.vaes.vt.edu/arec/alson-h-smith/arec-updates/fire-blight-workshop.html

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: N. J. Boeckman, M. C. Borba, S. G. Acimovic (2024): Evaluation of Giant Knotweed Extract, Regalia, and Antibiotics in Control of Shoot Blight and Fire Blight Canker Phases on Apple. Agronomy 2024, Special Issue: Detection and Control of Diseases and Pests in Fruits, 14(10), 2216: 1-14.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: American Phytopathological Society Annual Meeting - Plant Health 2024, 7/29/24 Memphis, TN, Poster: Firing Back: Novel Management Tools for Apple Shoot Blight and Cankers caused by Erwinia amylovora Nathanial Boeckman, Matheus C. Borba, Emmanuel Sempeles, Fernanda Ferreira, Julie Wong, Vivien Wong, and Srdjan G. Acimovic.