Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001
Performing Department
Prosser Irrigated Ag Res & Ext Center
Non Technical Summary
The impact of infection by virus-like agents (VLA; includes viruses, viroids, phytoplasmas, and systemic bacterial pathogens) on perennial specialty crop production was recognized long before their etiological agents were confirmed. In Washington State, awareness of the serious nature of virus infections was evident in the foreword to the report of a 1942 disease survey: "The increasing seriousness of the virus disease problem of fruit trees has been a matter of growing concern to orchardists, nurserymen, and investigators" (Reeves, 1943). The critical roles of grafting onto woody indicators for pathogen detection, and the need for replacement of infected trees with virus-tested trees were also discussed by Reeves (1943). It was demonstrated that aggressive tree removal in an effort to control Western X disease in Washington orchards was an effective tool to halt the increase in the numbers of infected trees in a defined growing region (Blodgett et al., 1949). To replace the infected trees that were removed, a reliable source of non-diseased trees was needed. This marked the genesis of the currently existing fruit tree foundation and certification programs that have ensured a continuous flow of virus-tested planting stock into nursery and production planting schemes over the past seventy years, which has led to a substantial reduction in economic loss from VLA infections in specialty crops.Today, the ability to provide virus-tested planting stock of vegetatively propagated specialty crops has become increasingly complex. The collective industries no longer depend on a few selections to support the industry. For example, the Clean Plant Center Northwest (CPCNW) at Washington State University receives an average of 139 temperate climate fruit tree selections each year for introduction and virus-testing (Eastwell, unpublished observation). The same intensive movement to establish niche markets with new selections is occurring in the grape and hop industries as well. Propagation source material originating from domestic and foreign locations is screened for VLAs before they are propagated and distributed throughout the industry. It takes many years for a single virus-tested plant to be expanded to an economically relevant planting that provides adequate product for marketing and sales initiatives. To meet the demand to have new distinctive product available in the market sooner, tremendous pressure is placed on programs to provide this service expeditiously. Rapid access to productive new clones is essential in allowing U.S. producers to remain competitive in the global marketplace. Consequently, the initial virus testing must be completed in a timely fashion. Similarly, much of the material distributed from the programs providing the virus-testing service enables plant breeders and developers to provide their product to foreign markets. Over the period 2011 to 2013, 9,914 out of the total of 36,979 or 27% of the distributions from the fruit tree program were to recipients outside of the U.S. Testing procedures used for the development of plant material must therefore be accepted by the regulatory agencies with authority in the recipient state or country. The goal of this project is to address issues associated with the demand for rapid detection and elimination of VLAs from planting stock. Overall, the three perennial specialty crops represented in this project constitute a large segment of U.S. agriculture. Nationally, the farm-gate value of these specialty crops are $11,162 million for temperate climate fruit and nut trees, $6,158 million for grapes and $249 million for hops (USDA National Agricultural Statistics Service, 2014). The ultimate goal of the CPCNW is to assist the industries it serves by providing propagation material as quickly as possible while maintaining the highest standards possible for plant health and quarantine security. Research is conducted to help achieve this goal.In addition to the vegetatively propagated perennial crops, the seed industry is also a critical component of U.S. agriculture. In 2012, the U.S. exported seed valued at $1,522 million, of which $70 million was produced in Washington State (Mertz et al., 2013). The CPCNW provides seed testing services for viruses to support Washington State quarantine regulations governing seeds for planting in the state and to satisfy requirements for products to be exported from Washington to many other states and countries. Two major seed crops serviced by the CPCNW are beans and peas with a combined production value of $174 million in Washington State (Mertz et al., 2013). Many other seed crops are tested on a sporadic basis.
Animal Health Component
40%
Research Effort Categories
Basic
25%
Applied
40%
Developmental
35%
Goals / Objectives
Provide appropriate virus diagnostic procedures to support agricultural production of specialty crops and their associated industries in the U.S.Evaluate platforms for the use of advanced technology to best capitalize on available technology while enhancing the biosecurity and economic viability of agricultural crop production.Determine the etiological agent(s) associated with graft transmissible agents of temperate climate fruit tree diseases.
Project Methods
1. Provide appropriate virus diagnostic procedures to support agricultural production of specialty crops and their associated industries in the U.S.Many of the pathogens of concern for field crops have been well characterized and reliable diagnostic methods are available. Many of these methods are based on serological methods such as ELISAs. Providing these routine diagnostic tests for seed industries supports regional quarantine and certification standards and helps protect specialty crop production. The CPCNW serves as a central location for the acquisition and ongoing monitoring of ELISA data that might reveal sudden changes in the epidemiology of important diseases. Many serological reagents are commercially available, and test procedures are validated using positive controls sourced commercially as well as controls maintained by the program at CPCNW. Samples are submitted by the seed industry as needed.Currently, the serological testing service is evaluating testing procedures to support U.S. pollen companies. In an effort to offset the negative impact of the decline of pollinating bee populations, pollen companies are expanding their markets while distributing a product that is free of serious pollen-borne viruses. The CPCNW serological testing services are providing the baseline information needed to advance the development of this market. The cooperating companies collect apple and/or cherry pollen in small batches. These are submitted for testing by both serological and molecular methods. This will provide data on the relative distribution of infection sources in commercial production as well as the relative sensitivity and reliability of the two detection methods. In combination, these will help define future protocols for the testing and safe distribution of pollen. The data are provided to regulatory agencies in other jurisdictions as the basis for forthcoming pollen collection and testing protocols.Ongoing research in collaboration with commercial specialty crop producers often results in valuable data regarding the distribution of emerging disease situations in U.S. production, which in turn provides opportunities for epidemiological studies. As putative agents are identified through advanced technology as well as other classical methods, their relationship with disease is evaluated by a suite of molecular assays to verify this association (MacDiarmid et al., 2013; Villamor et al., 2014b). The availability of genomic data provides the opportunity for translational research to produce diagnostic tools that can be quickly developed and offered by private enterprise, and applied in commercial agricultural production situations (e.g., Mekuria et al., 2014).Testing for and elimination of VLAs from propagation material that is subsequently distributed to U.S. producers is a seminal activity of the CPCNW. Testing follows protocols approved by USDA-APHIS-PPQ and recognized by many international agencies. Once testing is completed, foundation material is made available for propagation. This process is a crucial element in preventing the incursion of economically detrimental VLAs into U.S. production.2. Evaluate platforms for the use of advanced technology to best capitalize on available technology while enhancing the biosecurity and economic viability of agricultural crop production.High throughput sequencing does not define a monolithic technique for virus diagnosis. Multiple sample preparation procedures, sequencing techniques, and bioinformatic analyses exist. The most suitable combination of these methods remains to be determined. Because the majority of high throughput sequencing techniques rely on sample amplification, cross-contamination between samples remains a constant threat (MacDiarmid et al., 2013). Therefore, the adopted sample preparation strategy should minimize the number of manipulations that could lead to contamination and subsequent corruption of data. Total RNA, double stranded RNA, and small RNA represent three distinct populations that can be targeted for sample preparation, each with advantages and limitations. Each of these sample preparation strategies will be evaluated across several host plant species for their ability to enable accurate virus detection by PCR. Strategies in data analysis will also be evaluated so that a reasonable number of samples can be analyzed while still maintaining the necessary level of confidence of biosecurity (e.g., Ho & Tzanetakis, 2014). The results of the various processes will be evaluated by independent laboratories to gauge the robustness of high throughput sequencing as a general approach to virus diagnosis. Results of high throughput sequencing and data analysis will be compared with the currently accepted standards applied in Objective 1. The key question to address is whether this technology is at least as good as if not superior to existing protocols accepted by the international plant pathology and regulatory communities. Sufficient data will be collected to satisfy this need. As the current testing standard requires three years to complete, specimens for which testing has been initiated will also be subjected to high throughput sequencing and the results compared. Ambiguous results will be resolved by thorough supplemental testing using molecular methods such as PCR.3. Determine the etiological agent(s) associated with graft transmissible agents of temperate climate fruit tree diseases.A serious obstacle to the rapid domestic and/or international movement of plant propagation material, and for the management of diseases in commercial production settings is the abundance of VLAs that remain uncharacterized. A recent publication of the North American Plant Protection Organization (North American Plant Protection Organization, 2009) listed 94, 72, and 25 diseases of concern that occur in stone fruit, pome fruit, and grapevines, respectively. Of these, 39, 53, and 3 are caused by uncharacterized VLAs. A recent survey of research and regulatory programs in the continental U.S. revealed that many of these graft-transmissible diseases associated with uncharacterized origins no longer exist in virus collections (Simon Scott, personal communication). However, several remain problematic in commercial production. The diseases that still occur naturally in production areas and cause economic impact will be examined. The use of high throughput sequencing allows the entire biome of a plant sample to be examined. The results of data analyses provide important direction for future validation experiments to confirm the association of a particular agent with disease symptoms. This strategy was recently used with success to identify likely causal agents associated with several diseases of pome fruit trees (James et al., 2013), stone fruit trees (Mekuria et al., 2013a; Villamor et al., 2014b), and grapevines (Al Rwahnih et al., 2009). Representative voucher specimens have been catalogued in collections throughout North America. These recognized archetypal selections will form the foundation of high throughput sequence analyses. Once a putative causal agent is identified, other independent assays will be developed and applied to additional diseased samples from commercial production areas to substantiate the association between disease expression and presence of the suspected etiological agent. In many cases, the subject plant is infected with multiple agents, thus obscuring a true cause and effect relationship (Villamor et al., 2014b; Marais et al., 2015). Therefore, only a correlative association between the pathogen and disease can be established. Depending on the nature of the putative agent (size, virus genus, genome complexity, replication strategy), it may prove worthwhile to pursue development of an infectious clone to assist in firmly establishing the disease-VLA relationship.