Progress 04/09/07 to 04/08/12
Outputs
Progress Report 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. 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. This is the final report for project 1230-22000-022-00D which terminated April 2012. It was replaced with project 1230-22000-032-00D, entitled �New and Emerging Viral and Bacterial Diseases of Ornamental Plants: Detection, Identification, and Characterization�. Substantial results for each objective were realized over the 5 years of the project. Objective 1: Detection, identification and characterization of new and emerging viruses of ornamental plants. Using serological and molecular technologies, we have determined the identity of and characterized more than ten new and eight emerging viruses. We have partially or completely determined their genome sequences and have produced reagents and tools for their detection and diagnosis. The reagents and knowledge developed aid U.S. floriculture companies in establishing effective virus testing protocols that will improve clean stock production for new vegetatively- propagated annuals and perennials resulting in increased productivity and quality, and customer satisfaction. These reagents and tools should also enable state and federal regulatory officials to make timely and appropriate recommendations in safeguarding the movement of horticultural and agricultural products into and throughout the U.S. Objective 2: Genomic characterization and development of full-length infectious clones of selected viruses. We characterized and developed full-length cDNA clones of eight different viruses and developed infectious clones from 4 of them, including three Pelarspoviruses and the potexvirus Alternanthera mosaic virus, which has been engineered as an efficient vector for protein expression or Virus-Induced Gene Silencing in wide range of plant taxa. Understanding viral genome structures and functions, their mechanisms of pathogenicity and resistance, coupled with an increased knowledge of host interactions affecting viral replication and movement, should lead to the development of better disease control measures and increases in both productivity and quality of ornamental plants for industry and the consumer. Objective 3: Develop improved tools and evaluate methodologies for the identification, detection, and control of bacterial diseases of major significance to woody and floral ornamental crops. The development of a new, simple, fast and sensitive PCR assay for the detection and identification of Xylella fastidiosa (Xf) strains causing oleander leaf scorch disease has enabled further studies on the spread and presence of this strain in plant hosts and insect vectors. Our findings of new ornamental hosts of Xf, as well as our findings that red oak and box elder in the area of the nursery are infected with high numbers of Xf, will make the industry aware of the disease not just in their stocks, but also in the surrounding vegetation. An improved biovar differentiation culture test for Ralstonia solanacearum (RSr3b2) and a better understanding of the susceptibility of various ornamental species to RSr32b will help state and federal regulatory officials to make timely decisions to prevent and exclude the select agent pathogen from becoming established in the U.S. Accomplishments 01 Detection of interactions between viral proteins and host proteins. Viru infection causes significant losses in many crops, including ornamental plants. In collaboration with a colleague at Chungnam National Universit Korea, ARS scientists in Beltsville, MD have confirmed and characterized the interactions between several viral proteins and host plant proteins. Mutations of these viral proteins were used to determine how specific viral/host plant protein interactions affect disease symptom expression. One key finding indicated that the viral coat protein is the elicitor of systemic necrosis at low temperature. The results of this work should be especially beneficial to scientists examining other plant virus-host interactions, including those involved in viral pathogenesis, and may al be of value to plant breeders seeking to introduce plant virus resistanc
Impacts
(N/A)
Publications
- Hammond, J., Jordan, R.L., Vaira, A. 2011. Virus diseases of ornamentals. Encyclopedia of Life Support Systems. Available:
- Vaira, A., Lim, H., Bauchan, G.R., Owens, R.A., Dienelt, M.M., Reinsel, M. D., Hammond, J., Natilla, A. 2012. Lolium latent virus (Alphaflexiviridae) coat proteins: expression and functions in infected plant tissue. Journal of General Virology. 93:1814-1824.
- Valverde, R.A., Sabanadzovic, S., Hammond, J. 2012. Viruses that enhance the aethetics of some ornamental plants: beauty or beast?. Plant Disease. 96(5):600-611.
- Henderson, D.C., Hammond, J. 2012. CKC: isolation of necleic acids from a diversity of plants using CTAB and slica columns. Molecular Biotechnology. DOI: 10.1007/s12033-012-9494-y.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report 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. 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. Surveying commercial nurseries to determine the occurrence of viruses infecting phlox. Additional samples of various species of phlox have been obtained from several nurseries. Plants were tested by bioassay, ELISA, electron microscopy, and/or RT-PCR for various viruses known to infect phlox. Samples were tested by ELISA for six specific viruses and four groups of viruses; testing by RT-PCR for ten types of viruses is in progress. Alternanthera mosaic virus, Cucumber mosaic virus, and potyviruses were all detected by ELISA in multiple varieties and species of phlox. Phlox virus S, Ligustrum necrotic ringspot virus, and Helenium virus S were each detected from one or more phlox samples. Detection of interactions between viral proteins and host proteins. We have utilized yeast two-hybrid methods to identify interactions between viral proteins (Alternanthera mosaic virus coat protein, triple gene block 1 protein, or triple gene block 3 protein; Lolium latent virus coat protein) and host proteins from Nicotiana benthamiana or Arabidopsis thaliana cDNA libraries. The functions of some of the host proteins identified indicate potential involvement necessary for viral movement. The structural and movement-related proteins from two Pelarspoviruses, Pelargonium chlorotic ring pattern virus and Pelargonium line pattern virus, were sub-cloned into bacterial expression vectors. These proteins will be used as immunogens for polyclonal antisera production for use as both diagnostic reagents and as tools to confirm their novel viral protein translation strategies and to determine host interactions. Selection of single chain variable fragments (scFv) against Xylella fastidiosa subsp. pauca citrus strain 9a5c (Xf-9a5c) and Candidatus Liberibacter asiaticus (CaLas) by phage display. In a collaborative project with scientists in the Molecular Plant Pathology Lab, Beltsville, MD, we have made two separate libraries of scFv antibody fragments to Xf- 9a5c and CaLas. Some of the Xf-9a5c scFv antibodies recognized strain 9a5c but did not recognize Xf strains that cause Pierce�s disease of grapevine. ELISA data show that several different CaLas scFvs exhibited specificity for different proteins; including the enzyme producing polysialic acid capsule polysialic acid, a component of a type IV pilus, and the major outer membrane protein. Specific detection and identification of X. fastidiosa in woody ornamentals. We inoculated periwinkle plants with mulberry strains of X. fastidiosa and found that the bacterium can be detected by PCR and ELISA, as well as re-isolated from the periwinkle plants 5 months after inoculation. Accomplishments 01 Development of an improved method for extraction of total nucleic acids from diverse plant species. The best strategy to control plant pathogens is rapid identification and detection in quarantine, breeding programs, certification and production. Molecular techniques such as polymerase chain reaction (PCR)-based tests and newer microarray-based tests can be very fast and sensitive in detecting genetic materials (DNA or RNA) of these pathogens. A significant challenge in using these techniques, however, is the ability to prepare plant samples or extracts containing these genetic materials but free of other compounds that occur in plant cells, such as proteins and sugars, that can interfere with the detectio technique. ARS scientists in Beltsville, MD have developed a method to extract total nucleic acids from a wide variety of plant material. This method, named the �CKC� to signify the sequential use of CTAB, potassium acetate (KOAc), and column purification, was optimized for speed and nucleic acid purity in order to facilitate sample throughput. High quali RNA could be purified from all 60 plant species tested, as confirmed by amplification of specific host genes, or, for some virus-infected hosts, amplification of plant virus sequences. This technology will be useful t those who work in quarantine and certification programs that need to tes valuable plant material for pathogens by a variety of methods. The metho will also be useful for nucleic acid preparation for research applicatio including microarray analysis of gene expression from diverse plants. 02 Development of transgenic Gladiolus plants resistant to Cucumber mosaic virus (CMV). CMV is one of the most important plant viruses because it infects about 1000 plant species, including food crops and ornamentals. Infection of various flower bulb crops with CMV results in dramatic streaking of the flowers making the flowers unmarketable, and infected plants have decreased vigor resulting in a poor bulb yield. In order to develop CMV-resistant lines, ARS scientists in Beltsville, MD developed transgenic plants of Gladiolus �Peter Pears� and �Jenny Lee� that contai single-chain variable fragment antibodies (scFv) to CMV subgroup I or II isolates. When challenged with CMV, less than 30% of the plants from two plant lines with the CMV subgroup I scFv and three plant lines with the CMV subgroup II scFv were resistant as compared to non-transformed contr plants, which shows that this technology can provide some level of resistance to this pathogen. This work will facilitate the evaluation of virus resistance in transgenic Gladiolus plants to yield improved floral quality and productivity. 03 Development of genome-based diagnostic markers to detect and differentia strains of Xylella fastidiosa. X. fastidiosa causes many economically important plant diseases including Pierce�s disease of grapevine and citrus variegated chlorosis. It also causes bacterial leaf scorch of forest and/or landscape trees and shrubs such as oak, elm, mulberry, sycamore and oleander. ARS researchers in Beltsville, MD used comparati genomics to analyze publicly available genomic sequences of X. fastidios to identify highly specific diagnostic markers for target strains of X. fastidiosa and to design species-specific primers. Two primer pairs were designed that are specific to the oleander strain of X. fastidiosa; two primer pairs were specific only to the CVC strains; and one primer pair can be used to detect all strains of X. fastidiosa tested, but not other closely related bacterial species. These primers can also be used in multiplex PCR assays. This work will enable researchers to detect and differentiate strains/subspecies of X. fasitidiosa for use in epidemiological studies and in diagnosis and management of diseases caus by X. fastidiosa.
Impacts
(N/A)
Publications
- Huang, Q., Lakshman, D.K. 2011. Effect of clove oil on plant pathogenic bacteria and bacterial wilt of tomato and geranium. Journal of Plant Pathology. 92(3):701-707.
- Hammond, J., Reinsel, M.D. 2011. Mixed infection and novel viruses in various species of Phlox. Acta Horticulture Proceedings. 901:119-126.
- Hammond, J. 2011. Universal plant virus microarrays, broad spectrum PCR assays, and other tools for virus detection and identification. Acta Horticulture Proceedings. 901:49-60.
- Jordan, R.L., Guaragna, M.A., Putnam, M. 2011. Detection and molecular characterizations of new and emerging potyviruses of ornamental plants. Acta Horticulturae. 901:159-166.
- Radwan, O., Liu, Y., Clough, S.J. 2011. Transcriptional analysis of soybean roots response to Fusarium virguliforme, the causal agent of sudden death syndrome. Molecular Plant-Microbe Interactions. 24:958-972.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report 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. 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. Production of reagents for specific detection of new viruses that periodically emerge among ornamental crops aids growers in selection and production of healthy crops. Phlox virus M (PhlVM)-specific PCR primers have been produced. The PhlVM coat protein has been subcloned into a bacterial expression vector as a means of obtaining immunogen for antiserum preparation. Bacopa chlorosis virus (BaCV) is also difficult to purify; we have also subcloned the BaCV coat protein for a bacterial expression for antiserum production. Callicarpa americana (American beautyberry) plants with strong mosaic, and variegated C. dichotoma showing a mild mosaic were examined by electron microscopy. In both sets of plants, a novel type of virus-like particle was observed, with particles of variable length (100-600 nm) and 22-24 nm in width, with rounded ends. Purified virus particles will be used to generate virus-specific sequence and/or antiserum for virus detection and identification. Dwarf carnation plants infected with the Australian Carnation necrotic fleck virus (CNFV) isolate KF, were found to contain at least two viruses. The two genomes were cloned, sequenced in their entirety and in phylogenetic analyses demonstrated to represent two distinct closteroviruses. We propose to name one CNFV (14,825nt genome) and the second Carnation yellow fleck virus (CYFV; 15,602nt genome). Monoclonal antibodies and recombinant scFv antibodies were developed against Candidatus Liberibacter asiaticus, a non-culturable member of the alpha-proteobacteria consistently associated with huanglongbing (HLB) disease. A library of scFv antibodies was developed against Xylella fastidiosa subsp pauca strain 9a5c (causes citrus variegated chlorosis in Brazil and has �select agent� status in the United States) using phage display technology. ARS scientists at Beltsville, MD tested verbena �Quartz Mix XP�, ageratum �Hawaii Royal�, Phaseolus vulgaris, and beet in growth chambers by soil drenching method for their susceptibility to R. solanacearum race3, biovar 2. Improved standard biovar tests for Ralstonia solanacearum have been developed by ARS scientists at Beltsville, MD, that allow fast, unambiguous and economical differentiation of biovars of Ralstonia solanacearum. Grape, almond, plum, oak and maple strains of X. fastidiosa were used to mechanically inoculate oleander plants to determine if those ornamental and non-ornamental strains can infect and cause disease symptoms in oleander plants. Inoculated plants were tested at regular intervals for symptom development and presence of X. fastidiosa by Polymerase chain reaction (PCR). Summaries to document research conducted under Specific Cooperative Agreements or Reimbursable Agreements between the Floral and Nursery Plants Research Unit and others can be found in the following specific annual reports: Oregon State Univ. (#1230-22000-022-10S); Agdia, Inc (- 11T); Univ. of California, Riverside (-12S); Univ. of Wisconsin-Madison (- 13S); Univ. of Florida (-14S); Univ. of Utah (-15R, and -16S); Donald Danforth Plant Science Center (-17S); Washington Univ. (-18S); Oklahoma State Univ. (-19S); and, Cornell Univ. (-20S). Accomplishments 01 Development of variants of Alternanthera mosaic virus as efficient vecto for protein expression or Virus-Induced Gene Silencing (VIGS). Alternanthera mosaic virus (AltMV) infects a number of ornamental crops diverse taxonomic groups, including species of Alternanthera, Phlox, Portulaca, Angelonia, Torenia, Crossandra, Scutellaria, and Nandina; as well as the model species Nicotiana benthamiana and Arabidopsis thaliana AltMV therefore offers the ability to express foreign genes or as a Viru Induced Gene Silencing (VIGS) vector for a wide range of plant species. have previously developed infectious clones of AltMV variants differing pathogenicity and viral replication levels. We have now developed a series of viral vectors based on AltMV variants with either the efficien Triple Gene Block 1 (TGB1) suppressor of RNA silencing (effective for hi level protein expression), or the less efficient TGB1 suppressor of RNA silencing (effective for VIGS). The basic vectors can be used to initiat infection either by agroinfiltration, or by inoculation of in vitro transcribed RNA transcripts. A Patent Application relating to these nov viral vector systems has been filed by ARS scientists at Beltsville, MD. These AltMV vector variants will allow physiologists and geneticists to perform gene discovery and molecular marker development studies that should aid development of superior plants of a wide range of model and crop species. 02 Identification of amino acid mutations affecting the localization of the suppressors of RNA silencing. RNA silencing has become a major area of research, as it has become apparent that Virus-induced Gene Silencing (VIGS) plays an important role in regulation of gene expression in healt plants, as well as in plant defense against viral infection. ARS scientists at Beltsville, MD have previously developed infectious clones of Alternanthera mosaic virus (AltMV) variants differing in pathogenicit and demonstrated that a single amino acid in the Triple Gene Block 1 (TG protein had a major effect on viral replication levels through activity of TGB1 as a suppressor of RNA silencing. We have now utilized confocal microscopy to determine the subcellular localization of fusions of variants of the TGB1 protein of AltMV and other potexviruses. The wild- type TGB1 protein of AltMV, containing a Leucine residue at position 88 (L88), was shown to be localized to the nucleolus and is an efficient suppressor of RNA silencing. In contrast, TGB1 containing a mutation of L88 to Proline (P88) is a weak suppressor of RNA silencing and was shown to be localized to the nuclear envelope. Similar coupling of the L88>P88 mutation with nucleolar to nuclear membrane localizations and with the efficiency of RNA silencing suppression of two other potexviruses, Potat virus X and Plantago asiaticus mosaic virus, was also demonstrated. This mutation in an infectious clone of PVX also demonstrated the ability to alter the vector from an effective protein expression to a more efficien VIGS system. These results therefore provide important information about the mechanisms of RNA silencing in potexviruses. 03 Determination that two distinct members of the Flexiviridae require interactions with the chloroplast to effect systemic infection. Viruses must gain entry to the vascular system in order to establish a systemic infection in a host, but the interactions necessary to reach the vascula system are not well understood. We have previously shown that the Triple Gene Block 3 (TGB3) protein of Alternanthera mosaic virus (AltMV; genus Potexvirus) interacts with the chloroplast, and that mutations ablating TGB3, or deleting or substituting critical residues of TGB3, prevent chloroplast interaction and systemic infection. Lolium latent virus (LoL genus Lolavirus) is unusual among the members of the Flexiviridae in producing two co-C-terminal versions of the CP, which encapsidate the viral RNA in equimolar proportions. Mutation of the initiation codon for the larger form of LoLV CP (LoLV-K1) still allows infectivity and localized replication, but ablates systemic movement, whereas mutation o the initiation codon for the smaller form of CP (LoLV-K2) has no apparen effect on infectivity or systemic movement. The LoLV-K1 CP includes a predicted chloroplast transit peptide (cTP) in the N-terminal domain tha distinguishes the large CP from the smaller CP form. Different reversion and deletion mutants of the LoLV CP were fused to the fluorescent marker protein DsRed, and agroinfiltrated into leaves of Nicotiana benthamiana determine the effects on subcellular localization. The results indicate that the potexvirus AltMV and the lolavirus LoLV each require a specific interaction with the chloroplasts, mediated by a different viral gene product for each virus, as a means of establishing viral infection in th mesophyll as an intermediate step in establishing systemic infection. These results form the basis of further study of systemic movement withi the Flexiviridae, and may lead to strategies for blocking systemic viral infection by some of these viruses. 04 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 an 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 ear infections and/or in asymptomatic plants serving as reservoirs for transmission to symptomatic crops. 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 viru transgenes in genetically-engineered gladiolus. Primers include those to viral replicase or coat protein genes to determine the absolute (fg leve and relative copies of CMV genomic RNAs or transgenes contained in total RNA extracts from infected or transgenic plant tissues or purified virio The sensitive and accurate quantification property of this qRT-PCR assa could be useful to monitor viral replication kinetics, evaluate viral tolerance levels in new breeding programs, or to detect and identify CMV to help growers select healthy or virus-free plants in order to produce plants of higher quality and productivity. 05 Development of resistance to Cucumber mosaic virus (CMV) in Gladiolus plants transformed with either a defective replicase or coat protein subgroup II gene from Cucumber mosaic virus. CMV is one of the most important plant viruses because it infects about 1000 plant species, including food crops and ornamentals. Infection of various flower bulb crops with CMV results in dramatic streaking of the flowers making the flowers unmarketable, and infected plants have decreased vigor resulting in a poor bulb yield. There are two subgroups of CMV (I and II) that ar distinguished by serotype, biology, and molecular analysis. Research has continued on the production and evaluation of disease resistant transgen gladiolus through the incorporation of various viral and antiviral antibody genes. In collaboration with another FNPRU ARS scientist (ARS Project 1230-21000-047-00D), transgenic gladioli have been produced whic express CMV replicase, and/or subgroup I or II coat protein transgenes. Transgenic Gladiolus plants that contain either CMV subgroup I coat protein (CMV CP I), subgroup II coat protein (CMV CP II), subgroup I replicase (CMV Rep), a combination of the CMV CP I and CMV CP II, or a combination of the CMV CP II and CMV Rep genes were developed. These plants were multiplied in vitro and challenged with purified CMV I and I Gladiolus isolates using a hand-held gene gun. Three out of 19 independently transformed plant lines expressing the CMV Rep gene under control of the duplicated CaMV 35S promoter were found to be resistant t CMV I. Three out of 21 lines expressing the CMV CP II gene under contro of the Arabidopsis UBQ3 promoter were resistant to CMV II. Eighteen line expressing either the CMV CP I or a combination of CMV CP I and CP II genes were found to be susceptible to both CMV I and II. This work will facilitate the evaluation of virus resistance in transgenic Gladiolus plants to yield improved floral quality and productivity. 06 Differentiation of biovars of Ralstonia solanacearum using an improved method that is fast, unambiguous, and economical. Ralstonia solanacearum attacks over 450 plant species including ornamentals such as geranium. I is generally classified into 5 races based on host range and 5 biovars based on carbohydrate utilization. R. solanacearum race 3, biovar 2 caus destructive brown rot of potato, and is a quarantined and select agent pathogen in the U.S. Currently, biovars of R. solanacearum are differentiated by Polymerase chain reaction (PCR) assays using specific primers and biovar tests based on utilization of a carbohydrate panel. Standard biovar tests are done in 15 ml culture tubes containing bromothymol blue as a pH indicator in 3 ml basal medium supplemented wit a carbohydrate, and take weeks to complete. ARS scientists at Beltsville MD improved the biovar test by using phenol red as a pH indicator to produce an unambiguous color change at a higher pH. We also improved the tests by conducting the test in 0.5 ml PCR tubes or 0.2 ml PCR strip tub so less than one tenth of the medium is needed, which not only saves mon and space but also allows the test to be completed in about two days. Th improved biovar differentiation test will help state and federal regulatory officials to make timely decisions to prevent and exclude the brown rot pathogen from becoming established in the U.S.
Impacts
(N/A)
Publications
- Lim, H.S., Vaira, A.M., Domier, L.L., Lee, S.C., Kim, H.G., Hammond, J. 2010. Efficiency of VIGS and gene expression in a novel bipartite potevirus vector delivery system as a function of strength of TGB1 silencing suppression. Virology. 402:149-163.
- Norman, D.J., Huang, Q., Yuen, J.M.F., Mangravita-Novo, A., Byrne, D. 2009. Susceptibility of Geranium cultivars to Ralstonia solanacearum. HortScience. 44(5):1-5.
- Hammond, R., Hammond, J. 2009. Maize rayado fino virus capsid proteins assemble into virus-like particles in Escherichia coli. Virus Research. 147:208-215.
- Lim, H.S., Vaira, A.M., Reinsel, M.D., Bae, H., Bailey, B.A., Domier, L.L., Hammond, J. 2010. Localization of Althernanthera mosaic virus patholgenicity determinants to RdRp and TGB1, and separation of TGB1 silencing suppression from movement functions. Journal of General Virology. 91:277-287.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations Molecular biology of several novel Geranium viruses. Research continues on understanding the molecular biology and pathogenicity of several unique geranium viruses. The structural and movement-related proteins from Pelargonium chlorotic ring pattern virus (PCRPV) have been sub-cloned into bacterial expression vectors and the His-tagged proteins have been purified. These proteins are being used as immunogens for polyclonal antisera production for use as both diagnostic reagents (no antisera is available for the detection of PCRPV) and as tools to confirm the novel protein translation strategy predicted for this virus. A full length infectious cDNA clone of PCRPV has been developed and we have initiated site-directed mutagenesis and gene reassortment analysis to determine virus-specific sequences responsible, for example, for symptom expression in geranium and plant virus movement in the plant. Susceptibility of 4 additional ornamental species to Ralstonia solanacearum race 3, biovar 2. Four ornamental species were tested in growth chambers (poinsettia) and in an APHIS-approved greenhouse (portulaca, sage and oxalis) by the soil drenching method for their susceptibility to R. solanacearum race3, biovar 2. Disease symptoms were observed and the bacterium was detected by dilution plating from inoculated portulaca plants, but not from the other 3 plant species. Evaluation of pathogenic relationships among ornamental and non- ornamental strains and specific detection and identification of Xylella fastidiosa (Xf) bacteria. Grape, almond, plum, oak and maple strains of Xf were mechanically inoculated to oleander plants to determine if those ornamental and non- ornamental strains can infect and cause disease symptoms in this host. In this ongoing experiment, the inoculated plants are being tested every two months for symptom development and the presence of Xf is being determined by PCR. 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: �Biology and Pathogenesis of Ralstonia solanacearum Race 3 on Geraniums� with the Univ of Wisconsin-Madison (#1230-22000-022-08S and #1230-22000-022-13S); �Ralstonia solanacearum Bacterial Wilt Host Relationships� with the Univ of Florida (#1230-22000-022-09S and #1230-22000-022-14S); �Development of Sensitive Protocols for Detection of Bacterial Diseases in Ornamentals� with Oregon State Univ (#1230-22000-022-10S); �Development of new, superior plant virus detection methodologies and reagents� with Agdia, Inc (#1230-22000-022-11T); �Sensitive detection of single and mixed infections in ornamental plants at all stages of the propagation cycle� with the Univ of California, Riverside (#1230-22000-022-12S); and �Development and Validation of a Universal Plant Virus Microarray for Detection and Identification of Plant Viruses� (#1230-22000-022-15R). Significant Activities that Support Special Target Populations Three scientists from the Floral and Nursery Plants Research Unit (FNPRU) organized, developed and participated in a 2-day �Plant Biotechnology Workshop� at the Inter-American University at Barranquitas, Puerto Rico (IAUPR) in May 2008. More than 20 teachers and students were presented lectures regarding FNPRU plant biotechnology research areas (represented by the three scientists� Projects: 1230-22000-022-00D, 1230-22000-060-00D, 1230-22000-045-00D, and 1230-22000-047-00D). The teachers and students also participated in demonstrations and hands-on experiences in laboratory tools and protocols. Two of those teachers then spent 1 week in our labs in Beltsville in July 2009 to participate in additional hands- on experiences in our laboratories. This cooperative interaction will lead to additional teachers and students from Puerto Rico attending research internships in the FNPRU Beltsville labs to be trained further in the areas of plant virology, plant transformation and development of plant molecular markers. Technology Transfer Number of Active CRADAS: 1
Impacts
(N/A)
Publications
- Lim, H.S., Bragg, J.N., Ganesan, U., Ruzin, S.E., Schichnes, D., Lee, M., Vaira, A., Ryu, K., Hammond, J., Jackson, A.O. 2009. Subcellular localization of the barley stripe mosaic virsus triple gene block proteins. Journal of Virology 83(18):9432-9448. http://dx.doi.org/10.1128/JVI.00739- 09.
- Vaira, A.M., Hansen, M.A., Murphy, C., Reinsel, M.D., and Hammond, J. 2009. First report of Freesia sneak virus in Freesia sp. in Virginia, USA. Plant Disease. 93(9):965. Available http://dx.doi.org/10.1094/PDIS-93-9- 0965B.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations 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). Technology Transfer Number of Active CRADAS: 1 Number of Invention Disclosures submitted: 2
Impacts
(N/A)
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.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations 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: �Detection of Viruses in Verbena at All Stages of Its Propagation Cycle� with University of California-Riverside (#1230-22000- 022-02S); �Studies on the distribution and multiplication of tobacco mosaic virus 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); and, �Development of new, superior plant virus detection methodologies and reagents� with Agdia, Inc (#1230-22000-022-11T). Geranium viruses. Research continues on understanding the molecular biology and pathogenicity of several unique geranium viruses. The coat protein (p37) gene and a movement-related (p7) protein from Pelargonium chlorotic ring pattern virus (PCRPV) have been sub-cloned into bacterial expression vectors and the His-tagged proteins have been purified. These proteins are being used as immunogens for polyclonal antisera production for use as both diagnostic reagents (no antisera is available for the detection of PCRPV) and as tools to confirm the novel protein translation strategy predicted for this virus. The development of full length infectious cDNA clones of PCRPV and Pelargonium line pattern virus continues. When available, we will initiate gene reassortment analysis to determine virus-specific sequences responsible, for example, for symptom expression in geranium and plant virus movement in the plant. This research 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. Susceptibility of 4 ornamental species to Ralstonia solanacearum race 3, biovar 2. The four ornamental species, begonia, browallia, impatiens and pansy, were tested in growth chambers by soil drenching method for their susceptibility to R. solanacearum race3, biovar 2. Disease symptoms were not observed in the inoculated plants, and bacterial cells were 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, and ARS Strategic Plan Performance Measure 3.2.5. Accomplishments Complete sequence and development of infectious clones of Lolium latent virus and Alternanthera mosaic virus. We previously reported the first identification in the U.S. of Lolium latent virus (LoLV) in ryegrass germplasm. We have now determined the complete 7674nt sequence of LoLV isolate US1, and shown that the genome organization is similar to viruses of the genus Potexvirus and Foveavirus. However, phylogenetic analysis of the sequences of the polymerase and the capsid protein genes indicates that LoLV is distinct from members of the Potexvirus and Foveavirus genera, and should be regarded as an unclassified member of the Flexiviridae. We have also developed infectious cDNA clones of LoLV-US1, which will be used 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). A VIGS vector for turfgrasses will aid in gene discovery and functional genomics for breeding of superior turf types. We have previously reported the complete sequence of a Pennsylvania isolate of Alternanthera mosaic virus (AltMV) from creeping phlox (Phlox stolonifera), and partial sequences of additional Maryland isolates from creeping phlox and portulaca. We have now produced multiple infectious clones from a Maryland isolate of AltMV, which differ in symptom severity in Nicotiana benthamiana. Analysis of the sequence differences between the infectious clones, and gene exchange between these clones and other isolates differing in host range and symptom expression, will allow identification of host range and symptom determinants of AltMV. 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. 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 also tested multiple pansy lines for the presence of tobamoviruses, potexviruses, carlaviruses, and potyviruses by PCR, and for other viruses by inoculation of typical virus assay host plats, by electron microscopy of symptomatic pansies and inoculated plants, without detecting any other viruses. 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. The pansy ilarvirus has been mechanically transmitted to two herbaceous experimental hosts (N. benthamiana and N. occidentalis) with some difficulty. Purification attempts to date have not yielded clean virus, but have provided better RNA extracts from which to clone the genome. We are working to both extend to the 5� and 3� ends of RNA 1, and to obtain clones from RNA 2 and RNA 3. We expect to clone and express the coat protein gene in order to prepare a virus-specific antiserum. 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. In other experiments, we have shown by electron microscopy that regions of pansy leaves affected by Pansy mottle syndrome have abnormal proliferation of membranes, presumed to be extensions of the endoplasmic reticulum. Addition of approximately 50ppm, 100ppm, or 200ppm of 1,3-dichlorobenzene to the potting media of seedling transplants, or to tissue culture growth media used to germinate seed in vitro, failed to induce symptoms of pansy mottle syndrome. Additional tests will be carried out in combination with heat and other physiological stresses in order to gain greater understanding of the causes of Pansy mottle syndrome. The research in this accomplishment contributes to ARS National Program 303, Component 1, Problem Statement 1B, Component 2, Problem Statement 2A, 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 6546nt region of the expected ~10,000nt viral genome (AY864851), 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. 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. Novel carlaviruses in different species of 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 determined the complete nucleotide sequence of Phlox virus S, isolated from Phlox stolonifera (Creeping phlox), and partial sequences of additional novel carlaviruses from Phlox divaricata (Sweet William phlox) and a hybrid phlox cultivar. We have now determined the complete 9058nt sequence of the �WP carlavirus� from Phlox divaricata, for which the name �Phlox virus B� is proposed. We have also determined the 3�-terminal 1687nt of the carlavirus from hybrid phlox, deposited in GenBank as accession EF507476 under the name �Phlox virus M�. 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. Detection of additional viruses 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. Tobacco ringspot nepovirus, an unidentified tobamovirus, and two distinct types of potyvirus have also been 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. 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. 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. Cloning and sequencing of the coat protein gene of Vallota mosaic virus We have cloned and sequenced the 3�-terminal region of the potyvirus Vallota mosaic virus (ValMV) from Cyrtanthus elatus (GenBank accession EF441726), and shown that ValMV is most closely related to Ornithogalum mosaic virus (OrMV) and Ornithogalum virus 2 (OrV2). 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. Characterization of Freesia sneak ophiovirus from Lachenalia We have previously reported detection of an ophiovirus in commercial Lachenalia. We have now identified the virus in Lachenalia 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 cloned and sequenced almost all of RNA 2 in addition to portions of RNA 1 and all of RNA 3, and a part of a presumed RNA 4 (previously identified in only two ophioviruses infecting lettuce). 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. Detection and identification of Columbian datura virus in Brugmansia. Several Angel�s Trumpet, Brugmansia spp, exhibiting mosaic and, later, veinal necrosis, tested positive with our potyviral broad-spectrum monoclonal antibody (McAb PTY-1). This symptomatic plants were also tested using other potyvirus-specific and broad-spectrum monoclonal antibodies and by electron microscopy. Plants were negative using antisera to 16 other viruses. Total RNA extracts served as templates for RT-PCR with potyvirus 'generic' primers which amplify highly conserved fragments from the 3' terminus of most potyviruses which includes the coat protein (CP) and 3' non-coding region (3'NCR). The resultant PCR amplicons were cloned, sequenced and molecularly compared to potyvirus sequences in the international databases. Phylogenetic analysis indicated that this potyvirus is Columbia datura virus and is only the second report of this virus infecting this host. 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. Control of bacterial plant pathogens by soil treatment with clove oil. Bacterial diseases often result in significant losses in the production and quality of ornamental crops, and are very difficult to control. We have evaluated the effect of clove oil against major groups of plant pathogenic bacteria including Agrobacterium tumefaciens, Erwinia carotovora pv caortovora, Pseudomonas syringae pv syringae, Ralstonia solanacearum race 1 and race 3, Rhodococcus fascians, Streptomyces spp., and Xanthomonas campestris pv pelargonii. The clove oil inhibited the growth of both gram-posive and gram-negative bacteria by either fumigation or direct contact, and its bactericidal effect was dose dependent. The bacteria also displyed different degrees of sentitivity to the clove oil with Ralstonia being the most sensitive and Rhodococcus the least. The clove oil is currently being tested in our greenhouse experiments. Our preliminary results showed that pre-plant fumigation of soil-less potting mix with a clove oil formulation effectively controlled bacterial wilt of geranium and tomato caused by R. solanacearum race1, biovar 1 by preventing symptom formation and bacterial colonization of the host plants. The discovery that the clove oil is effective in vitro against a wide array of plant pathogenic bacteria, especially R. solanacearum, and it may also be effective against bacterial wilt caused by R. solanacearum in tomato and geranium suggests that clove oil may offer an effective and environmentally sound approach to control bacterial diseases. This research is under National Program 303, Component 4, Problem Area 4a, �Biological and Cultural Control Technologies�, and Performance Measure 3.2.6. Effect of the growth regulator Paclobutrazol on growth of the bacterial pathogen Xylella fastidiosa. X. fastidiosa is a slow growing, xylem inhabiting, nutritionally fastidious and insect transmitted bacterium. The bacterium is associated with bacterial leaf scorch (BLS) and decline in many economically important landscape trees and shrubs including oak, elm, sycamore, maple and oleander. Application of a growth regulator Paclobutrazol (PBZ) to infected plants has been found to cause symptom remission of BLS. This study was undertaken to find out the effect of PBZ on in vitro growth of two X. fastidiosa isolates. We found no significant effect of PBZ on colony growth of X. fastidiosa at the manufacturer's recommended rate of 20 ug ml -1. However, significant reductions in bacterial growth were observed at a rate of 200 ug m1 -1, indicating that high levels of PBZ may have a direct effect on the growth of X. fastidiosa. This suggests that PBZ may provide a promising treatment for BLS in shade trees by its direct effect on the bacterium and its growth regulator effect on plants. Our work will be of value primarily to arborists and other members of the nursery and landscape industry, and to plant pathologists interested in control of X. fastidiosa. This research is under National Program 303, Component 4, Problem Area 4a, �Biological and Cultural Control Technologies�, and Performance Measure 3.2.6. Technology Transfer Number of Active CRADAS and MTAS: 2 Number of Invention Disclosures submitted: 1 Number of Web Sites managed: 1 Number of Non-Peer Reviewed Presentations and Proceedings: 9 Number of Newspaper Articles,Presentations for NonScience Audiences: 3
Impacts
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Publications
- Vaira, A.M., Kleynhans, R., Hammond, J. 2007. First report of Freesia sneak virus infecting Lachenalia cultivars in South Africa. Plant Disease. 91:770.
- Guaragna, M.A., Ndum, O., Jordan, R.L. 2006. Detection and characterization of two previously undescribed potyviruses in the terrestrial orchid Spiranthes cernua. Acta Horticuture. 722:209-218.
- Guaragna, M.A., Jordan, R.L., Putnam, M. 2006. First molecular evidence of the occurrence of a pea mosaic strain of Bean yellow mosaic virus in Verbena x hybrida. Acta Horticulture. 722:305-312.
- DeStefano, D.A., Grybauskas, A.P., Sherald, J.L., Momen, B., Huang, Q. and Sullivan, J.H. 2007. Effect of the growth regulator Paclobutrazol on growth of the bacterial pathogen Xylella fastidiosa. Arboriculture to Arboriculture and Urban Forestry. 33:246-252.
- Ling, K., Wechter, W.P., Jordan, R.L. 2007. Development of a One-Step Immunocapture Real-Time TaqMan RT-PCR Assay for the Broad Spectrum Detection of Pepino Mosaic Virus. Journal of Virological Methods 144:65- 72.
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