Source: OREGON STATE UNIVERSITY submitted to
UNBURDENING US NURSERIES: INTEGRATING NEW TECHNOLOGIES AND INNOVATIVE APPROACHES TO MANAGE BROAD HOST RANGE, GALL-FORMING BACTERIAL DISEASES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1004242
Grant No.
2014-51181-22384
Project No.
ORE00126
Proposal No.
2014-07904
Multistate No.
(N/A)
Program Code
SCRI
Project Start Date
Sep 1, 2014
Project End Date
Aug 31, 2018
Grant Year
2014
Project Director
Chang, J. H.
Recipient Organization
OREGON STATE UNIVERSITY
(N/A)
CORVALLIS,OR 97331
Performing Department
Botany / Plant Path
Non Technical Summary
The United States is the world's leading producer of floriculture products. Horticultural plants are high-value crops of significant worth because of their aesthetic qualities and uses in enhancing personal and community-shared environments. The crown gall and leafy gall diseases caused by Agrobacterium tumefaciens and phytopathogenic Rhodococcus, respectively, are a significant national problem. The symptoms are often grotesque and the abnormal growth phenotypes render diseased plants of no value, resulting in annual losses exceeding a million of dollars for some growers. These pathogens are a particular problem because management is challenging. These pathogens each infect more than a hundred plant species important to the industry. There are few commercially available controls that are effective against these pathogens. Early and accurate diagnosis of disease is difficult for a number of reasons. Their transmission and evolution is poorly understood. There are no rapid and sensitive methods for diagnosing plants. Finally, it can be challenging to train nursery workers in effective management because of the diversity in educational levels and cultural background.To address the needs of the nursery industry, we will rigorously test preventative products for control. We will also develop compounds from plant extracts as green alternatives for control. This novel approach has the added potential benefits of being more environmentally friendly and safer for nursery workers. To understand the epidemiology of these pathogens, we will determine the genome sequences of isolates collected from infected plant tissues and environmental reservoirs from nurseries located across the US. The genomic information will be used to understand how pathogens establish within production sites, migrate between sites, and change, possibly as a consequence of practices. This information will be used to inform and help develop protocols to limit pathogen spread. We will also use genome sequences to develop dipstick diagnostic tools that can be used on-site without specialized equipment or expertise. This will provide a more rapid diagnosis of pathogens and reduce response time for more effective control. We will advance our bilingual training and economics programs to develop a more educated and skilled workforce for increased retention and upward mobility, which is expected to help stimulate local economies. In summary, the ultimate goal is to implement change to help the economically important nursery industry better manage broad host range, gall-forming bacterial diseases.
Animal Health Component
35%
Research Effort Categories
Basic
50%
Applied
35%
Developmental
15%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21240101160100%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1160 - Pathology;
Goals / Objectives
The major goal of this proposal is to assist the nursery industry in managing broad host range, gall-causing bacterial pathogens. To address this goal, we have developed trans-disciplinary approaches that rely on expertise in bilingual education, diagnostics, economics, extension, genomics, natural product chemistry, plant pathology, and population genetics.The specific objectives are:1) Test and develop control compounds for broad host range and gall forming bacterial pathogens.2) Determine population structure, migration patterns, and evolution of A. tumefaciens and phytopathogenic Rhodococcus.3) Develop sensitive and easy-to-use diagnostic kits.4) Develop a website and database for using molecular data to rapidly identify species and strains of A. tumefaciens and phytopathogenic Rhodococcus.5) Education outreach to advance in-depth bilingual programs, workshops, and courses focused on managing bacterial diseases, and develop materials to educate and train workers at all levels of the industry.6) Economic outreach to assess at the levels of the growers, retail and consumer, and community.
Project Methods
We developed trans-disciplinary approaches to achieve the goal of improving the management of diseases caused by these bacterial pathogens. We will test, and prescribe best use guidelines to detail optimal and effective use of disinfectants and preventative compounds for control. We will also plant extracts and synthesize a compound that was previously isolated from resistant plants as green alternatives for testing and development in the production setting. To gain insights into population structure, migration patterns, and evolution, we will employ a genomic epidemiology approach. Isolates of A. tumefaciens and phytopathogenic Rhodococcus will be sampled from various nurseries (infected tissues of various host species as well as potential environmental reservoirs) located in each of the regions in the US. This collection will be augmented with isolates currently available in our expansive culture collections. The genome sequences will be determined and analyzed, using tools in population genetics, to test hypotheses of population differentiation, presence of genetic bottlenecks, and sink or source populations. We will also develop facile, sensitive, and rapid molecular diagnostic dipstick approaches for on-site detection of A. tumefaciens and phytopathogenic Rhodococcus. Finally, through our bilingual education and economic programs, we will offer workshops and training to educate nursery workers for better management of bacterial diseases.Metrics for evaluation have been developed and progress will be evaluated via three different mechanisms. We will have once yearly meetings with an Advisory Committee to evaluate our program and discuss future directions and goals. The OSU Plant Clinic is renowned for its expertise in diagnosing crown and leafy gall diseases. The Plant Clinic has established a large network of clients. It is through this network, interactions with nursery growers, extension agents, publications, and grower meetings that we will cause change and also solicit evaluation of our research program. The key anticipated outcome of the proposed work is a change in practice to limit the introduction and spread of bacterial pathogens. Thus, we will work with the OSU Survey Research Center to develop two types of surveys. One is a follow up survey to assess how training is influencing worker knowledge and behavior. A second survey will infer manager opinions on changes in worker practices.

Progress 09/01/15 to 08/31/16

Outputs
Target Audience:The target audience is diverse and the same as that reported in the previous report. Changes/Problems:We had major problems testing transmission of Rhodococcus. The plants that we received, to be used for testing, came infected with Rhodococcus. This showed to us that many nurseries are unaware of Rhodococcus and its ubiquity. Additionally, we discovered that Rhodococcus is easily disseminated and can be recovered from watering devices and benches. We are attempting to repeat these studies and have worked with the greenhouse crew to be more careful in working with our plants. What opportunities for training and professional development has the project provided?In addition to training professionals in inter-disciplinary research skills and collaborating between research and extension, this project has provided opportunities for professionals (UG and Grad students/postdocs): to visit production sites, interact directly with stakeholders, to participate in formal teaching to high school students, and to mentor. How have the results been disseminated to communities of interest?Publications Trade journals Workshops Face-to-face interactions Conferences; basic and applied research-centric (MPMI and Crown Gall, APS, NPDN meeting) What do you plan to do during the next reporting period to accomplish the goals?1. Test extracts for in planta control against Rhodococcus. 2. Continue screening extracts for additional control compounds. 3. Continue sampling diseased tissues and sequence genomes from isolates. 4. Develop on site instruments to facilitate use of diagnostic dipsticks. 5. Introduce online modules for certification program. 6. Continue developing AgBiz and working with nurseries to test its efficacy.

Impacts
What was accomplished under these goals? Our accomplishments were recently described during a site visit so I will be brief and bullet accomplishments. 1) We have demonstrated that few commercially available control compounds have in vitro anti-bacterial activity against Agrobacterium and Rhodococcus but no in planta activity. 2) We have identified three extracts that have anti-bacterial activity. These were extracted from medicinal plants. We have determined the structure for one of the active compounds. All three show activity against Rhodococcus, but not Agrobacterium. 3) We have developed a dipstick method to rapidly detect pathogenic Agrobacterium. We have developed markers that detect all known genetic variants of plant pathogenic Rhodococcus. 4) We have successfully classified Ti plasmids into "genetic units" and identified correlations between Ti plasmid type, bacterial type, and host plant species. We have also identified novel combinations as well as derivatives of the Ti plasmid. 5) We have piloted a hybrid course, which involves face-to-face and online learning. We had a focus group to identify specific needs of the nursery industry; discussions were centered around training needs of front-line workers. 6) We continue to advance AgBiz specifically for the nursery industry. Key additions are chunking of budgets into smaller units to address the varied products in the industry and the inclusions of biotic and abiotic stresses as variables.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Davis EW II, Weisberg AJ, Tabima JF, Gr�nwald NJ, Chang JH. 2016. Gall-ID: tools for genotyping gall-causing phytopathogenic bacteria. PeerJ 4:e2222.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Tabima JF, Everhart SE, Larsen MM, Weisberg AJ, Kamvar ZN, Tancos MA, Smart CD, Chang JH, Gr�nwald NJ. 2016. Microbe-ID: an open source toolbox for microbial genotyping and species identification. PeerJ, 3rd ed. 4:e2279.
  • Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Almabruk KH, Chang, JH, Mahmud, T. 2016. Total Synthesis of (+/-)-Isoperbergins and Correction of the Chemical Structure of Perbergin
  • Type: Other Status: Published Year Published: 2016 Citation: Putnam, ML, Santamaria, L. (2016) Disinfectants: In search of a silver bullet. Digger. May; 41-45
  • Type: Book Chapters Status: Submitted Year Published: 2016 Citation: Savory, E. A.**, Chang, J. H.6, and Putnam, M. L. Rhodococcus fascians in The Laboratory Guide for Identification of Plant Pathogenic Bacteria, 4th Ed. APS press: St. Paul, MN.


Progress 09/01/14 to 08/31/15

Outputs
Target Audience:The target audience includes a diversity of groups of individuals: 1) frontline nursery workers, foremen, supervisors, managers, and sales managers of growers, producers, retailers and landscapers; 2) agricultural industry scientists, consultants, extension specialists/agents, diagnosticians and regulatory personnel, and government policy makers; and 3) plant pathologists, students, and educators. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided scientists opportunities to develop skills in computational biology, generate and analyze big datasets, curate databases, develop web-based tools, learn to work and communicate effectively across disciplines, and mentor graduate, undergraduate, and high school students. How have the results been disseminated to communities of interest?Information is disseminated via standard mechanisms in peer-reviewed journals (Microbe-ID and Gall-ID in preparation in open-access journals) and at conferences (posters and oral presentation). We also disseminate information in trade journals (see products) and via electronic means (Microbe-ID is available for download from Github and Gall-ID and AgBiz Logic are usable via the internet). We will also work directly with 10 nursery operations to use the AgBiz Logic decision tool to gather and collect cost of production data for their own operations and use these budgets in decision making strategies. What do you plan to do during the next reporting period to accomplish the goals?For the next reporting period, we will: 1. Complete testing transmission of bacteria from plant to plant and via cutting tools, as well as efficacy of commercially available control compounds. For the extracts from plant sources, we will test their efficacy as preventative compounds on plant hosts and develop simple methods that allow growers to make the extracts. 2. Begin sampling diseased tissues from nurseries across the US. The causative bacterium will be isolated and their genomes will be sequenced and analyzed to determine population structure and migration patterns. We will also leverage the whole genome sequences to develop target sequences as molecular diagnostic markers, which will be further advanced for on-site diagnostic kits. 3. Implement modules online as the next step towards developing a certificate program. 4. Begin integrating effects of disease and training/prevention in Agbizlogic and working with nurseries to test the software.

Impacts
What was accomplished under these goals? Aim I) We determined whether Rhodococcus fascians or Agrobacterium tumefaciens are carried on cutting tools, as has been assumed in the industry and by educators. Hosts tested were Veronica and Oenothera speciosa for R. fascians and Rosa sp. and Leucanthemum for Agrobacterium. Preliminary results suggest that R. fascians has the potential to be transmitted between herbaceous tissues via cutting tools whereas A. tumefaciens is not easily transmitted between woody tissues via cutting tools. We also tested whether bacteria can be recovered directly from cutting tools after cutting through infected plant material. One trial using rose and Agrobacterium has been completed. We were able to recover bacteria from the clippers used. We have completed assays to test the efficacy of six registered products in controlling against in vitro growth of bacteria. The products were tested at three concentrations with both bacteria at five different concentrations. An additional six products have been tested at three concentrations and two bacterial suspensions of R. fascians only. The products were applied to sterile paper discs which were then placed on lawns of bacteria, and any zones of inhibition were measured. Initial results show that three commercial products have at least some activity against R. fascians and one product has some activity against A. tumefaciens. We used three different methods to extract potentially active substances from macerated tissues from six plant sources. The extracts were tested in an assay similar to the one previously described, except that only one concentration of the extracts was tested against one concentration of R. fascians. Based on preliminary results, extracts from two plant sources showed strong inhibition of bacterial growth. Three plant sources showed weak to medium activity. One plant source showed none to weak activity. Aim II) In preparation for assaying environments for reservoirs of phytopathogenic Rhodococcus at major production facilities, we first determined whether R. fascians can be recovered from soil. Soil is hypothesized to be the environment where the pathogen persists over seasons. We examined natural, unpasteurized field soil and an artificial peat-based potting mix. Both media were inoculated separately with three concentrations of bacteria. After three days, the soils were assayed for R. fascians, using PCR and oligonucleotides specific to R. fascians as well as a culture-dependent method to detect live cells. Additionally, the spiked soil was allowed to air dry, after which recovery was attempted using PCR and culturing. We found that the populations of bacteria declined during the course of the six day experiment, but were recoverable under some conditions. We have determined the genome sequences for nearly 50 isolates of Agrobacterium that were obtained from a variety of culture collections (primarily the "Larry Moore" collection), and selected to maximize diversity based on host isolated from, geographic location of isolation, and date of isolation. The purpose of this was to develop a framework for accomplishing the goal of determining the population structure, migration patterns, and evolution of A. tumefaciens. Aim III) We have also developed Microbe-ID, which was used as a platform for Gall-ID. The latter website provides tools for using molecular markers as well as whole genome sequencing reads to group isolates into taxonomic units in order to infer their identity. The website also provides tools to align short reads to identify potential homologs of known virulence genes. The website also provides downloadable tools that automate the use of whole genome sequences to determine genetic relatedness. These tools are derived from the pipeline we developed to accomplish the goals of Aim II. Two manuscripts are in preparation. For educational outreach, we have worked with OSU PACE to advance current workshops into an online certificate program. We have identified the following topics for the certificate program: 1) Pesticide application, 2) Proper use of disinfectants, 3) Distinguishing between biotic and abiotic symptoms, 4) Basic biology of plant pathogens, and 5) Sanitation and management methods. The first topic will first be offered as a hybrid course in fall of 2015 and then repurposed for an online module. All courses will be offered in English and Spanish. We are working with the OSU survey group to develop assessment tools to understand usefulness of training workshops, for the certification program, and to help advertise the online courses. For economic outreach, we have advanced our AgBiz Logic tools and website and met with local nurseries to establish cost categories and sub-categories for nursery owners to easily construct nursery crop specific enterprise budgets. We met with the external advisory board on August 26th, 2015 and received feedback on our progress and future directions.

Publications

  • Type: Other Status: Accepted Year Published: 2015 Citation: Putnam, M.L. 2015. Crown gall  still confounding scientists and growers alike. (Accepted) Grower Talks.�
  • Type: Other Status: Published Year Published: 2015 Citation: Putnam, M.L. 2015. How to prevent leafy gall before you lose plants. GreenhouseGrower 33:57-60.
  • Type: Websites Status: Other Year Published: 2015 Citation: Gall-ID.cgrb.oregonstate.edu
  • Type: Websites Status: Published Year Published: 2015 Citation: http://www.agbizlogic.com/