2008 Annual Report 1a.Objectives (from AD-416) The overall goal of this project is to provide the means for growers to 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. 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. 1b.Approach (from AD-416) Characterize viruses of major significance to ornamental and nursery crops, including "new" currently uncharacterized or emerging viruses affecting key ornamental crops. The overall approach is to develop knowledge, tools, and reagents 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. Research will initially focus on those "new" currently uncharacterized or emerging viruses affecting key ornamental crops recently identified as significant to the floral and nursery industry, including those infecting petunia, impatiens, phlox and pansy. Viruses of serious consequence identified as significant to the floral and nursery industry in key ornamental crops also include, but are not limited to: Arabis mosaic virus and Cucumber mosaic virus, carlaviruses, carmoviruses, closteroviruses, fabaviruses, ilarviruses, pelarspoviruses, potexviruses, potyviruses, and tobamoviruses. Based on the knowledge and serological reagents, and molecular probes developed for the detection and management of diseases caused by the viruses characterized above, new virus-specific and broad-spectrum polyclonal and/or monoclonal antibody reagents, purification protocols, molecular nucleic acid hybridization probes, PCR primers, and improved associated protocols and diagnostic technologies will be developed. 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. Understanding viral genome structures and functions, the mechanisms of pathogenicity, and the mechanisms of resistance in plants will lead to the development of better viral disease control measures and increases in both productivity and quality of ornamental plants. Develop improved tools and evaluate methodologies for the identification, detection, and control of bacterial diseases of major significance to woody and floral ornamental crops. Conduct research on the host range and environmentally friendly control of bacterial wilt disease of geranium caused by the select agent pathogen Ralstonia solanacearum race 3, biovar 2. Conduct research on the identification and detection of Xylella fastidiosa in ornamental crops, study the genetic relationships among strains of X. fastidiosa, and determine the pathogenetic relationships between ornamental and non-ornamental strains of X. fastidiosa. 3.Progress Report Novel geranium viruses. Research continues on understanding the molecular biology and pathogenicity of several unique geranium viruses. Cloned and bacterially-expressed viral proteins and synthetic peptides have been produced and are being used as immunogens for antisera production for use as both diagnostic reagents and as tools to confirm the novel protein translation strategy predicted for these viruses. The development of full length infectious cDNA clones of Pelargonium line pattern virus and Pelargonium chlorotic ring pattern virus continues and the infectivity of RNA transcripts are currently under evaluation. This research contributes to ARS National Program 303, Component 1, Problem Statement 1B, and Component 2, Problem Statement 2A. Susceptibility of 4 ornamental species to Ralstonia solanacearum race 3, biovar 2. The four ornamental species, snapdragon, verbena, viola and Dusty Miller were tested in growth chambers by soil drenching method for their susceptibility to R. solanacearum race 3, biovar 2. Disease symptoms were not observed in the inoculated plants, and the bacterium was not detected by dilution plating and PCR from main stems of the inoculated plants. This research contributes to ARS National Program 303, Component 2, Problem Statement 2A. Evaluation of pathogenic relationships among ornamental and non-ornamental strains and specific detection and identification of Xylella fastidiosa. Grape, almond, plum, oak and maple strains of X. fastidiosa were mechanically inoculated to oleander plants to determine if those ornamental and non-ornamental strains can infect and cause disease symptoms in this host. The inoculated plants are being tested every two months for symptom development and the presence of X. fastidiosa is being determined by PCR. In a separate study, several sets of RAPD-PCR primer pairs specific to oleander strains of X. fastidiosa have been designed based on general PCR results and their specificity is under evaluation. This research contributes to ARS National Program 303, Component 1, Problem Statement 1B, and Component 2, Problem Statement 2A. Cooperative Agreements. Summaries to document research conducted under Specific Cooperative Agreements between the Floral and Nursery Plants Research Unit and others can be found in the following specific annual reports: ‘Studies on the distribution and multiplication of tobacco mosaic virus and other tobamoviruses within petunia’ with Ohio State University(#1230-22000-022-03S); ‘Biology and Pathogenesis of Ralstonia solanacearum Race 3 on Geraniums’ with the University of Wisconsin-Madison(#1230-22000-022-08S); ‘Ralstonia solanacearum Bacterial Wilt Host Relationships’ with the University of Florida(#1230-22000-022-09S); ‘Development of Sensitive Protocols for Detection of Bacterial Diseases in Ornamentals’ with Oregon State University(#1230-22000-022-10S); ‘Development of new, superior plant virus detection methodologies and reagents' with Agdia, Inc(#1230-22000-022-11T); and 'Sensitive detection of single and mixed infections in ornamental plants at all stages of the propagation cycle' with the University of California, Riverside(#1230-22000-022-12S). 4.Accomplishments 1. Analysis of infectious clones of Lolium latent virus and Alternanthera mosaic virus. Determination of virus:host interactions aids in understanding of virus infection processes and potential means of preventing virus infections and the economic damage that they cause. We previously developed infectious cDNA clones of LoLV-US1, (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). Directed mutagenesis of these clones has led to the identification of coat protein sequences necessary for systemic infection of Nicotiana benthamiana. We have also previously produced multiple infectious clones from a phlox isolate of AltMV, which differ in symptom severity in N. benthamiana. Gene exchange between these clones, and with clones of other isolates differing in host range and symptom expression, resulted in identification of replication level and symptom determinants of AltMV. Through directed mutagenesis, the Triple Gene Block (TGB) 3 (TGB3) protein has been shown to be dispensable for infection, but necessary for systemic movement. Laser scanning confocal microscopy was used to determine subcellular localization of AltMV proteins fused to the green fluorescent protein, expressed in leaves of N. benthamiana following agrobacterium-infiltration of the fusion constructs. Co-expression of different proteins demonstrated interactions between some of the viral proteins that affected subcellular localization. Interactions between AltMV proteins was also demonstrated by use of the yeast two hybrid expression system; in this system the TGB1 protein was shown to interact with itself and also with the viral polymerase; the coat protein was found to react only with itself. The research in this accomplishment contributes to ARS National Program 303, Component 1, Problem Statement 1B, and Component 2, Problem Statement 2A. 2. Detection of additional mixed virus infections in phlox. Phlox species in cultivation are propagated vegetatively, and many plants are infected by multiple viruses; virus infection causes reduction of both quality and productivity. We have previously detected a novel carlavirus, Phlox virus M (PhlVM), in hybrid annual phlox, and others have detected Angelonia flower break virus (AnFBV) in hybrid phlox. We have recently found PhlVM in mixed infection with AnFBV in hybrid phlox from commercial production; the mixed infection is more severe than either virus alone, causing severe mosaic and plant decline. Both viruses can be detected by virus-specific polymerase chain reaction assays, and AnFBV can be detected by ELISA with virus-specific antiserum. 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. The research in this accomplishment contributes to ARS National Program 303, Component 1, Problem Statement 1B, and Component 2, Problem Statement 2A, and ARS Strategic Plan Performance Measure 3.2.5. 3. Identification of Freesia sneak ophiovirus from Freesia We have previously reported detection of an ophiovirus in commercial Lachenalia from South Africa, which was identified as an isolate of Freesia sneak virus (FreSV) previously reported from freesia in Italy. In collaboration with a visiting scientist from the Istituto di Virologia Vegetale (IVV, CNR, Turin, Italy), we have now identified FreSV in a commercial freesia crop from Virginia showing symptoms of leaf necrosis. This is the first detection of FreSV in freesia in the United States. We are comparing the coat protein sequence of the Virginia freesia isolate to those of the South African Lachenalia isolate and the Italian freesia isolate. The research in this accomplishment contributes to ARS National Program 303, Component 1, Problem Statement 1B, and Component 2, Problem Statement 2A, and ARS Strategic Plan Performance Measure 3.2.5. 4. Development of a Real-Time RT-PCR assay for the detection of Cucumber mosaic virus. Cucumber mosaic virus (CMV) infects over 800 species in 85 families, has a worldwide distribution, and is considered as one of the most important viruses infecting horticultural and ornamental crops. A number of diverse strains of CMV have been reported and characterized and can be divided into two main subgroups by biological, serological properties and nucleotide sequence homologies. Although serological and biological methods for the detection of CMV are available, they may not offer the sensitivity for reliable detection of low levels of CMV in early infections and/or in asymptomatic plants serving as reservoirs for transmission to symptomatic crops. In addition, a sensitive, quantitative nucleic acid-based method is needed to determine the expression levels of virus-related transgenes in genetically-modified plants. We have developed a highly-sensitive, quantitative Real-Time Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) assay that can detect genetically diverse CMV strains and virus transgenes in genetically-engineered gladiolus. Primers include those to viral replicase or coat protein genes. Variations of the method use either the green fluorescent dye SYBR Green I or a single conserved TaqMan probe to determine the absolute (fg level) and relative copies of CMV genomic RNAs or transgenes contained in total RNA extracts from infected or transgenic plant tissues or purified virions. The sensitive and accurate quantification property of this qRT-PCR assay could be useful to monitor viral replication kinetics, the evaluation of viral tolerance levels in new breeding programs and to detect and identify CMV to help growers select healthy plants, or plants free of this most damaging virus, in order to produce plants of higher quality and productivity. This accomplishment contributes to ARS National Program 303, Component 1, Problem Statement 1A. 5. Complete sequence and characterization of a new potyvirus causing flower break in New Guinea Impatiens. New Guinea Impatiens (NGI) form a large part of the floral trade in ornamental plants, especially in the area of bedding and garden plants. NGI are generally vegetatively propagated and are popular as a spring ornamental because of the bright array of colors and large-flowered cultivars available. We previously reported the detection and partial characterization of a potyvirus from NGI plants (obtained from local commercial nurseries) showing virus-like color breaking symptoms on flower petals which tested positive for potyvirus in ELISA using our genus Potyvirus broad-spectrum reacting monoclonal antibody. Potyviruses collectively infect almost every major crop throughout the world, and in many crops a potyvirus may be the economically important viral pathogen. 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 9593nt viral genome (AY864851), and pair-wise 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 IFBV-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 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 accomplishment contributes to ARS National Program 303, Component 1, Problem Statement 1B, and Component 2, Problem Statement 2A. 6. Effective control of Ralstonia solanacearum bacterial wilt of tomato and geranium with clove oil. Bacterial diseases often result in significant losses in the production and quality of ornamental crops, and are very difficult to control. In collaboration with another FNPRU scientist (Project 1230-22000-019-00D), the effect of clove oil against bacterial wilt of tomato and geranium caused by R. solanacearum race 1, biovar 1 under greenhouse conditions was evaluated. Results showed that pre-plant fumigation of soil-less potting mix with a clove oil formulation effectively controlled bacterial wilt of tomato and geranium by preventing symptom formation and bacterial colonization of the host plants. Research is continuing on fine tuning pre-plant fumigation protocols to eliminate any phytotoxic effects clove oil may have on tested plants without affecting its effectiveness against R. solanacearum. The discovery that clove oil is effective on controlling bacterial wilt of tomato and geranium under greenhouse conditions suggests that clove oil has the potential to be an alternative control measure to combat bacterial wilt of tomato and geranium caused by R. solanacearum. This research is under National Program 303, Component 4, Problem Area 4a, ‘Biological and Cultural Control Technologies’, and Performance Measure 3.2.6. 6.Technology Transfer Number of Active CRADAs 1 Number of the New MTA (providing only) 2 Number of New Commercial Licenses Executed 2 Review Publications Huang, Q. 2007. Natural occurances of Xyella fastidiosa in a commercial nursery in Maryland. Canadian Journal of Plant Pathology. 29:299-303. Lim, H-S., Bragg, J.N., Ganesan, U., Lawrence, D.M., Yu, J., Isogai, M., Hammond, J., and Jackson, A.O. 2008. Triple gene block protein interactions involved in movement of Barley stripe mosaic virus. Journal of Virology. 82:4991-5006. Vaira, A.M., Maroon-Lango, C.J., and Hammond, J. 2008. Molecular characterization of Lolium latent virus, proposed type member of a new genus in the family Flexiviridae. Archives of Virology. 153:1263-1270. Jordan, R.L., Guaragna, M.A., Van Buren, T., and Putnam, M.L. 2008. First report of a new potyvirus, Tricyrtis virus Y, and Lily virus X Potexvirus, in Tricyrtis formosana in the United States. Plant Disease. 92:648. Kamo, K., Jordan, R.L., Hsu, H-T., and Hu, J. 2008. Antibodies in plants. Floriculture, Ornamental and Plant Biotechnology. 5:311-318. Huang, Q., Hartung, J.S. 2007. Construction of infectious clones for double stranded dna viruses of plants using citrus yellow mosaic cirus as an example. Methods in Molecular Biology. 451:525-534. Jordan, R., Hammond, J. 2008. Bean common mosaic virus and bean common mosaic necrosis virus. Encyclopedia of Virology, 3rd Ed. (B.W.J. Mahy and M.H.V. Van Regenmortel, Eds). 1:288-295.