2006 Annual Report 1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? The environmental horticulture industry, also known as the "Green Industry", is one of the fastest growing segments of the nation's agricultural economy and is comprised of a variety of businesses involved in production, distribution and services associated with ornamental plants, landscape and garden supplies and equipment. The National Gardening Association reported that 85 million U.S. households spent $39.6 billion at lawn and garden retail outlets in 2002. The USDA National Agricultural Statistics Service reported that in 2005 the wholesale value of floral and nursery crops was $15.22 billion with 12,258 growers employing 187,563 workers. Over one quarter of these growers (28%) had sales less than $100,000. Floral crop sales comprise one of the fastest growing segments within environmental horticulture, forming a $5.4 billion industry for 2005. The goal of this project is to provide the means for growers to reliably detect and then control viral and bacterial diseases of ornamentals using environmentally friendly practices. We will investigate and characterize viruses and bacteria of major significance in ornamental and nursery crops, and develop serological reagents, molecular probes, and diagnostic technologies. Accurate and sensitive diagnostic methods will allow identification of viral and bacterial diseases, and selection of healthy stock for propagation (exclusion of disease). There is no known effective natural resistance against viral and bacterial diseases in many ornamentals. We will therefore investigate the genome organization of important viruses of ornamentals, and determine which genes are involved in host range, pathogenicity and symptom induction. An understanding of the factors involved in viral and bacterial pathogenesis may allow development of effective means of interference in the disease process. Expression of virus-derived genes, antigen-binding peptides, or lytic peptides will be tested for introduction of genetically engineered resistance against selected viral and bacterial pathogens of important floral crops. Examining host/pathogen interactions of Ralstonia solanacearum will lead to increased understanding of the epidemiology of bacterial wilt disease in geranium. Botanical extracts effective against soil-borne fungi will be examined to determine if they can control bacterial wilt in this important crop. Studies of host and vector specificity of strains of Xylella fastidiosa are expected to result in development of strain-specific diagnostic methods, and epidemiological information. This knowledge will be used to target interventions for improved disease control. Many plant virus and bacterial diseases cause significant losses in the production and quality of ornamental crops, are very difficult to control, and new diseases occur as different crops are introduced or grown in new areas. Many crops are susceptible to multiple viruses, each of which may cause serious economic losses, and infected plant material may not be acceptable for export. Methods to reliably and rapidly detect and identify these viral and bacterial pathogens are necessary for the production of pathogen-free or pathogen-indexed plants. An understanding of the genome organization of important ornamental viruses and bacteria, as well as the mechanisms of infection and pathogenicity of viruses and bacteria, is needed in order to develop better methods of virus and bacteria disease control. Neither wild nor improved germplasm with virus or bacterial resistance is available for many genera of ornamental plants. Tools and genes need to be developed for use in the genetic engineering of plants to introduce effective resistance to plant viral and bacterial diseases and to understand the mechanisms of resistance. This research is needed to allow increases in both productivity and quality of ornamental plants in an environmentally friendly manner, thereby reducing the use of and reliance on chemical control of pests and diseases. Research is directed to understanding the genome organization of important ornamental viruses and bacteria as well as their mechanisms of infection and pathogenicity in order to develop better methods of disease control. Research is also directed at developing tools and genes for the introduction of viral, anti-viral and anti-bacterial gene constructs to transgenic plants to confer effective virus or bacterial resistance, and to understanding the mechanisms of resistance. By this means we hope to introduce the most effective means of resistance to the broadest spectrum of viruses and bacteria infecting ornamentals, and thus to allow increases in both productivity and quality and a decrease in the use of and dependence on pesticides. There are few sources of virus or bacterial resistance in the available germplasm of many ornamental species, yet virus and bacterial infection is a major constraint on both productivity and quality of many crops. Whereas pathogen-free plants of some species can be generated by growing them from seed, this option is not available for the many hybrid, self-incompatible, or vegetatively propagated cultivars. Pathogen-free plants of other species may be obtained by tissue-culture propagation combined with either heat or chemical treatments, but such plants are readily re-infected when grown in typical commercial conditions. Use of genetically engineered crops offers a novel and effective strategy for the control of viral and bacterial diseases. One problem that limits the commercialization of such crops is the use of promoters that have licensing restrictions such as the widely studied 35S promoter from Cauliflower mosaic virus (CaMV). It is, therefore, essential to find a promoter that will not pose a licensing problem for the development and commercialization of transgenic plants. Our research is directed to isolate an effective promoter from Citrus yellow mosaic virus (CYMV) for expression of disease resistant genes in ornamental crops that may lead to the development of genetically engineered ornamental crops for bacterial disease resistance. Such disease control strategy is both cost-effective and environmentally sound. Bacterial diseases caused by Agrobacterium, Erwinia, Pseudomonas, Xanthomonas, Xylella and Ralstonia spp., for example, often result in significant losses in the production and quality of ornamental crops and are very difficult to control. Many crops are susceptible to multiple viruses, each of which may cause serious economic losses, and infected plant material may not be acceptable for export. At least 125 different viruses have been identified that infect and cause disease in ornamental plants. Virus diseases may be transmitted by a number of different biological vectors, such as aphids, whiteflies, or thrips. Different isolates of a particular virus may differ significantly in both serological and biological characteristics that complicate disease diagnosis and identification. Identification of the virus and vector is important for development of practical disease prevention or control methods, and to minimize pesticide usage. At the final 'production' stage of growing and distributing ornamental plants, losses due to viral and bacterial infections can range from 10 to 100%, depending upon the virus- or bacteria-host combination. Viruses of serious consequence recently identified by the floral and nursery industry in key ornamental crops include, but are not limited to: potyviruses, tospoviruses, fabaviruses, pelarspoviruses, closteroviruses, potexviruses, carlaviruses, cucumber mosaic virus (CMV), arabis mosaic virus (ArMV), dahlia mosaic virus (DMV), and plum pox virus (PPV). There is therefore a need for research on these new and emerging virus and virus-like problems. Ralstonia solanacearum causes bacterial wilt, a very destructive disease that is distributed worldwide in tropical and warm temperate regions. In 1999, race 3 of R. solanacearum was isolated for the first time in the U.S. from imported geraniums in Wisconsin, but very little is known about this race in geranium. Our research is directed at studies on host range, epidemiology, and control of the geranium race of R. solanacearum. These studies are important steps towards a better understanding of the pathogen and will result in better quarantine measures to control and prevent the disease it causes from becoming established in the United States. Xylella fastidiosa has been found to cause bacterial leaf scorch diseases of woody ornamentals for 20 years, as well as Pierce's Disease (PD) of grapevine, but our knowledge of the bacterium in landscape trees is still very limited. Recent surveys indicate that the disease is spreading and becoming more severe in a number of important landscape trees such as oak, elm and sycamore in many states of the U.S. Bacterial leaf scorch of oleander, a relatively new disease that was reported in 1999, is also an emerging problem in California, Arizona and Texas where oleander is used as a popular landscape plant. Our research is directed to study genetic diversity and relationships among woody ornamental and other strains, to develop simple, specific and sensitive detection methods for shade tree strains, and to investigate pathogenic relationship between the shade tree and PD strains, in order to effectively control the diseases. The project has five specific objectives: (1) Investigate and characterize viruses of major significance to ornamental and nursery crops (annuals, perennials, woody ornamentals, bedding plants, and bulb crops). These viruses include, but are not limited to: potyviruses (including plum pox virus), tospoviruses, fabaviruses, pelarspoviruses, closteroviruses, cucumber mosaic virus, arabis mosaic virus, dahlia mosaic virus, and 'new' currently uncharacterized or emerging viruses affecting key ornamental crops. Develop serological reagents, molecular probes and diagnostic technologies for the detection and management of diseases caused by the viruses listed above; (2) Determine the genome organization of selected important ornamental viruses and develop full-length infectious clones to determine the genes or gene products involved in pathogenicity and their mode of action; (3) Develop and utilize anti-viral and anti-bacterial genes in the production of transgenic floral crops exhibiting induced resistance to selected major viral and bacterial diseases. Identify and develop alternative plant expression promoters; (4) Conduct research on the host range, epidemiology and non-pesticidal control of bacterial wilt disease of geranium caused by Ralstonia solanacearum race 3; and, (5) Develop knowledge and tools for the detection and control of bacterial leaf scorch disease of woody ornamental crops caused by Xylella fastidiosa. Characterize the genome of ornamental strains of X. fastidiosa, their relationships with hosts and vectors, and develop methods for disease control. The research to be undertaken falls under National Program 303, Plant Diseases. The proposed research addresses the development of more effective means for the detection and identification of plant virus and bacterial diseases affecting ornamentals and to utilize those methods to allow selection of pathogen-free or pathogen-indexed plants. Understanding viral and bacterial genome structures and functions, the mechanisms of viral and bacterial pathogenicity, the mechanisms of virus and bacterial resistance, and developing tools and genes for conferring virus and bacterial resistance in plants will lead to the development of better control measures and increases in both productivity and quality of ornamental plants for industry and the consumer. The research in this project contributes to Components I (Identification and Classification of Pathogens), III (Cultural Control), IV (Pathogen Biology, Genetics, Population Dynamics, Spread and Relationship with Hosts and Vectors), and V (Host Plant Resistance to Disease). 2.List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2003) - Begin to identify and characterize the causal agents of new and emerging viral diseases in economically important ornamental crops (Obj. 1). - Develop full genome length viral clones to key ornamental viruses (Obj. 2). - Develop transgenic plants expressing single-chain antibody (scFv) constructs (Obj. 3). - Develop additional scFv constructs to non-structural viral gene products (Obj. 3). - Begin to develop strong viral promoter regions for transient GUS expression (Obj. 3). - Determine host range and inoculum threshold of R. solanacearum race 3 (Obj. 4). - Begin genetic relationship studies among ornamental and non-ornamental X. fastidiosa strains (Obj. 5). Year 2 (FY 2004) - Identify and characterize the causal agents of new and emerging viral diseases in economically important ornamental crops; begin to develop diagnostic reagents or tools for diagnosis (Obj. 1). - Continue to develop full genome length viral clones; begin to develop infectious clones (Obj. 2). - Develop a new method for inoculation of Cucumber mosaic virus to gladiolus (Obj. 3). - Begin resistance evaluation of transgenic plants expressing viral or anti-viral transgenes (Obj. 3). - Begin to determine strong promoter regions of CYMV by transient GUS expression (Obj. 3). - Finalize host range and inoculum threshold of R. solanacearum race 3; evaluate plant growth temperatures effects in disease development (Obj. 4). - Determine genetic relationships among X. fastidiosa strains; develop a DNA extraction protocol for detection of X. fastidiosa from plant materials (Obj. 5). - Publication of research documenting recent accomplishments. Year 3 (FY 2005) - Identify and characterize a second round of the causal agents of new and emerging viral diseases in economically important ornamental crops (Obj. 1) - Optimize detection regime protocols for virus diagnosis; transfer technology and reagents to industry (Obj. 1). - Continue to develop full genome length viral clones; begin to develop infectious clones (Obj. 2). - Continue resistance evaluation of transgenic plants expressing viral or anti-viral transgenes (Obj. 3). - Continue to determine strong promoter regions of CYMV by transient GUS expression (Obj. 3). - Begin host range study for R. solanacearum race 3, biovar 2 (Obj. 4). - Develop PCR detection of X. fastidiosa strains; determine natural occurrence of X. fastidiosa in a commercial nursery (Obj. 5). - Publication of research documenting recent accomplishments (All Objectives). Year 4 (FY 2006) - Further characterize the new and emerging viruses (comparative virology) (Obj. 1). - Begin to develop diagnostic reagents or tools for diagnosis for second set of viruses; transfer technology and reagents to industry (Obj. 1). - Continue to develop full genome length viral clones; begin to create infectious clones and begin to evaluate gene exchange mutants to determine key viral determinants. (Obj. 2). - Evaluate and establish a method using a plant virus vector for production of tospovirus nucleocapsid proteins for production of specific diagnostic reagents (rabbit polyclonal antiserum and mouse monoclonal antibody production). (Obj. 2). - Finish resistance evaluation of transgenic gladioli expressing CMV coat protein and replicase genes using the biolistic inoculation method developed. (Obj. 3). - Continue resistance evaluation of transgenic gladioli expressing CMV-specific scFv antibody genes. (Obj. 3). - Continue studies on tissue specificity and level of expression of strong CYMV promoter regions (Obj. 3). - Continue host range study for R. solanacearum race 3, biovar 2 (Obj. 4). - Complete studies on natural occurrence of X. fastidiosa in a commercial nursery (Obj. 5). - Continue pathogenic relationship between the agricultural strains, such as grape strain, and ornamental strains, such as oak or oleander strain; transfer detection protocols and reagents to industry (Obj. 5). - Develop CRADA with appropriate commercial grower company for technology transfer. - Publication of research documenting recent accomplishments (All objectives). Year 5 (FY 2007) - Continue characterization of previously identified viruses (comparative virology) (Obj. 1). - Identify and characterize additional new and emerging viruses as needed; begin to develop diagnostic reagents or tools for diagnosis (Obj. 1). - Begin to develop diagnostic reagents or tools for diagnosis for second set of viruses; transfer technology and reagents to industry (Obj. 1). - Begin to examine differences in host gene expression in response to engineered infectious clones (Obj. 2). - Continue resistance evaluation of transgenic gladioli expressing CMV-specific scFv antibody genes. (Obj. 3). - Begin to create transgene constructs to combine resistance to multiple viruses (Obj. 3). - Continue studies on tissue specificity and level of expression of strong CYMV promoter regions (Obj. 3). - Continue host range study for R. solanacearum race 3, biovar 2 (Obj. 4). - Determine inoculum threshold of R. solanacearum race 3, biovar 2 (Obj. 4). - Continue pathogenic relationship study for strains of X. fastidiosa (Obj. 5). - Transfer detection protocols and reagents to industry (All objectives). - Publication of research documenting recent accomplishments (All objectives). - Begin to prepare new project plan as directed by National Program Staff. Year 6 (FY 2008) - Continue characterization of previously identified viruses (comparative virology) (Obj. 1). - Continue to identify and characterize additional new and emerging viruses as needed; begin to develop additional diagnostic reagents or tools for diagnosis (Obj. 1). - Continue evaluation of host virus interactions in response to engineered infectious clones (Obj. 2). - Begin to determine resistance to multiple viruses in transgenic lines expressing multiple constructs; evaluate possible recombination events and synergistic effects (Obj. 3). - Evaluate transgenic geranium for broad-spectrum bacterial resistance (Obj. 3). - Continue to explore the potential use of the CYMV promoter in ornamental crops (Obj. 3). - Evaluate plant growth temperature effects on disease development caused by R. solanacearum race3, biovar 2 (Obj. 4). - Explore biological and cultural control measures for R. solanacearum (Obj. 4). - Publish pathogenic relationship study for strains of X. fastidiosa (Obj. 5). - Transfer additional detection protocols and reagents to industry (All objectives). - Publication of research documenting recent accomplishments (All objectives). - Finalize new project plan through ARS-OSQR for implementation. 4a.List the single most significant research accomplishment during FY 2006. Detection and characterization of several novel carlaviruses in different species of Phlox We have previously detected two carlaviruses, from Phlox stolonifera (creeping phlox) and Phlox divaricata, and identified them as novel members of the carlavirus group based on the sequences of the 3’-terminal region of the viral genomes; virus-specific polymerase chain reaction (PCR) assays were developed for each virus. The complete 8590nt sequence of the novel carlavirus from creeping phlox has now been determined; sequence comparisons of all regions of the genome confirm that this is a novel virus, for which the name ‘Phlox virus S’ is proposed. A virus-specific polyclonal antiserum has been prepared against purified ‘Phlox virus S’, and this antiserum will be made available to diagnosticians. Approximately 5000nt of the sequence of the carlavirus (‘WP carlavirus’) from Phlox divaricata has also been sequenced, and similarly confirms its status as a distinct virus. The ‘WP carlavirus’ has been partially purified from mechanically infected snapdragon plants, although the degree of purity is not sufficient for preparation of a virus-specific antiserum. Recently a third distinct carlavirus was detected by electron microscopy and PCR from a new hybrid phlox cultivar, and the sequence of the 3’-terminal region shown to be distinct from any sequences available in GenBank. No alternate host has yet been identified for this third carlavirus. Methods to detect and identify the viruses infecting phlox should allow growers to select healthy plants, or plants free of the most damaging viruses, in order to produce plants of higher quality and productivity. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. 4b.List other significant research accomplishment(s), if any. Annotation and analysis of the 8x draft genome of a geranium isolate of Ralstonia solanacearum race 3, biovar 2, strain UW551 Ralstonia solanacearum causes bacterial wilt, a soilborne vascular disease that is distributed worldwide in tropical and warm temperate regions. The disease attacks over 450 plant species including ornamentals, and is the major constraint on production of many economically important crops including potato and tomato. R. solanacearum is generally classified into 5 races and 5 biovars based on host range and carbohydrate utilization. Race 3, biovar2 (R3B2) causes brown rot, which ranks among the most destructive diseases of potato in Africa, Asia, and Central and South America. R3B2 strains are listed as Select Agents in the United States and are quarantined pathogens in Europe and Canada. In this collaborative study with fifteen other university and agency scientists, the 8x draft genome of R3B2 strain UW551 (a geranium isolate) was determined and the unique R3B2 genome components were identified (see UW551 Project link at http://genomics.biotech.ufl.edu). This knowledge of the complete genome will help to gain a better understanding of the pathogen and to develop race-specific detection methods. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. Novel Bacopa Ilarvirus Characterized Bacopa plants (Sutera cordata cv. Snowflake) with virus-like symptoms including chlorosis and leaf dimpling were obtained from nurseries in Maryland. A virus was mechanically transferred to, established and maintained in Nicotiana tabacum. The virus infects Chenopodium quinoa, Cucumis sativus, Hyoscyamus niger, N. tabacum, but not to Rutgers tomato or several other inoculated species. In N. tabacum, faint chlorotic spots developed 2-3 days after inoculation, later developing into necrotic spots, lines and rings. Symptoms were more apparent in plants grown at temperatures between 60 and 72 degrees F, but would disappear above 75 degrees F. No serological reactions were observed with antiserum to Broad bean wilt virus (previously reported from bacopa) or Tobacco streak virus. Partial purification of the virus resulted in a banding profile in sucrose gradients typical of multicomponent viruses. RT-PCR using the Ilarvirus Group primers (Agdia, Inc., Elkhart, IN) yielded a ca. 450-bp product which shared 80-87% nucleic acid and 83% amino acid identities with Parietaria mottle ilarvirus. Cloning and sequencing of almost the entire RNA 1, more than the 5 half of RNA 2, and the 5 half of RNA 3 of the bacopa virus revealed that it is a distinct ilarvirus and the name Bacopa chlorosis virus was proposed. Development of a virus-specific RT-PCR assay and antiserum will allow selection of plants free from this virus as a source of propagation material for growers. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. Cloning and sequencing of a U.S. isolate of Lolium latent virus We previously reported partial sequencing of an isolate of Lolium latent virus (LLV) in a sample of ryegrass germplasm under quarantine, and identification of LLV for the first time in U.S. ryegrass germplasm. We have now prepared a virus-specific antiserum against a purified preparation of the U.S. isolate of LLV, and will use this antiserum to survey US germplasm more widely; the antiserum will also be made available to USDA-APHIS for screening germplasm accessions in quarantine, and to diagnosticians and turf breeders to aid in selection of healthy turf germplasm for propagation and breeding. We have also cloned and sequenced almost the complete genome of the U.S. isolate for comparison to the U.K. isolate, and to develop an infectious clone for use as a viral vector for gene expression and virus-induced gene silencing (VIGS; collaboration with project ‘Genetic Enhancement of Turfgrass Germplasm for Reduced Input Sustainability’, 1230-21000-045-00D; and a visiting scientist from the Istituto di Virologia Vegetale, CNR, Turin, Italy). Knowledge of the presence of this virus in the US, and virus-specific reagents for detection and identification, should aid in selection of healthy turf germplasm for propagation and breeding and a VIGS vector for turfgrasses will aid in gene discovery and functional genomics for breeding of superior turf types. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. Production of tospovirus-specific antibodies from a potyvirus-based vector In collaboration with scientists in Taiwan, common epitopes on a conserved region of NSs proteins among members of Watermelon silver mottle virus (WSMoV) serogroup were identified. Three monoclonal antibody (MAb)-secreting mouse hybridoma cell lines were produced using the Zucchini yellow mosaic virus (ZYMV)-expressed WSMoV NSs protein purified from squash. The three MAbs target at the aa 89 to 125 region of WSMoV NSs protein. All three MAbs also reacted with high-temperature recovered gloxinia HT-1 isolate of Capsicum chlorosis virus (CaCV), and Calla lily chlorotic spot virus (CCSV). In collaboration with investigators in India, the three MAbs were successfully used in field surveys of Peanut bud necrosis virus (PBNV), and Watermelon bud necrosis virus. Sequence comparison of the deduced MAb-recognized region with the reported tospovirus NSs proteins revealed the presence of a consensus sequence at the aa 98 to 120 position of NSs proteins, sharing 86 to 100% identities among WSMoV, CaCV, CCSV, and PBNV. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. Association of X. fastidiosa with asymptomatic nursery plants X. fastidiosa is associated with bacterial leaf scorch and decline in many economically important landscape trees and shrubs. Since the natural occurrence of X. fastidiosa in commercial nurseries is unknown and such information is greatly needed to ensure the production of healthy plant materials, we conducted a survey in 2003 and 2004 in a wholesale and production nursery in Maryland using antibody- and DNA-based methods to determine the natural occurrence of X. fastidiosa in nursery plants and wild and cultivated plants surrounding the nursery. Our results indicate a possible association of X. fastidiosa with the nursery plants crape myrtle, Japanese stewartia, Schipka laurel and Japanese flowering cherry, as well as with the nursery surrounding plants mimosa and sassafras, previously unknown hosts of the bacterium. It is possible that the source of the X. fastidiosa in the nursery plants came from red oak and box elder plants on the edge of the nursery that showed leaf scorch symptoms and harbored high populations of the bacterium. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. 4d.Progress report. A novel ilarvirus is seed-transmitted in pansy Pansy is one of the most important bedding plants in much of the U.S.A. In the 2005 season many growers in multiple states experienced the highest frequency of ‘Pansy Mottle Syndrome’ (PMS) observed in recent years, in many cultivars. While the cause of PMS is not known with certainty, a viral association was suspected. We identified the presence of an ilarvirus in pansy plants showing PMS symptoms by PCR utilizing Ilarvirus-Group primers obtained from Agdia; the PCR product was cloned and sequenced, and shown to be distinct from other ilarvirus sequences available in GenBank, and from Bacopa chlorosis virus (currently under investigation in our laboratory). The PCR product from four separate pansy varieties from different sources was essentially identical, indicating that there is little diversity in the viral population. Testing of further pansy samples of many different varieties has shown that the novel ‘Pansy ilarvirus’ is present in many plants without PMS symptoms, and absent in many plants showing pronounced PMS symptoms. The ‘Pansy ilarvirus’ therefore is not a unique incitant of PMS, but may contribute to the development of PMS, which appears to be associated with multiple stresses. We have shown that the ‘Pansy ilarvirus’ is carried in the seed of many varieties of pansy, from multiple seed producers. Plants grown from seed lots of some varieties tested have not shown any signs of seed transmission; additional tests are under way to determine whether these varieties are resistant to the virus, or were just derived from un-infected parental plants. We are continuing efforts to identify a suitable purification host to enable production of a virus-specific antiserum; in parallel we are working to clone the complete genome of the ‘Pansy ilarvirus’. If we are unable to purify the virus adequately to use a virus preparation to develop a virus-specific antiserum, an alternative would be bacterial expression of the cloned viral coat protein gene. Availability of a virus-specific antiserum would allow breeders and seed producers to select healthy plants for breeding and seed production, thus allowing elimination of this virus from seed lots. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. Detection and characterization of a new potyvirus causing flower break in New Guinea Impatiens New Guinea Impatiens form a large part of the floral trade in ornamental plants, especially in the area of bedding and garden plants. New Guinea Impatiens (I. hawkeri) are generally vegetatively propagated and are popular as a spring ornamental because of the bright array of colors and large-flowered cultivars available. Several New Guinea Impatiens plants (obtained from local Maryland commercial nurseries) showing virus-like color breaking symptoms on flower petals tested positive for potyvirus in ELISA using our genus Potyvirus broad-spectrum reacting monoclonal antibody. Potyviruses, members of the genus Potyvirus, collectively infect almost every major crop throughout the world, and in many crops a potyvirus may be the economically important viral pathogen. Some of the biological and molecular characteristics of this new potyvirus, for which we propose the name Impatiens flower break virus (IFBV) have been determined. Phylogenetic analysis of the molecularly cloned and sequenced 3'-terminal region 1710nt fragment, and pairwise comparisons of the nucleotide and deduced amino acid sequences of IFBV coat protein with other potyviruses, revealed that IFBV is a new species in the genus Potyvirus. Western-blot analysis and RT-PCR tests using Impatiens virus-specific primers have shown this virus can be detected in several cultivars exhibiting flower color breaking. The virus has also been mechanically transmitted to healthy impatiens and produces flower break symptoms in several different cultivars. This is also the first report of a potyvirus infection associated with flower color breaking in New Guinea Impatiens. This information will be useful to growers and nurseries in their screening assays to detect and control this virus in the parent propagation stock lines and subsequently in the large scale plant production phases. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. Engineering Cucumber mosaic virus resistance in gladiolus Research has continued on the production and evaluation of disease resistant transgenic gladiolus through the incorporation of various viral and antiviral antibody genes. In collaboration with the Floral and Nursery Plants Research Unit, Project 1230-21000-037-00D, transgenic gladioli have been produced which express Cucumber mosaic virus (CMV) replicase, coat protein, or antiviral antibody transgenes. Studies include two varieties of gladiolus, different types of gene constructions and different promoters. Using the biolistic inoculation method developed in the laboratory, studies are underway to determine the degree of protection when transgenic plants are challenge-inoculated with CMV. Initial challenge inoculation of the 59 transgenic lines containing coat protein or replicase gene or both showed that 9 lines were resistant to CMV. Initial screening of transgenic gladioli containing antibody genes was just completed. Reactions to CMV inoculation will be tested in the next few months. This work will facilitate the evaluation of virus resistance in transgenic gladiolus plants to yield improved floral quality and productivity. This research in this accomplishment contributes to ARS National Program 303, Component V and ARS Strategic Plan Performance Measure 3.2.5. Further characterization of an ophiovirus from Lachenalia. We have previously reported detection, by electron microscopy and PCR using generic ophiovirus primers, of an ophiovirus in commercial Lachenalia. The complete coat protein gene (encoded by RNA. 3)and a major portion of RNA 1 of the Lachenalia ophiovirus have now been cloned and sequenced, in collaboration with a visiting scientist from the Istituto di Virologia Vegetale (IVV, CNR, Turin, Italy). The coat protein gene of the Lachenalia ophiovirus is almost identical to that of an ophiovirus from Freesia (tentatively named Freesia sneak virus) previously discovered at the IVV lab in Italy. The sequence of RNA 1 of the Lachenalia virus is also very similar to the available sequence of the Freesia ophiovirus, suggesting that the Lachenalia virus is an isolate of the same virus. The virus is difficult to purify, and so we expect to express the Lachenalia ophiovirus coat protein gene in bacteria for use as antigen to prepare a virus-specific antiserum for virus detection and diagnosis. Such an antiserum would be useful for diagnostic labs and growers, to enable selection of healthy propagation stock. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. Detection of additional viruses in phlox Tobacco ringspot nepovirus, an unidentified tobamovirus, and two distinct types of potyvirus were detected in various types of phlox by electron microscopy, various serological methods, and/or by polymerase chain reaction methods followed by molecular cloning and sequencing. Phlox species in cultivation are propagated vegetatively, and many plants are infected by multiple viruses; virus infection causes reduction of both quality and productivity. One class of potyvirus sequences were identified as Spiranthes mosaic virus-3, previously reported only from the terrestrial orchid Spiranthes cernua, and for which no alternate host was previously known; the other class of potyvirus sequence represents a novel type not represented in the GenBank database. Some phlox plants were found to be infected by both classes of potyvirus sequence. A tobamovirus was detected by electron microscopy, and gave weak positive results with Tobacco mosaic virus-specific ImmunoStrips; the weak serological reaction and host plant reactions suggest that this may be a previously undescribed tobamovirus. Tobacco ringspot virus was detected by ELISA, and the identity confirmed by sequencing of a cloned portion of the genome generated via the polymerase chain reaction. Methods to detect and identify the viruses infecting phlox should allow growers to select healthy plants, or plants free of the most damaging viruses, in order to produce plants of higher quality and productivity. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. Virus detection in other cultivars of Verbena [This report portion serves to document research conducted under Specific Cooperative Agreement #1230-22000-012-01S between the Floral and Nursery Plants Research Unit and Oregon State University; please see that annual report for a full summary]. In previous research, a potyvirus infecting Verbena x hybrida cv. ‘Lavender Shades’ was identified as a pea mosaic isolate of bean yellow mosaic virus (BYMV), although the host range did not match that of typical BYMV isolates. We solicited other Verbena cultivars to determine if the previously reported results from the temperature-dependent detection experiments with Verbena ‘Lavender Shades’ would be consistent across different genotypes. We assayed 14 different cultivars of Verbena using double-stranded RNA (dsRNA) analysis and also tested them using group-specific RT-PCR assays (Agdia, Elkhart IN) to ensure the plants were virus free. Several ‘healthy’ and symptomatic cultivars were found to contain viruses. Products from these reactions were cloned and sequenced. ‘Silver Ann’ contained a potexvirus that showed similarity with Clover yellow mosaic virus (with a nucleic acid identity of 82%) and a ‘new’ undescribed potyvirus species. The cultivar ‘Taylor Town Red’ tested positive for potyvirus which different from the one found in ‘Silver Ann’. ‘ES Burg 1136-3’ had reduced vigor and tested positive for trichovirus and contained 7.5 and 6.7 kb dsRNAs. ‘Lanai Scarlet 921-1’ and two other cultivars were symptomless but tested positive for carmovirus and contained four dsRNAs. The carmovirus in the symptomless verbenas is most similar to Angelonia flower break virus. Further characterization of these viruses is ongoing. These results will be useful to propagators, who will be best able to detect infected plants during cycles of active growth, and thus reject infected plants for further propagation. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. Detection of Viruses in Verbena at All Stages of Its Propagation Cycle [This report portion serves to document research conducted under Specific Cooperative Agreement 1230-22000-012-02S between the Floral and Nursery Plants Research Unit and the University of California-Riverside; please see that annual report for a full summary]. A novel tymovirus affecting ornamental plants in California was characterized. We have previously obtained about 30 tymovirus isolates from plants of Diascia, Verbena, Nemesia, Torenia, and Phlox from commercial nurseries in California. The California Department of Food and Agriculture (CDFA) had previously Quarantine-rated these tymoviruses as a potential threat to California agriculture. After a survey of nurseries showed that such tymovirus isolates were widespread (76% of plants tested from nine cities within five counties), the Quarantine status was lifted. We have now further examined these isolates by RT-PCR using primers specific for Nemesia ring necrosis virus (NeRNV), a tymovirus recently described in Europe. Cloning is also currently underway from tymovirus isolates that do not yield a PCR product using NeRNV-specific primers but do yield a PCR product using the Agdia “Tymovirus Group” primers; the sequence of this region shows significant differences from the NeRNV sequence. Further sequence from these isolates will allow determination of whether these isolates represent a distinct novel tymovirus, or a divergent strain of NeRNV. Angelonia flower break virus (AnFBV) was also detected of in California. AnFBV is a carmovirus recently described from Angelonia from Florida, Maryland, and Israel. A few Angelonia and Verbena plants from a California nursery were found by a commercial diagnostic lab to be infected with AnFBV. PCR products from one Angelonia and five Verbena plants were sequenced and the CA isolate from Angelonia had higher homology (96% identity) to the MD isolate than to the Israel and FL isolates (88-90% identity). The verbena isolates were more closely related to the Israel and FL isolates (average 95% identity) than the MD isolate (average 88% identity). This is the first formal detection of AnFBV in CA, and the results show that there is diversity among California isolates. CDFA has been informed of these results, and we anticipate performing a state survey to avoid imposition of Quarantine status on AnFBV in California. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. Studies on the distribution and multiplication of tobacco mosaic virus within petunia [This report portion serves to document research conducted under Specific Cooperative Agreement 1230-22000-012-03S between the Floral and Nursery Plants Research Unit and Ohio State University; please see that annual report for a full summary]. Detection and characterization of tobamoviruses from petunia. Petunia producers have previously reported frequent occurrence of tobamoviruses, especially in vegetatively propagated petunia, in which latent infections have been detected in plants previously subjected to multiple rounds of tissue culture and virus indexing. Thirteen new samples of tobamovirus-infected petunia were obtained from commercial nurseries in Ohio; twelve were symptomatic, and one asymptomatic at the time of receipt. All 13 plants were detected as infected by TMV Immunostrip assay, and by ELISA with commercially available tobamovirus antiserum and more specific TMV antiserum. Eight additional petunia cultivars exhibiting virus symptoms that were collected from variety trials on The Ohio State University campus have been tested and confirmed to be tobamovirus-positive, including one isolate that causes flower break. Additional symptomatic and asymptomatic petunia cultivars from the variety trials are being screened for presence of tobamoviruses. New tobamovirus isolates are being established in the greenhouse and are being screened in several petunia cultivars to determine if these virus isolates cause non-symptomatic infections in certain cultivars under certain conditions. The coat protein (CP) gene of each of these isolates is being cloned for sequence analysis and comparison to characterized isolates. The relationship of each isolate to TMV, Tomato mosaic virus (ToMV), Tobacco mild green mosaic virus (TMGMV), a petunia-specific tobamovirus, and other recognized tobamovirus species will be determined to aid in the design of molecular probes. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. Biology and Pathogenesis of Ralstonia solanacearum Race 3 on Geraniums [This report portion serves to document research conducted under a Specific Cooperative Agreement 1230-22000-012-08S between the Floral and Nursery Plants Research Unit and the University of Wisconsin-Madison; please see that annual report for a full summary]. Determined that the standard real time PCR diagnostic method for R. solanacearum R3b2 can give false positives with R. solanacearum strains known to be present in Florida. Following several months of testing, it was determined that the plants were actually infected with a biovar 1 strain, originally from the Caribbean but now distributed in Florida that can cause a delayed positive response in the existing real-time PCR assay. Also determined that effluent from actively and latently infected geranium plants does not invariably contain R. solanacearum cells. Preliminary experiments conducted in the field in Guatemala and more thorough experiments conducted under controlled conditions in growth chambers both found that latently infected plants containing as many as 107 cfu/gm did occasionally release effluent with no detectable R3b2 cells. These results suggest, given the zero-tolerance for this pathogen, that a method that is only 80% accurate would not form an acceptable diagnostic method for an offshore testing protocol. Completed collection and characterization of R. solanacearum 59 strains from around Guatemala; including isolates from sea level to the highlands, with emphasis on the higher altitudes where ornamental cutting production is located. All isolates were characterized using both phenotypic biovar tests and molecular analyses that allowed us to place them phylogenetically in the larger R. solanacearum species complex and to develop a map showing the distribution of the pathogen around the country. Only three distinct strains were found: Race 2-type sequevar 6 Musa strains from lowland bananas and plantains; biovar 1 sequevar 12 (Asian origin) strains from tomato, eggplant, and nightshade growing in the central plateau; and typical R3b2 strains from potatoes, geraniums, and highland screen house salad tomatoes. Completed analysis and annotation of the genomic sequence of R3B2 strain UW551 (a geranium isolate) as part of a consortium lead by Dean Gabriel (U. Florida). This research finding will provide a direct benefit to the $300 million/year geranium industry and will help safeguard the $1.2 billion/year US potato industry. This research in this accomplishment contributes to ARS National Program 303, Components I, III and IV, and ARS Strategic Plan Performance Measure 3.2.5. Ralstonia solanacearum Bacterial Wilt Host Relationships [This report portion serves to document research conducted under a Specific Cooperative Agreement 1230-22000-012-09S between the Floral and Nursery Plants Research Unit and the University of Florida; please see that annual report for a full summary]. Discovered that certain cultivars of geranium can act as asymptomatic carriers of R3B2 without any symptom expression. No symptoms were expressed in these plants over a two month period. However, large populations of the bacteria could be found in the vascular system. These cultivars could act as asymptomatic carriers of the pathogen. Previously, 61 cultivars of geraniums, including zonal, regal, ivy, and scented, were tested for resistance to Ralstonia solanacearum (Rs). R3B2 was found to infect zonal, some ivy and scented cultivars; however, it was not able to infect cultivars of regal geranium. This year we expanded this research by wound inoculating the same cultivars with lower concentrations of bacteria. We have now determined that certain cultivars of geranium can act as asymptomatic carriers of R3B2 and that certain scented cultivars appear to be immune to this pathogen. Over 125 strains of Rs collected over a seven year period from tomato, potato, pepper, pothos, anthurium and other ornamentals were genetically compared using rep-PCR primers BOX, ERIC and REP. Populations could be distinguished by biovar and to an extent country of origin and original host. Primarily because of their ability to survive and spread in temperate environments, R3B2 strains are listed by the USDA as Select Agents in the United States and are quarantined pathogens in Europe and Canada. However, to our knowledge, the relative cold survival tolerance of R3B2 strains as compared with other R. solanacearum strains has not been investigated and is unknown. Using a freeze-thaw assay, we determined that the survival of R3B2 strain UW551 is >100X higher than that of race 1 biovar 3 strain GMI1000. All R3B2 strains and some R1B1 strains were more cold tolerant than R1B3 strains by this assay. These results demonstrate that other races of Rs entering the US may be as dangerous to US agriculture as R3B2. This research finding will provide a direct benefit to the $300 million/year geranium industry and will help safeguard the $1.2 billion/year US potato industry. This research in this accomplishment contributes to ARS National Program 303, Components I, III and IV, and ARS Strategic Plan Performance Measure 3.2.5. Development of Sensitive Protocols for Detection of Bacterial Diseases in Ornamentals [This report portion serves to document research conducted under a Specific Cooperative Agreement 1230-22000-012-10S between the Floral and Nursery Plants Research Unit and Oregon State University; please see that annual report for a full summary]. Developed PCR primers for Agrobacterium tumefaciens and Rhodococcus fascians disease detection. We have devised a primer set that specifically detects the p450 gene that is required for virulence in Rhodococcus fascians and a primer set that specifically detects the virA gene required for pathogenicity in Agrobacterium. The efficacy of this assay was tested with all three primer sets and template preparations from 23 different isolates of R. fascians, 7 different isolates of pathogenic Agrobacterium, and two unrelated bacteria also isolated from leafy galls (Ochrobactrum anthropi and Stenotrophomonas maltophilia). In all cases the p450 primers specifically detected the R. fascians isolates and the virA primers specifically detected the pathogenic Agrobacterium. The fas1 set detects a region in the fas1 gene which is also required for virulence in Rhodococcus fascians. This set was tested against 24 R. fascians isolates, 28 different strains of agrobacteria, four species of Pseudomonas, and three other plant-associated bacteria from our collection. In all cases R. fascians was correctly identified; the primers did not react with DNA from bacteria that were other than R. fascians. We have also developed a set of primers in the virB region of A. tumefaciens, a gene involved in virulence. We were able to detect Rhodococcus fascians and virulent Agrobacterium in plant washes. Overall, 61 of 62 template preparations from symptomatic plants tested positive for the 16S marker (98.4% effective test). Of these 16S positive preparations, R. fascians was detected in 53 of 55 (96.4%) samples of symptomatic plants inoculated with 10 different strains of R. fascians. All samples were negative for the virA marker (diagnostic for virulent Agrobacterium). These procedures have been applied effectively to detect R. fascians in 8 different OSU Plant Clinic submissions, and to detect virulent Agrobacterium in two different OSU Plant Clinic submissions. Testing of these primer sets are continuing and are being compared to results obtained from plant inoculations and isolations. This research in this accomplishment contributes to ARS National Program 303, Components I, III and IV, and ARS Strategic Plan Performance Measure 3.2.5. Development of new, superior plant virus detection methodologies and reagents [This report serves to document research conducted under a Cooperative Research and Development Agreement between the Floral and Nursery Plants Research Unit and Agdia, Inc.; please see that annual report for a full summary]. The overall goal of this cooperative research is the development of knowledge, serological reagents, molecular primers and probes, control antigens and diagnostic technologies for the specific detection of selected key viruses infecting ornamental plants. In this first year of the agreement scientists from Agdia visited with the Floral and Nursery Research Unit (FNPRU) scientists at Beltsville, MD to discuss the scope of the research and plan the work. The Lead FNPRU Scientist visited Agdia at Elkhart, IN to continue those discussions and to give a seminar on current research. Monthly conference calls with the three FNPRU scientists and the 4-7 Agdia researchers were also held to update both parties on the current status of the projects. One of the Agdia technicians spent two weeks in the Beltsville labs learning recombinant DNA cloning and expression of His-tagged viral proteins in prokaryotic systems. Specific transfer of knowledge and reagents to Agdia included the following: purified virus preparations (for the development of polyclonal antisera); five recombinant DNA clones, from 4 different viruses, expressing viral coat proteins for potential use as positive control antigen proteins in ELISA assays (to eliminate the use of virus preparations); and, technical information on their development and use. Other cooperative research included the determination that the lack of reactivity of our broad-spectrum reacting potyvirus monoclonal antibody (previously developed by FNPRU and licensed to Agdia for commercial use) to an identified potyvirus was due to the inclusion of two additional amino acids in the antibody’s coat protein binding site. This knowledge will enable us to develop alternative protocols for better detection using this reagent. Other reagents and tools for the specific detection of selected key viruses infecting ornamental plants will be developed, transferred and implemented. This research in this accomplishment contributes to ARS National Program 303, Component I, and ARS Strategic Plan Performance Measure 3.2.5. 5.Describe the major accomplishments to date and their predicted or actual impact. A new carmovirus in Angelonia A novel carmovirus causing flower break in many cultivars of the bedding and pot plant Angelonia was purified, cloned, fully sequenced, and a partial host range established, in collaboration with scientists at USDA-ARS (Fort Pierce, FL; project "Domestic, Exotic and Emerging Diseases of Citrus, Vegetables and Ornamentals", 6618-22000-026-00D) and the Agricultural Research Organization (The Volcani Center, Bet Dagan, Israel). Several companies producing Angelonia had reported an apparently seedborne viral disease causing foliar mosaic and flower break; at least one company halted an Angelonia breeding program as a result. At Beltsville, one isolate of the new virus was purified, and parts of the viral genome including the viral coat protein gene were cloned and sequenced for comparison to Florida and Israeli isolates; the virus was found to infect several species in the Solanaceae and Scrophulariaceae; a characteristic pattern of virus-related double-stranded RNA molecules was detected in extracts from infected plants and used to aid in characterization of the viral genome. Infection of test plants was confirmed by serological tests using a) virus-specific antiserum prepared by the Israeli cooperator; b) virus-specific antiserum prepared by Agdia, Inc. from virus prepared by the Fort Pierce lab; and by a polymerase chain reaction assay using generic carmovirus primers available from Agdia. The Fort Pierce laboratory has cloned and obtained the complete genomic sequence of the Florida isolate, while the Volcani Center lab has cloned and sequenced the coat protein gene of the Israeli isolate as well as carrying out biochemical and serological analyses of the virus; comparison of the coat protein sequence of the three isolates confirms that they represent the same virus, which represents a novel species in the genus Carmovirus. The availability of reagents and assays to detect the virus, and knowledge of other plant species which are susceptible to the virus affecting Angelonia, should allow growers and breeders to identify healthy plants for propagation, and to breed superior new cultivars of this attractive and popular crop that are free from infection by this virus. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. Detection and characterization of new and emerging ornamental plant viruses. Diseases caused by viruses continue to seriously affect the production and quality of ornamental plants. New diseases, either caused by a new virus or by an existing virus on unreported crops, are being identified every year. The floral industry is further complicated by the active trade of plant material worldwide. Growers have reported problems with previously unreported viruses in several economically important ornamental crop species exhibiting virus-like symptoms. Other than natural or engineered resistance (which is rare), the prevention of disease through the development of more effective means for the detection and identification of plant virus and bacterial diseases affecting ornamentals, and utilization of those methods to allow selection of pathogen-free or pathogen-indexed plants is the best method of controlling viral diseases. Research to develop tools, reagents and knowledge that will aid U.S. floriculture companies in establishing effective virus testing protocols that will improve clean stock production for new vegetatively-propagated annuals and perennials is continuing. The initial focus is on those "new" currently uncharacterized or emerging viruses affecting key ornamental crops recently identified as significant to the floral and nursery industry. Using serological and molecular technologies (cloning and sequencing of virus genes) we have determined the identity and performed the initial characterization of several of these new and emerging viruses, and have produced reagents and tools for their detection and diagnosis. This includes: new potyviruses infecting and causing flower break in commercial New Guinea Impatiens (Impatiens flower break virus), mosaic and ringspots in toad lily Tricyrtis formosana (Tricyrtis virus Y), and, mosaic in Navelwort Omphalodes (Omphalodes virus Y); a new ilarvirus infecting Bacopa; a new carmovirus causing flower break in many cultivars of the bedding and pot plant Angelonia; two novel carlaviruses in different species of phlox (Phlox stolonifera and P. divaricata) with foliar mosaic; a new ophiovirus in commercial Lachenalia; the first report of Lolium latent virus among US ryegrass germplasm; the first report of Cucumber mosaic virus in Allamanda; and, Calla lily chlorotic spot virus and Calla lily latent virus were identified in diseased Zantedeschia plants and shown to be new, distinct Tospovirus and Potyvirus members, respectively. Other unknown or previously undescribed emerging viruses causing diseases in Amorphophallus, Bacopa, Impatiens, Lachenalia, Phlox, Scaveola, and Viola are currently under investigation. Identification of these new and emerging viruses and availability of reagents for detection will allow growers and breeders to test propagation stock in order to select healthy plants, breed new virus-free cultivars, resulting in increased productivity and quality, and customer satisfaction. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. Heterologous tospovirus proteins expressed from potyvirus vector In collaboration with scientists at the National Chung Hsing University and the Taiwan Agricultural Research Institute, Taichung, Taiwan, a plant virus vector engineered from an infectious clone of Zucchini yellow mosaic virus (ZYMV) was developed and used to express nucleocapsid proteins (NPs) of various tospoviruses, including Tomato spotted wilt virus, Impatiens necrotic spot virus, Peanut bud necrosis virus, Watermelon bud necrosis and watermelon silver mottle virus (WSMoV). The ORFs of NPs were in frame inserted in between the P1 and HC-Pro genes of the ZYMV vector. Six histidine residues and an NIa protease cleavage site were added at the C-terminal region of the inserts to facilitate purification and processing of the free form of the expressed NPs. Approximately 1.2 to 2.5 mg NPs per 100 g tissues were purified from infected leaf extracts prepared from zucchini squash. In addition, the S RNA encoding the NSs of WSMoV was successfully expressed in squash and purified from infected leaf tissues. The purified WSMoV NSs protein was used in production of rabbit antisera and mouse MAbs. Various deletions of the NSs ORF were constructed and expressed by the ZYMV vector for epitope analysis. Results indicate that MAbs target at amino acid positions 89-125 of WSMoV NSs protein. Serological analysis and sequence alignment indicate that the MAbs-targeted region of NSs protein is highly conserved among members of WSMoV. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. Detection and identification of new and emerging ornamental potyviruses Viruses infecting ornamental plants reduce vigor, and decrease foliage and flower quality. Many crops are susceptible to multiple viruses, each of which may cause serious economic losses, and infected plants may not be acceptable for sale or export. The potyvirus group contains over 200 definitive and possible members which cause significant losses in various crops. Here we report the cloning, sequencing and identity of potyviruses detected by our potyviral broad-spectrum monoclonal antibody (McAb PTY-1) in various ornamental plants exhibiting mosaic, ringspot, or flower break symptoms. Symptomatic ornamental plants that were tested positive with McAb PTY-1 were also tested using other potyvirus-specific and broad-spectrum monoclonal antibodies and by electron microscopy. Total RNA extracts served as templates for RT-PCR with potyvirus 'generic' primers which amplify highly conserved ~335, 700 or 1300-1600bp fragments from the 3' terminus of most potyviruses which includes the coat protein (CP) and 3' non-coding region (3'NCR). PTY-1 McAb immunocaptured (IC) virions were also used in IC-RT-PCR reactions. The resultant PCR amplicons were cloned, sequenced and molecularly compared to potyvirus sequences in the international databases (using bioinformatics software). Cloned CP and 3'NCR amplicons from McAb PTY-1 positive ornamental plants revealed: the identification of several new potyviruses - Impatiens flower break virus (in New Guinea Impatiens), Tricyrtis virus Y (in toad lily Tricyrtis formosana), and, Omphalodes virus Y (in Navelwort Omphalodes); the first report of Dasheen mosaic virus in Spiranthes cernua and Bean yellow mosaic virus (BYMV) in Verbena; and, previously identified re-occurring potyviruses infecting the terrestrial orchid Spiranthes cernua (Spiranthes mosaic virus 2 and Spiranthes mosaic virus 3), Crown of Thorns, Euphorbia splendens (Euphorbia ringspot virus); Amorphophallus (Dasheen mosaic virus); Daisybush, Osteospermum (Lettuce mosaic virus); George lily, Cyrtanthus (Vallota mosaic virus); and Kafir lily (BYMV). Using a broad-spectrum reacting potyvirus McAb and 'generic' potyvirus primers in IC- and RT-PCR, five new potyviruses, two emerging potyviruses and five reoccurring potyviruses infecting at least eight different ornamentals have been identified and molecularly characterized. Methods to detect and identify new and emerging potyviruses infecting ornamentals should allow growers to select healthy plants, or plants free of the most damaging viruses, in order to select and produce plants of higher quality and productivity. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. New, quick and efficient procedure for detecting the bacterial leaf scorch pathogen Xylella fastidiosa causes significant economic losses in many agriculturally important plants and many economically important landscape trees and shrubs. A quick, simple and efficient procedure for detecting Xylella fastidiosa in potential insect vectors was developed. The procedure employs a commercially available kit for the extraction of high-quality DNA from the insect, followed by one-step polymerase chain reaction amplification using previously published oligonucleotide primers specific to X. fastidiosa. The procedure does not require the use of phenol, chloroform or alcohol for the precipitation of nucleic acids. Also it does not need additional purification or enrichment steps, and can be completed in less than a day. It is well suited for testing large numbers of samples, and may also be applied to plant samples. The procedure was used successfully in detecting X. fastidiosa in two potentially important leafhopper species, Graphocephala versuta and G. coccinea, and in a treehopper species Entilia concisa, collected from a nursery where bacterial leaf scorch disease caused by X. fastidiosa occurs. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. Characterization of two new U.S. isolates of Pepino mosaic virus Improved knowledge of economically important viral pathogens such as Pepino mosaic virus (PepMV) will result in better quarantine measures to control and prevent these pathogens from entering into and/or spreading within the United States. In collaborative research between the Floral and Nursery Plants Research Unit and a commercial diagnostic company (Agdia, Inc) domestic samples of symptomatic tomato leaves from Arizona, California, Colorado, Florida, Oklahoma and Texas were found to be infected with PepMV using a potexvirus group-specific primer set in RT-PCR and in serological assays using rabbit polyclonal antiserum raised against both tomato and pepino isolates of PepMV. Complete viral genome sequences were determined from cloned cDNAs generated using group- and virus-specific primers on total RNA extracted from infected leaves, and sequence analysis indicated the presence of two unique U.S. isolates of PepMV; both of which are distinct from each other as well as from published sequences of tomato and pepper isolates of PepMV from other countries, including Peru, Spain, France and England. A third, more European-like, isolate was also detected in MD. PepMV, first described in pepino crops in Peru, is currently the cause of epidemics in tomato in Europe and is now also confirmed in North America; however, the information and tools from this research will lead to the development of superior reagents and technologies for the detection and control of this virus and the ability to produce virus-free plants. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. New method for virus-inoculation of gladiolus This new method of inoculation of gladiolus with CMV was developed using the Bio-Rad Helios Gene Gun System. This method circumvents the traditional use of aphids to transmit CMV, a virus that is mechanically transmissible to many plant species but generally only with difficulty to gladiolus. The biolistic procedure successfully transmitted three CMV isolates, two from serogroup I and one from serogroup II, to Nicotiana benthamiana leaves and to gladiolus corms and cormels. Infection rates of 100% were obtained. The technique will enable us to evaluate the resistance of previously transformed gladioli expressing CMV CP, replicase and antiviral antibody genes. This research in this accomplishment contributes to ARS National Program 303, Component V, and ARS Strategic Plan Performance Measure 3.2.5. Cucumber mosaic virus infecting Allamanda In collaboration with scientists at the Taiwan Agricultural Research Institute, Taichung, Taiwan an isolate of Cucumber mosaic virus (CMV) was identified in Allamanda cathartica showing severe mosaic, rugosity and leaf distortion symptoms. The virus measured approximately 28 nm and induces symptoms similar to those incited by CMV on indicator plants. The virus reacted with CMV subgroup I-specific MAbs in both ELISA and immunoblots. With primers specific to the 3'half of RNA3, a 1,115 bp DNA fragment was obtained by reverse transcription-polymerase chain reaction (RT-PCR) from total RNA extracted from diseased allamanda or inoculated Nicotiana benthamiana. Sequence analysis indicates that the coat protein ORF shares 91.8 to 98.9% identities with those of CMV in subgroup I and II. Results of MspI-digested restriction fragment length polymorphism patterns of the RT-PCR fragment and nucleotide sequence analysis indicate that the CMV isolate from allamanda belongs to subgroup IB. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. Characterization of Calla lily chlorotic spot tospovirus Calla lily chlorotic spot virus (CCSV) was serologically and molecularly characterized. Rabbit antisera and mouse monoclonal antibodies (MAb) were produced. The antisera produced to CCSV and to Watermelon silver mottle virus (WSMoV) reacted strongly with homologous, but weakly with heterologous antigens in ELISA and in immunoblotting. MAbs produced to CCSV and WSMoV reacted only homologously but not heterologously. The CCSV S RNA was determined to be 3,172 nucleotides in length with an inverted repeat at 5' and 3' ends, and two open reading frames (ORF) encoding a nucleocapsid protein (NP) and a non-structural (NSs) protein in an ambisense arrangement. This research was done in collaboration with scientists at the Taiwan Agricultural Research Institute, Taichung, Taiwan. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. Detection and characterization of re-emerging ornamental plant viruses Diseases caused by viruses seriously affect the production and quality of ornamental plants. The coat protein (CP) and movement protein (MP) genes of a previously described tobamovirus-like agent infecting hibiscus in Taiwan was molecularly cloned and characterized. Results show that the nucleic acid and amino acid sequences of this virus share high sequence homology with Hibiscus latent Singapore virus (HLSV) but less with Hibiscus latent Fort Pierce virus (HLFPV), indicating that the hibiscus isolates that occur in Taiwan are different strains of HLSV. A commercially-grown Euphorbia milli 'Crown of Thorns' plant exhibiting ring-shaped chlorotic spots and leaf and flower deformations tested positive for potyvirus in ELISA using our genus Potyvirus broad spectrum reacting PTY-1 monoclonal antibody. Sequence analysis of cloned RT-PCR amplicons revealed potyviral similarities but confirm the classification of this poorly described virus as a new potyvirus member. Since no antisera against this virus is available, synthetic peptides and bacterially-expressed His-tagged cloned CP from this virus are under evaluation as sources of antigen for EuRSV-specific antisera production for future use in diagnosis. Chrysanthemum virus B has been serologically detected in a new host, Argyranthemum. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. First report of bacterial leaf scorch pathogen in Japanese beech bonsai Xylella fastidiosa causes significant economic losses in many agriculturally important plants and many economically important landscape trees and shrubs. In collaboration with scientists in the Fruit Lab at Beltsville, Xylella fastidiosa was found to be associated with a leaf scorch disorder in a 76-year-old Japanese beech bonsai. This is the first time that X. fastidiosa has been found associated with the high-valued bonsai and with beech tree. Our work will be an important step leading to the control of the disorder and preservation of the horticultural masterpiece. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. Genetic relationships among alternate host strains of the bacterial leaf scorch pathogen Genetic relationships between strains of X. fastidiosa isolated from economically important hosts and alternative hosts are currently unknown and greatly needed for the development of control strategies for diseases caused by the pathogen. In order to determine how X. fastidiosa strains from alternative hosts are related genetically to other hosts, we first isolated X. fastidiosa from several alternative hosts including porcelain berry and wild grape, in collaboration with the National Park Service at Washington, DC. We then determined their genetic relationships with each other and with strains from grape, peach, plum, oak, mulberry, maple and oleander. We found for the first time that these alternative host strains of X. fastidiosa are more closely related to the oak strain. Our study suggests that suppression of these plants may be important for the control of the disease caused by X. fastidiosa in oak. This research in this accomplishment contributes to ARS National Program 303, Components I and IV, and ARS Strategic Plan Performance Measure 3.2.5. Alternanthera mosaic virus in commercial phlox cultivars A potexvirus infecting creeping phlox from a commercial nursery in Pennsylvania was identified as Alternanthera mosaic virus (AltMV), previously reported only from a weedy species of Alternanthera from Australia. The complete 6607nt genomic sequence of the phlox isolate of AltMV was determined. Additional isolates of AltMV were detected in two cultivars of phlox and from trailing Portulaca plants from other commercial nurseries in Maryland; the 3'-terminal region of the genomes of these isolates was also determined and compared to the Pennsylvania and Australian isolates. This research in this accomplishment contributes to ARS National Program 303, Components I and IV and ARS Strategic Plan Performance Measure 3.2.5. New and re-emerging viruses infecting Ornithogalum and Lachenalia Productivity and quality of the bulbous ornamentals Ornithogalum and Lachenalia is reduced by potyvirus infections. The coat protein gene and 3' untranslated region of several potyvirus isolates from these crops were cloned and sequenced by the Floral and Nursery Plants Research Unit in order to determine which viruses were present, and to allow development of reagents for detection. The sequences of cloned cDNA of potyvirus isolates from several lines of Ornithogalum and Lachenalia revealed three sequence types that correspond to Ornithogalum mosaic virus and two other distinct types that are similar to viruses recently reported from Ornithogalum in Japan. Developing reagents for detection and differentiation of these potyviruses will allow growers to select healthy material for propagation; it may also be possible to use these sequences to introduce virus resistance into these crops, in which no natural resistance has been identified. This research in this accomplishment contributes to ARS National Program 303, Components I and IV and ARS Strategic Plan Performance Measure 3.2.5. 6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? A display booth describing the programs of the National Arboretum, and research of the Floral and Nursery Crops Research Unit, was presented at the Mid-Atlantic Nursery Trade Show in Baltimore (January, 2006). A Floral and Nursery Crops Research Unit ‘Open House’ for the Green Industry was held at the U.S. National Arboretum, May 2006. Research was featured at a Maryland Technology Showcase designed to connect ARS research with stakeholders who might be interested in commercial applications. The successful 20-year partnership between FNPRU virologists and the company Agdia, which has resulted in the development of diagnostic plant virus test kits used throughout the world, was highlighted. The event was organized by ARS’s Office of Technology Transfer, the Maryland Technology Development Corporation, and the Tech Council of Maryland. A poster entitled “Evaluating non-chemical alternatives to control fungal and bacterial plant pathogens” was also presented. A new Trust Fund Agreement entitled “Development of improved pathogen detection methodologies and reagents based on PCR and hybridization technologies” and a new CRADA entitled "Development of new, superior plant virus detection methodologies and reagents" were signed with Agdia, Inc. Seminars and workshop-labs entitled "Biotechnology and Plant Viruses" were presented to the 'Biotechnology Junior Practicum' Applied Research Labs, Columbia, MD, November, 2005 and February-May, 2006. Participated in multiple conference calls with other plant virus researchers and ornamentals industry collaborators, in order to discuss progress made on viruses affecting ornamental crops. Participated in the National Floriculture Forum, met with other ornamental plant researchers and several growers; discussed viral and other disease problems and other issues relating to ornamental crops. Contacted by, and provided information to, Canadian, New Zealand, and U.S. scientists interested in Pepino mosaic virus. Continue to advise agricultural diagnostics company on the use of FNPRU-generated and supplied immunological reagents (polyclonal and monoclonal antibodies) and technologies. Prepared and submitted summaries of research and participated in discussions on viruses of ornamentals for the 'Ornamentals Virus Discussion Group' of other researchers and industry personnel at the annual American Phytopathological Society meeting (July, 2006). Participated in, and made oral and poster presentations on FNPRU and collaborative research on viruses and bacterial diseases affecting ornamental crops at the second Floral and Nursery Crops Researchers Workshop, and discussed results and prospective future research areas with other researchers and floral and nursery industry representatives. Responded to many industry inquiries and concerns about Pansy Mottle Syndrome and the newly discovered Pansy ilarvirus, including meetings and calls with grower representatives from the United Kingdom and the Netherlands. Responded to various nursery industry queries about specific virus problems affecting ornamental crops. Invited to attend, and participated in an Indo-US Interactive Workshop on Plant Molecular Virology, New Delhi, India (February 11-12, 2006). Deposited, released and/or updated viral, bacterial, and mouse antibody gene fragment nucleotide sequences at GenBank (National Center for Biotechnology Information, National Institute of Health) with the following accession numbers (in numerical order), making them available to the global research community free of charge: - AY864849 [Lily virus X, strain TF, 3' terminal genome portion (3010 bp)] - AY864851 [Impatiens flower break virus, 3' terminal genome portion (6543 bp)] - DQ221212 [Angelonia flower break virus, isolate AnFBV-MD, coat protein gene (1096bp)] - DQ333886 [Lolium latent virus, isolate LLV-UK, partial RdRp gene (1118bp)] - DQ355837 [Ryegrass mosaic virus, isolate RGMV-US1, partial NIb gene (175bp)] - DQ355838 [Ryegrass mosaic virus, isolate RGMV-US2, partial NIb gene (176bp)] - DQ523597 [Impatiens necrotic spot virus, isolate INSV-B, nucleocapsid gene (957 bp)] - DQ523598 [Impatiens necrotic spot virus, isolate INSV-Igg, nucleocapsid gene (1003 bp)] - DQ523599 [Tomato spotted wilt virus, isolate TSWV-L, nucleocapsid gene (896 bp)] - NZ_AAKL00000000 [Ralstonia solanecearum UW551 race 3 biovar 2, 8x genome draft (5,895,318 bp)] 7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Participated in the Floral and Nursery Crops Research Unit ‘Open House’ for the Green Industry, May 9, 2006 in Washington DC. Presented “Overview of ornamentals research at the U.S. National Arboretum” and interacted with stakeholders (nurserymen, urban foresters, gardening public). Attended the Second Floral and Nursery Crops Researchers Workshop held June 12-15, 2006 in Portland, OR, and presented talks and posters entitled: “Detection and characterization of new and emerging viruses affecting ornamental crops”, “Detection and characterization of novel ilarviruses from Bacopa and Pansy”, “New viruses affecting Phlox, Portulacca, Angelonia and Lachenalia”. Interacted with stakeholders and other researchers; and, visited several floral and woody ornamental nurseries in the area. Attended the American Phytopathological Society annual meeting held July 29-August 2, 2006 in Quebec City, Canada, and presented three papers entitled: “Molecular characterization of several new and emerging potyviruses of ornamental plants”; “Partial genomic sequence and characterization of a novel carlavirus isolated from Phlox divaricata”; and, “Molecular and biological characterization of a novel ilarvirus in Bacopa”. Presented an invited seminar “Detection and identification of novel viruses affecting ornamental crops” at the Volcani Center, Bet Dagan, Israel, to Israeli researchers and growers of ornamentals (April, 2006). Presented an invited seminar “Detection and identification of new and emerging viruses affecting ornamental crops” to researchers and diagnosticians at Agdia, Inc (July, 2006). Review Publications Huang, Q., Bentz, J. and Sherald, J.L. 2006. Fast, easy and effecient DNA extraction and one-step PCR for the detection of Xyllela fastidiosa in potential insect vectors. 88:77-81. Chen, T.C., Hsu, H.T., Jain, R.K., Huang, C.W., Lin, C.H., Liu, F.L., and Yeh, S.D. 2005. Purification and serological analyses of tospoviral nucleocapsid proteins expressed by Zucchini yellow mosaic virus vector in squash. Journal of Virological Methods. 129:113-124. Hammond, J., Reinsel, M.D., Maroon-Lango. 2006. Identification and full sequence of an isolate of Alternanthera mosaic potexvirus infecting Phlox stolonifera. Archives of Virology. 151:477-493. Adkins, S.T., Hammond, J., Gera, A., Maroon Lango, C.J., Sobolev, I., Harness, A., Zeidan, M., Spiegel, S. 2006. Biological and molecular characterization of a novel carmovirus isolated from angelonia. Phytopathology. 96:460-467. Jordan, R.L., Guaragna, M.A. 2006. Molecular characterization of several new and emerging potyviruses of ornamental plants. Phytopathology. 96:S56. Hammond, J., Reinsel, M.D. 2006. Partial genomic sequence and characterization of a novel carlavirus isolated from Phlox divaricata. Phytopathology. 96:S45. Maroon-Lango, C.J., Aebig, J., Hammond, J., Hsu, H.T. 2006. Molecular and biological characterization of a novel ilarvirus in bacopa. Phytopathology. 96:S73.