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NE1006: Eradication, Containment and/or Management of Plum Pox Disease (Sharka)

Duration:
October 01, 2001 to September 30, 2006
Administrative Advisor(s):
Bruce McPheron (PEN)
NIFA Reps:
Thomas A. Bewick

Statement of Issue(s) and Justification:

Plum pox disease or "Sharka" is one of the most devastating and economically important diseases of stonefruit worldwide. The disease is caused by the Plum Pox Virus (PPV) which is naturally spread by several species of aphid. Although the virus is endemic to Eastern Europe it has been spread throughout Europe, the Mediterranean region and several locations in the Western Hemisphere by the unwitting use of infected propagation material. The first report of PPV in North America came from a peach orchard in Adams County Pennsylvania in 1999 and in the summer of 2000 it was found in Ontario Canada. Extensive surveys have presumably delimited the disease situation in the U.S. and Canada but the source of virus introduction into North America still has not been identified. Most economically important species of Prunus are susceptible to PPV, including peaches, nectarines, plums, and apricots (8,21). A wild species, (Prunus spinosa), occurs widely in Europe and functions as a natural symptomless PPV reservoir making eradication of the virus impossible in many countries. The virus has a large experimental host range including 134 species of plants in 16 families that have been identified by artificial inoculation (3). The complete natural host range and the role of alternate weed hosts, in virus survival and spread is not fully known especially in countries where the virus has recently been introduced. Interstate and international cooperation is needed to either eliminate or control PPV in North America. In the U.S., PPV is currently limited to Pennsylvania with approximately 5,400 Ac of stone fruits grown on small family farms. Depending on the year, Pennsylvania ranks as the fourth or fifth largest peach producing state. In 1998 Pennsylvania peach production was 65 million bushels valued at $21 million. This does not include the value to subsidiary agricultural industries or the total value to the small communities located throughout southern Pennsylvania and Maryland that are dependent on these crops. Total peach production in the eastern U.S. encompasses about 82,000 acres with a value in 1999 of $191 million. Canada has approximately 38,000 acres of stone fruits currently threatened and the occurrence of PPV in Ontario is a direct threat to stone fruit industries in New York, Michigan, and Ohio. Movement of propagation materials between Canada and New York has already occurred, but PPV has not yet been detected in this material. Other major peach producing states, such as South Carolina and Georgia, are equally threatened by the possibility of accidental movement of infected propagation material or introduction of infectious aphids. If infected propagation materials or the virus were to reach California, the results could be devastating. California currently grows approximately 265,000 Ac of susceptible stone fruits. Unlike the situation in Pennsylvania, very large commercial orchards are grown in close proximity under ideal environmental conditions for rapid aphid spread. The total value of the stone fruit industry in the U.S. is valued at $1.8 billion annually. With the ability to curtail fruit production anywhere from 10-100%, depending on the cultivar, PPV is of serious economic concern. One of the objectives of this project is to develop the infrastructure supporting multi-state and international collaborations to solve problems associated with PPV control and to provide mechanisms for increased communication and sharing of educational and scientific information. In addition, diverse facilities are needed to study this quarantined pathogen, as well as, personnel with diverse areas of expertise. It is beyond the scope of any one agency or institution to support this diversity of needs. Because PPV is a recently introduced invasive species to North America, many personnel at State Regulatory Agencies, as well as, University and County Extension personnel are unfamiliar with Plum Pox disease. Many cultivars of peach, nectarine, plum, and apricot do not show obvious symptoms when first infected and are difficult to detect and identify when first encountered. Nevertheless, excellent tools have been developed to detect and identify PPV. One of the most practical and economical is a commercially available ELISA kit that is Plum Pox specific. A number of excellent illustrated bulletins and web-sites have also been developed to assist with identification of visual symptoms. Virtually any virology lab in the country is equipped with the basic tools that would allow participation in survey work for virus detection. Despite the extensive survey work that has taken place since PPV was first found in North America it is too early to say that all sources of infected budwood and all infected orchards have been identified. Multistate cooperation is needed to continue the search and to establish a means of surveillance for possible escapes and future infestations. Even if eradication is totally successful there remains the possibility of future introductions. Furthermore, the action taken by NE-501 to deal with the PPV problem can provide a plan of action that may be applicable to other plant viruses of quarantine concern. How the U.S. fruit industry deals with PPV will depend on many factors. If PPV is still localized near the initial infection foci, it may be possible to eradicate the disease by enforced tree removal. Whether or not this will be effective depends on whether the virus is spreading to new orchards and homeowner plantings, whether the virus has invaded wild plant species and whether local aphids are efficient PPV vectors. If infected budwood has been used for propagation of trees in areas outside the quarantine area then other foci of infection in geographically distant regions may exist. If PPV is determined to be widespread in wild plant species and/or efficient vector species is identified, then eradication of PPV becomes impossible. In this scenario, management of PPV to optimize yield and profit and reduce PPV effects will be necessary. Therefore, education of all personnel involved in the fruit industry to recognize newly arriving potential invasive fruit pathogens is needed. To prevent the reintroduction of PPV requires increased education of the dangers associated with transport of restricted plant tissues. A portion of this project is directed at developing and presenting educational programs concerning PPV and other potential foreign stone fruit pathogens to concerned members of industry and the public. Standardized controls adopted nationally are needed to prevent spread of PPV to new geographical areas. Likewise, certification standards are needed to guarantee PPV-free production, transport, and sale of nursery stock and budwood. Even if PPV is eradicated within the next few years, there is a high probability that reintroduction will occur in the future. Alternatively if PPV becomes established in the U.S., it must be controlled to economically acceptable levels. One objective of this project will be to develop standards for PPV testing and certification of nursery stock among states. A portion of the research effort in this program will be directed at understanding how PPV spreads from orchard to orchard within growing regions and how the virus and its vectors survive overwinter. This information will be used in development of epidemiological models to understand how to control the disease and in the development of disease management systems (Decision Support Systems). The objectives for this proposal were developed during a meeting of participants in the NE-501 Northeastern Multistate Research Project entitled, "Eradication, Containment, and/or Management of Plum Pox (Sharka) Disease" held Oct. 31 to Nov. 1, 2000, in Gettysburg PA. Participants included major stone fruit growers in Adams County, PA, stone fruit nursery owners in Adams Co., county extension agents, fruit specialists and researchers from 18 major stone fruit growing states, and representatives from several state and federal regulatory agencies. Plum pox disease or "Sharka" is one of the most devastating and economically important diseases of stonefruit worldwide. The disease is caused by the Plum Pox Virus (PPV) which is naturally spread by several species of aphid. Although the virus is endemic to Eastern Europe it has been spread throughout Europe, the Mediterranean region and several locations in the Western Hemisphere by the unwitting use of infected propagation material. The first report of PPV in North America came from a peach orchard in Adams County Pennsylvania in 1999 and in the summer of 2000 it was found in Ontario Canada. Extensive surveys have presumably delimited the disease situation in the U.S. and Canada but the source of virus introduction into North America still has not been identified.

Most economically important species of Prunus are susceptible to PPV, including peaches, nectarines, plums, and apricots (4,11,13). A wild species, (Prunus spinosa), occurs widely in Europe and functions as a natural symptomless PPV reservoir making eradication of the virus impossible in many countries. The virus has a large experimental host range including 134 species of plants in 16 families that have been identified by artificial inoculation (1). The complete natural host range and the role of alternate weed hosts, in virus survival and spread is not fully known especially in countries where the virus has recently been introduced.

Interstate and international cooperation is needed to either eliminate or control PPV in North America. In the U.S., PPV is currently limited to Pennsylvania with approximately 5,400 Ac of stone fruits grown on small family farms. Depending on the year, Pennsylvania ranks as the fourth or fifth largest peach producing state. In 1998 Pennsylvania peach production was 65 million bushels valued at $21 million. This does not include the value to subsidiary agricultural industries or the total value to the small communities located throughout southern Pennsylvania and Maryland that are dependent on these crops. Total peach production in the eastern U.S. encompasses about 82,000 acres with a value in 1999 of $191 million. Canada has approximately 38,000 acres of stone fruits currently threatened and the occurrence of PPV in Ontario is a direct threat to stone fruit industries in New York, Michigan, and Ohio. Movement of propagation materials between Canada and New York has already occurred, but PPV has not yet been detected in this material. Other major peach producing states, such as South Carolina and Georgia, are equally threatened by the possibility of accidental movement of infected propagation material or introduction of infectious aphids. If infected propagation materials or the virus were to reach California, the results could be devastating. California currently grows approximately 265,000 Ac of susceptible stone fruits. Unlike the situation in Pennsylvania, very large commercial orchards are grown in close proximity under ideal environmental conditions for rapid aphid spread. The total value of the stone fruit industry in the U.S. is valued at $1.8 billion annually. With the ability to curtail fruit production anywhere from 10-100%, depending on the cultivar, PPV is of serious economic concern. One of the objectives of this project is to develop the infrastructure supporting multi-state and international collaborations to solve problems associated with PPV control and to provide mechanisms for increased communication and sharing of educational and scientific information. In addition, diverse facilities are needed to study this quarantined pathogen, as well as, personnel with diverse areas of expertise. It is beyond the scope of any one agency or institution to support this diversity of needs.

Because PPV is a recently introduced invasive species to North America, many personnel at State Regulatory Agencies, as well as, University and County Extension personnel are unfamiliar with Plum Pox disease. Many cultivars of peach, nectarine, plum, and apricot do not show obvious symptoms when first infected and are difficult to detect and identify when first encountered. Nevertheless, excellent tools have been developed to detect and identify PPV. One of the most practical and economical is a commercially available ELISA kit that is Plum Pox specific. A number of excellent illustrated bulletins and web-sites have also been developed to assist with identification of visual symptoms. Virtually any virology lab in the country is equipped with the basic tools that would allow participation in survey work for virus detection.

Despite the extensive survey work that has taken place since PPV was first found in North America it is too early to say that all sources of infected budwood and all infected orchards have been identified. Multistate cooperation is needed to continue the search and to establish a means of surveillance for possible escapes and future infestations. Even if eradication is totally successful there remains the possibility of future introductions. Furthermore, the action taken by NE-501 to deal with the PPV problem can provide a plan of action that may be applicable to other plant viruses of quarantine concern.

How the U.S. fruit industry deals with PPV will depend on many factors. If PPV is still localized near the initial infection foci, it may be possible to eradicate the disease by enforced tree removal. Whether or not this will be effective depends on whether the virus is spreading to new orchards and homeowner plantings, whether the virus has invaded wild plant species and whether local aphids are efficient PPV vectors. If infected budwood has been used for propagation of trees in areas outside the quarantine area then other foci of infection in geographically distant regions may exist. If PPV is determined to be widespread in wild plant species and/or efficient vector species is identified, then eradication of PPV becomes impossible. In this scenario, management of PPV to optimize yield and profit and reduce PPV effects will be necessary.

Therefore, education of all personnel involved in the fruit industry to recognize newly arriving potential invasive fruit pathogens is needed. To prevent the reintroduction of PPV requires increased education of the dangers associated with transport of restricted plant tissues. A portion of this project is directed at developing and presenting educational programs concerning PPV and other potential foreign stone fruit pathogens to concerned members of industry and the public.

Standardized controls adopted nationally are needed to prevent spread of PPV to new geographical areas. Likewise, certification standards are needed to guarantee PPV-free production, transport, and sale of nursery stock and budwood. Even if PPV is eradicated within the next few years, there is a high probability that reintroduction will occur in the future. Alternatively if PPV becomes established in the U.S., it must be controlled to economically acceptable levels. One objective of this project will be to develop standards for PPV testing and certification of nursery stock among states. A portion of the research effort in this program will be directed at understanding how PPV spreads from orchard to orchard within growing regions and how the virus and its vectors survive overwinter. This information will be used in development of epidemiological models to understand how to control the disease and in the development of disease management systems (Decision Support Systems).

The objectives for this proposal were developed during a meeting of participants in the NE-501 Northeastern Multistate Research Project entitled, "Eradication, Containment, and/or Management of Plum Pox (Sharka) Disease" held Oct. 31 to Nov. 1, 2000, in Gettysburg PA. Participants included major stone fruit growers in Adams County, PA, stone fruit nursery owners in Adams Co., county extension agents, fruit specialists and researchers from 18 major stone fruit growing states, and representatives from several state and federal regulatory agencies. Participants from the Ontario Ministry of Agriculture and the Canadian Food Inspection Service also participated. Following the first day of research reports, the second day was dedicated to workshop sessions in which all attendees (approx.100), including growers, and nursery representatives also participated in a USDA sponsored meeting held in Harrisburg, PA in Dec. 1999 to develop priorities for PPV control. The objectives of this proposal were derived from these extensive discussions and meetings among all stakeholders in the stone fruit industry.

Related, Current, and Previous Work:

Up until the 1990s PPV was exclusively a problem of Europe and the Mediterranean Region. Few U.S. scientists worked on this disease and those that did were generally working in collaboration with Europeans. Therefore much of what is known regarding the basic biology of this virus including strains, host range and vector transmission has come out of studies in Europe. All field experiments must be conducted in countries where PPV is endemic as research using the live virus in the United States is restricted to approved quarantine facilities.

Previous Work:

Biology of the pathogen: PPV is a member of the virus Family Potyviridae. Virus particles are flexous rods, approximately 730 X 12 nm in size. The genomic single-stranded RNA makes up about 5% of the particle mass; and 2000 copies of a 37 kDa structural protein coats and protects the RNA molecule (1, 21). Virus concentration is generally not evenly distributed in infected trees and makes virus detection difficult. Symptom expression varies widely with the host species, the cultivar, age of the infected plant, and the environment. When infected, some plants are almost symptomless, while others show diagnostic chlorotic vein banding or rings on leaves, and chlorotic rings on misshapen fruits (4, 13). Many stone fruit cultivars suffer severe fruit drop before harvest resulting in up to 100 % crop loss. Regardless of symptom production, PPV infection reduces the trees productive life. PPV is transmitted tree to tree or between orchards by aphids. Human activity is responsible for long distance spread. PPV-infected budwood, seedlings, or rootstocks used for propagation can be transported by man; thus by-passing natural barriers such as mountain ranges, forests, or oceans that would naturally limit virus spread by aphids. Buds taken from infected trees will transfer the virus to healthy trees when grafted.

Strain characterization and identification: Plum pox virus occurs as 4 major strains designated PPV-D, -M, -C, and -EA. Strains differ structurally and biologically. The most common strains in Europe are PPV-D and -M which infect apricots, plums, and peaches; but not cherry. PPV-C infects cherries, in addition to other Prunus species. PPV-EA was characterized from isolates in Egypt (1). In France, PPV-M spreads more quickly in peach than does PPV- D and is, therefore, more difficult to control. Recently, a genetic variant of PPV-D was detected in peach and is now causing more epidemics in French peach orchards (3). Only PPV-D has been identified in Spain, Chile, and the U.S. Plum pox virus is usually identified using enzyme linked immunosorbent assays (ELISA). Similar to other tree fruit viruses, PPV concentration can be low at certain times of the year and is notoriously unevenly distributed in trees (13). Recently, monoclonal antibodies have been developed for the universal detection of all PPV strains and for identifying the four serogroups (2). Nucleic acid hybridization tests (PCR) have been developed to universally or specifically identify PPV strains based on characteristics of the potyvirus genome (2,7,12). Development of immunocapture PCR (IC-PCR) increased the sensitivity of PCR to about 5000 times that of ELISA (24). However, cost and ease of use have made ELISA the preferred method for detection. Virus infecting peach (Prunus persicae cv. Encore) in Adams Co., PA was structurally and serologically similar to the old PPV-D strain from France (4).

Vector Transmission: The species of aphids functioning as important PPV vectors varies from country to country. In Spain, four aphid species were identified as the major vectors, Aphis spiraecola, A. fabae, A. gossypii, and Myzus persicae (14). Of these, only M. persicae actually colonizes and lives on Prunus. The other species are called migrant species because they do not colonize and reproduce on Prunus. Instead, they tend to occur in large populations migrating from other crops. Aphids acquire PPV during very brief test probes on the leaf surface. The PPV acquisition and transmission process can occur very rapidly within seconds. Therefore, PPV is spread most efficiently by migrant aphids moving rapidly from tree to tree while searching for suitable hosts (9,10). In France, about 14 species have been shown capable of efficiently transmitting PPV (9,10), and the dominant aphid species varied from region to region. No correlation was found between ability to transmit PPV and the ability to colonize Prunus species. Eleven aphid species are known to colonize peach in the U.S. (22), but nothing is known of their PPV vectoring ability. Of the 14 aphid species testing positive for PPV transmission in France, 8 of these species occur in Pennsylvania, all but 2 as migrant species. The efficiency of PPV transmission decreases rapidly with distance between the infected source plant and the next potential host. Rarely are these types of viruses transmitted beyond 100 meters, and there are few documented cases of PPV being transmitted over 500 meters from an infected plant.


Current Work:

Current Distribution of PPV in Pennsylvania: Since PPV was not known in the US until 1999, most virus testing for stone fruit viruses did not include tests for PPV. Evidence suggests that PPV was introduced years earlier because it takes several years following infection to observe symptoms. Once PPV was identified, the Pennsylvania Department of Agriculture (PDA) and USDA-APHIS initiated actions to inhibit further spread of the virus. Initial surveys suggested that infected orchards were localized in only two townships of Adams County. In addition, PPV was not detected in stone fruit nurseries, lessening the chance that virus had been accidentally spread out of the area. A quarantine prohibiting movement of stone fruit seedlings and budwood out of infected areas was enacted. Mechanisms for grower compensation for lost trees also were enacted and the following spring and summer orchards containing infected trees were destroyed and burned (almost 900 acres). ELISA surveys of all orchards in Pennsylvania were initiated the spring of 2000. Expanded surveys did detect a few infected trees in orchards near to the already quarantined area and the quarantine was expanded to include parts of two additional townships in both Adams and Cumberland Counties. Surveys of commercial orchards in other counties in Pennsylvania are continuing. A national survey in 2000 supported in part by USDA-APHIS and conducted by several states (mostly CA, GA, OR, SC and WA) failed to detect PPV in other major stone fruit growing regions of the US. However, in some states, surveys focused on nursery crops and commercial orchards will be tested more intensely during 2001.

Current Distribution of PPV in Canada: In June of 2000, orchards at Niagara-on-the-Lake, Ontario were tested by the Canadian Food Inspection Agency (CFIA) and verified to be infected with PPV-D. Evidence suggested that PPV had been introduced to Ontario years earlier, possibly as early as 1992. How PPV entered Canada and where it originated is unknown. Unfortunately, budwood from PPV-infected cling peach cultivars was selected for seedling production in Ontario and the resulting infected trees were shipped to several locations throughout the region, over a distance of 250 km. Thus, human activity greatly expanded the range of PPV infections in Canada. At the end of the 2000 growing season, 57 infected sites were identified in Canada. Currently, Canadian growers and government regulatory agencies are working to eradicate PPV from Canada. Over 13,000 PPV-infected fruit trees have been removed from all infected sites, and plans are being considered to establish quarantine zones and appropriate buffer zones around infected areas. As in Pennsylvania, the full extent of the PPV epidemic may not be accurately determined until this year's continued surveys are concluded. The close proximity of the Ontario infected sites to New York's stone fruit producers will require added vigilance to prevent introductions of PPV into upstate New York by aphids or accidentally (illegally) by humans. To date, no PPV has been detected in stone fruits of New York.

Weed Assays: Plant virologists have identified 134 species of plants in 16 different families that can serve as alternate hosts of PPV. However, these host range studies have relied primarily on artificial inoculation of plants under greenhouse conditions. There is little information available on the role of common weeds serving as virus reservoirs. In Pennsylvania leaf samples from nearly 7,500 weeds and native trees were collected from sites adjacent to PPV infected orchards and assayed for PPV. To date, all weed samples have been negative for PPV however, these studies should be ongoing.

Aphid Studies: Aphids have never been considered a major problem in peach production and therefore information is lacking on numbers and types of aphids present in stone fruit orchards. Research in Europe has shown that at least 20 species of aphid are capable of transmitting PPV. Available data shows that at least 8 of these vector species are present in Pennsylvania. A project to trap, identify and monitor peak population cycles of aphids in Pennsylvania peach orchards is in progress.

Transgenic Work: High levels of natural resistance have not been identified in Prunus species and germplasm. In a collaborative effort over the last nine years, between scientists at ARS, Kearneysville, WV; APHIS, Beltsville, MD; ARS, Frederick, MD; and INRA, Bordeaux, France, a transgenic plum pox resistant plum tree, clone C5, was developed and tested (18,19,20). This plum tree when graft-inoculated with PPV maintains the virus at nearly undetectable levels. In field plantings in Europe graft inoculated trees of C5 only show mild and transient symptoms in early spring and late fall. After 4 years of observation, C5 trees in the field in Europe appear to be immune to PPV by natural aphid inoculation. When used as a parent C5 transfers PPV resistance to its progeny (13). Thus C5 is the only plum that is highly resistant to PPV and can reliably transfer resistance to its progeny as a simply-inherited trait. Although this technique works well in plum it has not worked in peach. A reliable transformation protocol for peach still needs to be developed. Development of cultivars highly resistant to PPV through genetic engineering and/or conventional breeding programs will be the best means for ensuring continued viability of the stone fruit industry in the U.S.

Objectives

  1. Determine the distribution and incidence of Plum Pox Virus throughout the Northeast region, and nationally (including Canada).
  2. Fill gaps in knowledge about PPV survival and spread through basic and applied research.
  3. Develop PPV management strategies.
  4. Develop traditional and innovative delivery systems for information transfer to stone fruit researchers and extension personnel, fruit growers, and fruit industry representatives on current knowledge of plum pox virus.

Methods:


Objective 1: Determine the distribution and incidence of Plum Pox Virus throughout the Northeast region, and nationally (including Canada).

A national PPV survey has already been initiated in the United States and a similar program has been started in Canada. These programs have been instrumental in helping to identify major focal points of infection. However the possibility that additional as yet unidentified pockets of infection exist elsewhere in the country is real. All major stonefruit-producing states have a vested interest in participating in a continued effort to scout for PPV. In cases where the level of infection is currently very low or possibly where there has been undocumented movement of Prunus material it could be difficult to find. Therefore survey work should continue over several years.

A PPV survey protocol has been published on the web by USDA / APHIS. The availability of a commercial DURVIZ PPV ELISA (DASI) test kit further serves to standardize the survey procedures in every state. New reports of PPV in the United States need to be confirmed by the USDA/APHIS lab in Beltsville, MD by PCR. This step will confirm the virus as PPV and identify the serotype to determine if it is the same or different from that found in Pennsylvania and Canada.

In addition to the survey of commercial orchards it is also important to assay for PPV in weeds from infected areas. Knowing the incidence of plum pox in plant populations is a key factor in developing an epidemiological model since the percentage of plants that are infected influences the probability that an aphid has acquired the virus (8,17). Based on the degree of aggregation of diseased plants a 95% confidence interval for disease incidence can then be determined (16). These data can predict the number of samples that are needed to conclude with 95% confidence, that disease incidence is below a pre-determined value (e.g., < 0.1%). This information is useful for the evaluation of eradication efforts and to determine if disease persistence will or will not occur.

Objective 2: Fill gaps in knowledge about PPV survival and spread through basic and applied research.

Despite extensive research in Europe many questions regarding the biology of PPV remain unanswered. In North America the aphid vector(s) have not yet been identified therefore there is no information regarding transmission efficiency, vector-host relationships, or the range of potential alternate weed hosts.

2.1 Potential weed reservoirs of PPV:

Surveys of indigenous plants from undisturbed sites adjacent to infected orchards will determine if PPV has crossed over into wild plant populations. This information is critical to evaluate the success of the eradication program and to uncover potential hidden reservoirs of PPV. If eradication is not successful, knowledge of wild alternate hosts will be essential for the development of a virus management program.

In addition, seed of undercover weeds will be obtained from the National Seed Storage. The seeds will be germinated and checked by ELISA for any seed-borne potyviruses. The weeds will be inoculated with PPV-PA and various PPV isolates by mechanical abrasion, aphid inoculation, or by grafting to Prunus indicators such as GF305 peach seedlings, or P. tomentosa. Inoculated test plants will be observed for symptoms (30 days for weed hosts, and 3 months for woody indicator hosts) and then tested by ELISA and RT-PCR. Any weed host identified as a host of PPV will be used as a source of inoculum for aphid vector experiments from weed hosts to Prunus to determine their epidemiological roll as a natural host of PPV.

2.2 Identification of potential aphid vectors:

Various collection techniques such as yellow sticky traps, pan traps, sweep nets, and suction traps will be employed and evaluated to optimize methods for aphid capture, identification, and studies of seasonal dynamics. An aphid taxonomist will provide an initial aphid identification and verification. From these surveys a list of potential aphid vectors will be assembled for use in developing future control strategies for aphid vectors of PPV. Initial collections will be made in stone fruit orchards and later emphasis will shift to identify migrant aphid species associated with hosts other than commercially cultivated plants that are occurring in and around orchards (i. e., wild and ornamental Prunus, weeds).

Potential vector species will be established and maintained at Penn State University. Aphids will be transported to the BL3-P facility in Frederick, MD and used in transmission experiments. Colonies of aphids will also be developed of those aphids that are known vectors of PPV. These colonies will be tested for their ability to transmit the PA isolate of PPV Vectors will be determined by mass inoculation techniques such as the arena test or unrestricted probing test. Those determined to be vectors will be further tested for transmission efficiency using individual aphids.

2.3 Mechanisms regulating aphid transmission of PPV:

Vector and nonvector species of aphids can be compared for their ability to bind PPV isolates to their stylet food canals. Aphids allowed to test probe on membranes containing highly concentrated virus preparations with HC will be fixed and embedded for ultrastructural studies and immunohistochemical studies (5). Preliminary work will focus on using a variety of polyclonal and monoclonal antibodies to PPV for localizing specific regions of the aphid feeding apparatus binding PPV in a specific manner correlated to virus transmission. Antisera produced to PPV HC will be used to identify sites specifically binding HC to the aphid in treatments not containing PPV. Results of this study would identify sites in different aphid species associated with regulating PPV transmission. Once specific regions of the aphid associated with virus binding specificity are identified, experiments will focus on identifying components of PPV and the aphid involved in HC-virus recognition and binding to the aphid. Antiidiotypic antibodies (15,23) can be produced and selected for ability to compete with HC for binding to aphid styles when acquired through artificial membranes; and used in immuno-affinity columns to purify aphid stylet proteins or peptides regulating PPV transmission.

2.4 Characterization of the North American isolate(s) of PPV:

The original isolate(s) of PPV-PA identified in Prunus persica cvs Encore or Redskin and from Prunus domestica will be bud-grafted onto P. tomentosa (Nanking cherry) or P. persica cv. GF 305. Flushes growing from these buds will be used for inoculum to inoculate herbaceous species either by mechanical abrasion or aphid vectors. Published methods for the purification of PPV virions will be utilized to obtain highly purified preparations. All stages of the purification will be monitored by bioassay onto tobacco or pea. Highly purified virus preparations will be used for electron microscopy, antibody production, determination of chemical properties such as coat protein size, RNA concentration, particle size and titer. Comparative studies of PPV-PA biology will be conducted with PPV strains of known biology maintained in containment. Physical and biological characteristics such as post-inoculation incubation periods, optimal temperatures for infection, symptom development, thermal inactivation point and transmission efficiency will be determined.

Objective 3: Develop PPV management strategies.

The appearance of PPV in North America has created a need for the development of effective management strategies including the production of virus-free propagation material. In the event that PPV is successfully eradicated from the United States there must be safeguards against the possible reintroduction and spread of this disease. However, in the event that eradication is unsuccessful, disease management strategies must be developed to keep stonefruit production profitable.

3.1 Development of national virus certification standards:

The appearance of PPV in the U.S. has shown that the national importation program should not be relied upon as the only safeguard against the introduction of exotic diseases. Input from each state is needed to draft standards for certification of Prunus nursery stock incorporating new testing and inspection protocols to effectively monitor for introduction of PPV and other exotic pathogens.

3.2 Identification of sources of clean propagation material:

An important step in the management of PPV and other viruses is to insure that new orchards use virus-free planting stock. To this end tree nurseries must have access to virus tested mother blocks. Multistate cooperation is needed to identify and exchange clean propagation material that is true to type.

3.3 Development of management strategies to monitor and restrict virus movement:

In the event that PPV becomes established, new ideas for restricting virus movement need to be developed and tested at field stations around the country. Examples include new ideas for orchard ground cover management to reduce aphid populations or to limit their movement. The testing of new or unregistered aphicidal compounds and seeking registration for those that prove effective.

In areas where PPV must be managed an economical survey strategy to monitor for the appearance of the virus needs to be developed. One suggestion that needs to be tested is the identification of a sensitive host plant that can be planted at strategic locations to serve as sentinels. Frequent testing of sentinel plants may forewarn of increased virus pressure.

Objective 4: Develop traditional and innovative delivery systems for information transfer to stone fruit researchers and extension personnel, fruit growers, and fruit industry representatives on current knowledge of plum pox virus.

The Plum Pox Virus is a recent introduction to North America. Because this is a new problem most people in the fruit industry from growers to scientists are not knowledgeable about the disease and good educational materials have not been readily available. A successful eradication program will rely heavily on the educational tools available to growers and the public. In the event that PPV becomes a management issue timely delivery of research results will be necessary for growers to make informed decisions. Since PPV is an issue that is important to stone fruit produces across the United States and Canada it is important that there is wide spread participation in the development and implementation of educational programs. In order to reach a wide audience and make materials readily available traditional and novel delivery systems including grower meetings, extension publications, videos, and the worldwide web will be used.

Measurement of Progress and Results:

Outputs:
Outcomes or projected Impacts:
Milestones:

(2002):Finish collection of epidemiological data and calculation of confidence intervals; finish characterization of the North American isolate of PPV; hold educational meetings for stone fruit producers in the major fruit production regions. Create and/or enhance educational tools that provide the latest information on plum pox.

(2003):Determine if weeds or native trees serve as alternate hosts of PPV; identify the aphid vectors of PPV; publish educational materials for growers and consumers

(2005):Establish PPV management strategies; promote new interstate virus certification protocols

(2006):Evaluate the success of the eradication program

Projected Participation:

Include a completed Appendix E form

Outreach Plan:

1. World Wide Web site for Plum Pox education and research.

2. Decision Support Systems for Plum Pox Virus Management.

3. Educational Plum Pox Video Production.

4. Grower Educational Meetings.

5. Extension Demonstration Trials and Applied Field Research Experiments.

6. Hard Copy Extension Publications.

Organization and Governance:

The technical committee will consist of at least one voting member from each of the participating states (Appendix E), the administrative advisor, and the CSREES representative. Each year the technical committee will elect a chairperson, secretary, and at least one member-at-large to serve as an executive committee. The regional Technical committee will meet annually to report on the research results obtained, discuss and exchange information and ideas and to plan and coordinate next years work relating to the objectives of this proposal. A coordinator for each of the objectives may be designated to facilitate the coordination and reporting of the research being conducted by the collaborators. The technical committee may invite other scientists with experience in epidemiology, aphid biology, immunology, plant virology and others to participate in the annual meeting to provide specific information and strengthen the discussion.

Literature Cited:

Attachments:

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s:/Bruce McPheron

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