Progress 02/26/07 to 02/25/12
Outputs
Progress Report Objectives (from AD-416): Establish molecular biomarkers of evolutionary divergence for spiroplasma and phytoplasma detection and identification. Expand the molecular scheme for phytoplasma classification through genome-based selection of "constellations of identifier genes". Identify and characterize plant pathogenic mollicute genes involved in pathogenesis and symptom induction for eventual establishment of identifiers of pathotypes. Determine the correlation of chromosomally-integrated repetitive sequences and extrachromosomal DNAs in genome plasticity and biological diversity of phytoplasmas. Approach (from AD-416): Genome-based information will form the basis for improving detection, identification, and classification of plant pathogenic, wall-less prokaryotes (class Mollicutes). Gene sequencing, bioinformatics methods, recombinant DNA technology, phylogenetic and gene evolution analyses, enzyme and bacterial growth assays, gene function analyses, and biochemical methods will be employed with inoculations of susceptible plants, tissue culture and plant transformation, to investigate the structure and function of DNA sequences and gene products as molecular markers for detection and identification of genus-, species-, and strain- level evolutionary divergence. Phytoplasmas associated with newly emerging diseases will be identified, and molecular criteria for improving descriptions of 'Candidatus Phytoplasma' species and for developing a formal taxonomy will be sought. DNA sequences enabling genome plasticity and biological diversity will be investigated as potential molecular biomarkers for making finer distinctions at species, strain, and population levels. Agrobacterium-mediated stable transformation and plant viral vector-mediated transient expression technologies will be used to introduce antimicrobial peptide genes and other defense-related genes into plants, enhancing natural plant defense and mitigating phytoplasmal and spiroplasmal diseases. Plant diseases caused by phytoplasmas and spiroplasmas are responsible for economic losses in U.S. and world agriculture. Knowledge from genomics will make it possible to devise improved and novel technologies for pathogen detection and classification to aid efforts to reduce crop losses due to plant diseases through improved crop management, production of certified disease-free germplasm, and implementation of plant quarantine regulations. We characterized, described, and proposed new �Candidatus Phytopasma� species to encompass strains causing X-disease, one of the most significant limiting factors in peach production in the U.S. We determined the interrelatedness of phytoplasmas causing grapevine yellows (GY) disease in eastern U.S., and discovered that GY disease is present in more states than previously known. We identified molecular markers for identification of insect vectors and for devising strategies to stem the spread of GY disease. We designed group specific primers for amplification of, and completed characterization of, secY, from 23 phytoplasma strains and updating sequences of rp and 16S rRNA genes of uncharacterized strains belonging to 16SrIX group; developed virtual RFLP analysis based on the secY gene for differentiation of closely related strains; completed characterization of 16S rRNA, rp and secY gene sequences from 16Sr IX group and new 16SrIII group strains. We identified additional phytoplasmas that are associated with potato purple top wilt and other diseases in Moscow region in Russia and potential leafhopper vectors that transmit these phytoplasmas. We continued to characterize phytoplasmas associated with blueberry stunt and cranberry false blossom diseases in New Jersey. We completed partial genome sequencing of four phytoplasma strains representing four subgroups. We made further progress in identifying and characterizing new phytoplasmas associated emerging diseases in Asia with analysis of multiple genetic loci. We investigated the interactions between potato purple top (PPT) phytoplasma and its plant host, tomato. We found that PPT phytoplasma infection caused a significant reduction in endogenous levels of gibberellin acid (GA3), and the decrease in GA3 content was correlated with a down regulation of genes encoding key enzymes responsible for biosynthesis of bioactive GA and GA precursors. Exogenous application of GA3 was able to alleviate the characteristic �big bud� symptoms induced by the phytoplasma. We investigated phytoplasma infection-induced transcriptional reprogramming of host genes and their effects on disease symptom expression. Improving customer service, we further expanded the MPPL online, interactive Phytoplasma Classification Database. This web site is used worldwide by scientists, students, professors, quarantine agencies, and diagnostic companies to aid their phytoplasma classification and taxonomy including delineation of new phytoplasma groups/subgroups and discovery of new �Candidatus Phytoplasma� species. Accomplishments 01 Enhanced the functionality of iPhyClassifier, the first interactive onli phytoplasma identification and classification tool. We updated the 16S rRNA gene sequence database to include sequences from new phytoplasma species and phytoplasma lineages. We included the newest reference RFLP pattern types that represent recently delineated phytoplasma groups and subgroups. A simple operation of iPhyClassifier on user input sequences can quickly lead to identification of phytoplasma strains under study. The online phytoplasma research tool has been used by scientists all ove the world and by government agencies for implementation of new quarantin regulations. 02 Discovered that grapevine yellows (GY) disease is present in the states Pennsylvania and Maryland. GY is a destructive disease complex that affects a wide variety of grapevine cultivars in all major wine producti countries, but the extent of GY occurrence in the U.S. had not been explored. The findings from this study will help viticulturists to devi measures to stem the spread of GY diseases in U.S. vineyards. 03 Developed a practical approach to increase plant defense against phytoplasma infection. Recent outbreaks and continued spread of diseas caused by phytoplasmas in potato, tomato, and other vegetable crops in t U.S. accentuates the need for practical strategies to mitigate the impac of the phytoplasmal diseases. The purpose of the current study was to investigate whether exogenous application of salicylic acid (SA) on healthy tomato seedlings would enhance the plants� defense against a subsequent potato purple top (PPT) phytoplasma infection. Our results showed that a twice-applied SA pretreatment, at four and two days prior phytoplasma inoculation, significantly decreased the rate of PPT phytoplasma infection and reduced disease symptoms. Our results also revealed that SA-pretreatment caused transcriptional reprogramming of defense-related genes. With the dosage and treatment regime implemented in the present study, no noticeable phytotoxic effects or other negative impact on vegetative growth or reproductive development was observed in treated plants throughout our experiment. Our findings encourage field trials of SA pretreatment as a possible approach to protect crops from phytoplasmal diseases. 04 Discovered a new phytoplasma species. In collaboration with scientists Malaysia, U.K. and University of Florida, this work was aimed at expandi knowledge regarding phytoplasmas associated with emerging diseases in periwinkle, coconut palm, and oil palm trees in Malaysia. We conducted gene ancestry and computer-assisted DNA fingerprint analyses to determin the genetic relationship between these new phytoplasmas and previously known phytoplasmas. We concluded that these Malaysian phytoplasmas possess distinct genetic features and represent a new species, �Candidat Phytoplasma malaysianum�. We also determined that these phytoplasmas represent three distinct lineages and a new phytoplasma group. In addition, we identified molecular markers that can be used for quick identification of phytoplasmas belonging to this new group. This work will interest diagnostics laboratories, research scientists and extensio personnel who are concerned with phytoplasma disease management, and government agencies that implement plant quarantine regulations to preve the introduction of foreign pests and diseases into U.S. agriculture. 05 Completed updating classification of phytoplasma strains in the 16SrIX group based on sequences of three conserved biomarker genes, 16S rRNA, ribosomal protein (rp) and secY genes. Phylogenetic analyses using thes three genes revealed at least six distinct genetic lineages distinguishe among the constituent members in the 16SrIX group. Some of these distinc lineages cannot be readily differentiated based on analysis of 16SrRNA gene sequences alone. The relative genetic distances among these closely related lineages were better assessed by including additional, more variable genes (e.g. rp and secY genes). The present study underscores a need to include additional, less conserved gene markers for classificati A recording system was proposed to designate each distinct rp and secY subgroups in the 16SrIX group. Therefore, including an additional gene, such as rp or secY, in assessing the genetic distance of a potential candidate for a new taxon should alleviate ambiguity from analysis of th 16SrRNA gene alone. 06 Conducted first comprehensive survey of phytoplasma strains associated with recurring blueberry stunt disease in New Jersey. Recently, the recurrence of blueberry stunt disease became evident in many old and new farms in New Jersey. The phytoplasmas causing the disease in New Jersey had not been previously characterized. We conducted a state-wide survey the causal pathogens of blueberry stunt disease in all blueberry cultiva in several major blueberry production counties throughout New Jersey. Sequence and phylogenetic analyses of 16S rRNA, ribosomal protein and se genes indicated that phytoplasma strains predominantly belonging to aste yellows group, subgroup 16SrI-E, were present in diseased blueberries in New Jersey, but a new phytoplasma belonging to group 16SrIX was also surprisingly discovered in infected plants.
Impacts
(N/A)
Publications
- Lee, I., Bottner-Parker, K.D., Zhao, Y., Bertaccini, A., Davis, R.E. 2012. Differentiation and classification of phytoplasmas in the pigeon pea witches'-broom group (16SrIX): an update based on multiple gene sequence analysis. International Journal of Systematic and Evolutionary Microbiology. doi:10.1099/ijs.0.038273-0.
- Wu, W., Cai, H., Wei, W., Davis, R.E., Lee, I., Chen, H., Zhao, Y. 2012. Identification of two new phylogenetically distant phytoplasmas from Senna surattensis plants exhibiting stem fasciation and shoot proliferation symptoms. Annals of Applied Biology. 160:25-34.
- Wu, W., Ding, Y., Wei, W., Davis, R.E., Lee, I., Hammond, R., Zhao, Y. 2012. Salicylic acid-mediated elicitation of tomato defense against infection by potato purple top phytoplasma. Annals of Applied Biology. 161:36-45.
- Nejat, N., Vadamalai, G., Davis, R.E., Harrison, N.A., Sijam, K., Dickinson, M., Abdullah, S., Zhao, Y. 2012. Candidatus Phytoplasma malaysianum, a novel taxon associated with virescence and phyllody of Madagascar periwinkle (Catharanthus roseus). International Journal of Systematic and Evolutionary Microbiology. doi:10.1099/ijs.0.041467-0.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) Establish molecular biomarkers of evolutionary divergence for spiroplasma and phytoplasma detection and identification. Expand the molecular scheme for phytoplasma classification through genome-based selection of "constellations of identifier genes". Identify and characterize plant pathogenic mollicute genes involved in pathogenesis and symptom induction for eventual establishment of identifiers of pathotypes. Determine the correlation of chromosomally-integrated repetitive sequences and extrachromosomal DNAs in genome plasticity and biological diversity of phytoplasmas. Approach (from AD-416) Genome-based information will form the basis for improving detection, identification, and classification of plant pathogenic, wall-less prokaryotes (class Mollicutes). Gene sequencing, bioinformatics methods, recombinant DNA technology, phylogenetic and gene evolution analyses, enzyme and bacterial growth assays, gene function analyses, and biochemical methods will be employed with inoculations of susceptible plants, tissue culture and plant transformation to investigate the structure and function of DNA sequences and gene products as molecular markers for detection and identification of genus-, species-, and strain- level evolutionary divergence. Phytoplasmas associated with newly emerging diseases will be identified, and molecular criteria for improving descriptions of 'Candidatus Phytoplasma' species and for developing a formal taxonomy will be sought. DNA sequences enabling genome plasticity and biological diversity will be investigated as potential molecular biomarkers for making finer distinctions at species, strain, and population levels. Agrobacterium-mediated stable transformation and plant viral vector-mediated transient expression technologies will be used to introduce antimicrobial peptide genes and other defense-related genes into plants, enhancing natural plant defense and mitigating phytoplasmal and spiroplasmal diseases. Plant diseases caused by bacteria called phytoplasmas and spiroplasmas are responsible for economic losses in U.S. and world agriculture. Improved and novel technologies for pathogen detection, identification, and classification are needed to reduce crop losses through improvement of crop management, production of disease-free germplasm, and implementation of plant quarantine regulations. We investigated the interactions between potato purple top (PPT) phytoplasma and tomato. In infected plants, levels of plant hormones were altered, and expression profiles of hormone biosynthesis and degradation genes were changed. Application of certain hormones impeded disease progression and attenuated symptoms. Results suggest that levels and ratios of plant hormones are markers of early phytoplasma infection. The findings should aid development of practical strategies to manage phytoplasmal diseases. We made further progress in piecing together the highly complex, virus- ridden genome sequence of Spiroplasma kunkelii, cause of corn stunt disease - a major factor in corn production in the Americas. The results will aid understanding of phloem-limited bacterial plant pathogens and their pathogenicity and insect vector specificity. Grapevine yellows disease, caused by phytoplasma, is rapidly increasing in importance in mid-Atlantic states, and may be spreading in other regions of the U.S. We initiated work to provide needed base data for viticulturists and scientists to devise management approaches to combat grapevine yellows disease. We have enhanced online phytoplasma identification and classification by updating the gene database in the online iPhyClassifier to include phytoplasmas associated with emerging diseases worldwide. Our iPhyClassifier quickly leads to identification of multiple unknown phytoplasma strains at once, providing suggestions on their classification and taxonomic assignment. This online research tool impacts research worldwide and is being used by scientists in research and by government agencies in implementation of quarantine regulations. We investigated alternative hosts and potential leafhopper vectors of phytoplasmas associated with potato purple top wilt (PPTW) in Russia. We tested weeds and crop plants from different potato-growing regions and found that many possibly serve as alternative hosts for PPTW phytoplasma. In the Moscow region, we found four different leafhopper species that that may be vectors of potato-infecting phytoplasmas. We initiated work to develop rapid, automatic analysis of single nucleotide polymorphisms (SNPs) in gene sequences for finer differentiation and characterization of phytoplasmas by providing additional molecular markers. The progress should aid differentiation of closely related strains that are ecologically distinct and/or originate from different geographical regions. Accomplishments 01 Molecular markers and technology for rapid detection and differentiation of quarantine phytoplasmas in grapevine. Grapevine yellows (GY) is a destructive disease complex that affects a wide variety of grapevine cultivars in all major wine production countries. Known etiological agents of GY diseases are phytoplasmas affiliated with at least four distinct genetic groups and five species. Several GY phytoplasmas have been listed as quarantine pests in the United States, European Union, China, and many other countries. Since disease symptoms induced by different GY phytoplasmas are very similar, visual inspections at ports are not sufficient for diagnosis of various GY diseases. In the present study, we identified a series of molecular markers that can effectively distinguish three quarantine phytoplasmas. Selected markers were used t devise a DNA fingerprinting-based procedure for simultaneous detection a differentiation of three major GY phytoplasmas in affected plants. The findings and the new technology resulting from this study will help phytoplasma researchers, plant doctors, and quarantine personnel to identify and distinguish GY phytoplasmas more efficiently. 02 New phytoplasma associated with little leaf disease in azalea. We discovered that little leaf disease in azalea is associated with infecti by a phytoplasma belonging to the aster yellows phytoplasma group. Analysis of multiple molecular markers revealed that this azalea-infecti phytoplasma represents a new, genetically distinct branch of phytoplasma By revealing a new plant disease and a previously unknown phytoplasma lineage, the work will impact plant pathological research, aid in designing new technologies to detect phytoplasmas in the newly delineate lineage, and develop strategies to prevent spread of the phytoplasma. 03 Two new phylogenetically distant phytoplasmas from sunshine trees exhibiting the stem fasciation and shoot proliferation symptoms. In the summer of 2008, a new disease of sunshine tree was observed in a natural forested area of southwest China. The disease was characterized by enlargement and flattening of stems, and excessive branching of shoots. Using DNA fingerprinting technology, we found that the diseased sunshine trees were infected by two mutually distinct phytoplasmas classified in two groups named 16SrV and 16SrXII, respectively. The 16SrV phytoplasma has a wide distribution in northern China but this is the first report that the phytoplasma has reached to one of the southernmost areas of the country. Phytoplasmas classified in group 16SrXII are considered seriou quarantine pests in China, and no 16SrXII phytoplasma had been formally reported in China. Findings from this study signal a significant expansion of both geographical distribution and host range of 16SrXII an 16SrV phytoplasmas. This report will be of interest to diagnostics laboratories, research scientists and extension personnel who are concerned with phytoplasma disease management. The information is also important to international quarantine agencies for implementation of new quarantine regulations to prevent further spread of the phytoplasmas. 04 New phytoplasma species. Phytoplasmas infect edible fruit-bearing plant Passion fruit in Brazil, where it is widely grown commercially, is valu as a nutritious source of vitamins, and is used in producing fruit juice Using DNA-based molecular methods for detection and identification, we discovered that diseased plants of passion fruit were infected by two different phytoplasmas, and one is a novel phytoplasma species, for whic we propose the name �Candidatus Phytoplasma sudamericanum�. The results provide new information for detection and identification of the two previously unknown phytoplasmas. This advance is significant in part because the complete plant host ranges of the two phytoplasmas are not y known, and because the phytoplasmas might have potential to invade passi fruit and other agricultural crops in the U.S. This work will interest scientists and students studying plant diseases, diagnostics companies a centers involved in pathogen detection, companies producing disease-free fruit trees, fruit growers and juice producers, and government agencies that implement plant quarantine regulations to prevent the introduction foreign pests and diseases into U.S. agriculture. 05 New phytoplasma lineages causing emerging diseases in fruit trees and woody ornamentals. Using molecular methods for detection and identification, we found two new phytoplasmas that are the apparent caus of diseases in fruit trees and wood ornamentals. The two phytoplasmas a related to a phytoplasma associated with disease of clover in North America and Europe, but both phytoplasmas represent previously undescrib lineages. Finding these phytoplasmas expands the known biodiversity of phytoplasmas discovered to infect woody plants. The data provide means for detecting and identifying the same phytoplasmas in cherry, lilac, an other plants around the world. Concepts presented in this report open n avenues for understanding the evolution of new phytoplasma lineages. Thi report will be of interest to diagnostics laboratories, research scientists, quarantine regulatory agencies, and growers. 06 Molecular characterization of phytoplasma affecting vegetable crops. Using DNA fingerprinting and bioinformatic tools, we found that phytoplasmas apparently cause a new disease, which we have named onion proliferation disease. We discovered that two different phytoplasmas wer associated with the disease. While studying how to identify one of the phytoplasmas, we discovered that differences in a single base (single nucleotide polymorphism, SNP) was a powerful molecular marker of phytoplasma evolution. Our results provide molecular markers useful for following the spread of the onion disease phytoplasmas to new geographic regions. This report will be of interest to diagnostics laboratories, research scientists, international quarantine agencies including APHIS, and scientists researching the evolution of bacteria, including phytoplasmas. By revealing a new vegetable disease and its apparent caus the work immediately impacts food security and efforts to attain sustainable agriculture systems. 07 New species of phytoplasma causing disease in Costa Rica. A new diseas occurred in soybean, sweet pepper, and passion fruit in Costa Rica. In collaboration with scientists from Costa Rica, we detected a new previously unknown phytoplasma in soybean, sweet pepper, and passion fru We named the phytoplasma as a new species, �Candidatus Phytoplasma costaricanum�. By revealing a new disease and its apparent cause, the work immediately impacts food security and efforts to attain sustainable agriculture systems.
Impacts
(N/A)
Publications
- Wei, W., Cai, H., Jiang, Y., Lee, I., Davis, R.E., Ding, Y., Yuan, E., Chen, H., Zhao, Y. 2011. A new phytoplasma associated with little leaf disease in azalea: multilocus sequence characterization reveals a distinct lineage within the aster yellows phytoplasma group. Annals of Applied Biology. 158:318-330.
- Jomantiene, R., Zhao, Y., Lee, I., Davis, R.E. 2011. Phytoplasma infecting cherry and lilac represent two distinct lineages having close evolutionary affinities with clover phyllody phytoplasma. European Journal of Plant Pathology. 130:97-107.
- Lee, I., Bottner-Parker, K.D., Zhao, Y., Villalobos, W., Loreira, L. 2011. "Candidatus phytoplasma costaricanum" a new phytoplasma associated with a newly emerging disease in soybean in Costa Rica. International Journal of Systematic and Evolutionary Microbiology. 60:2887-2897.
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Progress 10/01/09 to 09/30/10
Outputs
Progress Report Objectives (from AD-416) Establish molecular biomarkers of evolutionary divergence for spiroplasma and phytoplasma detection and identification. Expand the molecular scheme for phytoplasma classification through genome-based selection of "constellations of identifier genes". Identify and characterize plant pathogenic mollicute genes involved in pathogenesis and symptom induction for eventual establishment of identifiers of pathotypes. Determine the correlation of chromosomally-integrated repetitive sequences and extrachromosomal DNAs in genome plasticity and biological diversity of phytoplasmas. Develop new practical strategies for the enhancement of plant defenses against diseases caused by phytoplasma and spiroplasma. Approach (from AD-416) Genome-based information will form the basis for improving detection, identification, and classification of plant pathogenic, wall-less prokaryotes (class Mollicutes). Gene sequencing, bioinformatics methods, recombinant DNA technology, phylogenetic and gene evolution analyses, enzyme and bacterial growth assays, gene function analyses, and biochemical methods will be employed with inoculations of susceptible plants, tissue culture and plant transformation, to investigate the structure and function of DNA sequences and gene products as molecular markers for detection and identification of genus-, species-, and strain- level evolutionary divergence. Phytoplasmas associated with newly emerging diseases will be identified, and molecular criteria for improving descriptions of 'Candidatus Phytoplasma' species and for developing a formal taxonomy will be sought. DNA sequences enabling genome plasticity and biological diversity will be investigated as potential molecular biomarkers for making finer distinctions at species, strain, and population levels. Agrobacterium-mediated stable transformation and plant viral vector-mediated transient expression technologies will be used to introduce antimicrobial peptide genes and other defense-related genes into plants, enhancing natural plant defense and mitigating phytoplasmal and spiroplasmal diseases. Plant diseases caused by bacteria called phytoplasmas and spiroplasmas are responsible for economic losses in U.S. and world agriculture. Knowledge from genomics will make it possible to devise improved and novel technologies for pathogen detection and classification to aid efforts to reduce crop losses due to plant diseases through improved crop management, production of certified disease-free germplasm, and implementation of plant quarantine regulations. This reporting year, our research has progressed on several fronts. We completed characterization of secY gene sequences from 54 phytoplasma strains belonging to 12 groups, developed virtual RFLP analysis based on the secY gene for differentiation of closely related strains, identified and characterized phytoplasmas associated with potato purple top wilt and similar diseases in Russia, and identified potential insect vectors transmitting potato purple top diseases in Russia. We investigated practical strategies to increase natural resistance of crops to phytoplasma infections. We found that exogenous application of salicylic acid (SA) through root drenching or foliar spray can provide partial protection to tomato plants against potato purple top (PPT) phytoplasma infection: it decreases infection rate, slows down disease progression, and reduces disease symptoms. Our results suggest that early onset of SA signaling and induction of systemic acquired resistance may be the molecular basis of SA-mediated protection of tomato against PPT. We examined involvement of plant growth regulators in phytoplasmal disease symptom modulation. We found that, following graft infection of tomato plants with potato purple top (PPT) phytoplasma, endogenous levels and distribution patterns of several growth regulators were altered. Exogenous applications of growth regulators were able to modify symptoms. The findings provide a clue to understanding mechanisms of phytoplasma pathogenesis and symptom expression. We discovered a new phytoplasma associated with western juniper trees in northwestern U.S. The phytoplasma represents a novel �Candidatus Phytoplasma� lineage and a new 16Sr subgroup. We determined that novel phytoplasma strains, that we discovered in diseased cherry trees and diseased lilac in Europe, represented two unique phytoplasma types, each distinct from each other and both related to but distinct from a clover-infecting phytoplasma that is found in Europe and North America. Improving customer service, we expanded the MPPL online, interactive Phytoplasma Classification Database. This Web site is used worldwide by scientists, students, professors, quarantine agencies, and diagnostic companies to aid their phytoplasma classification and taxonomy including delineation of new phytoplasma groups/subgroups and discovery of new �Candidatus Phytoplasma� species. We made further progress in piecing together the highly complex, virus-ridden genome sequence of Spiroplasma kunkelii, cause of corn stunt disease - a major factor in corn production in the Americas. Accomplishments 01 Characterization of new genes in plant disease-causing phytoplasma bacteria. We completed characterization of phytoplasma strains based on secY genes, which encode proteins important in bacterial secretion syste We designed technology for secY gene- amplification, and developed virtual RFLP (DNA fingerprint) analysis based on the secY gene. This progress resulted in finer differentiation of closely related phytoplasm strains. The accomplishment provides an additional biomarker useful for differentiation of biologically and/or ecologically distinct phytoplasma strains that are involved in diverse phytoplasmal diseases. 02 Diverse potato-infecting phytoplasma in Russia. Identified and characterized phytoplasmas associated with potato purple top wilt and similar diseases in Russia. Phytoplasmal diseases have long been suspected to occur in several potato-growing regions in Russia on the basis of symptoms and the presence of insect vectors. However, phytoplas etiologies of these diseases had never been verified by molecular means. In this reporting year, MPPL scientists, in collaboration with scientist from Russia, found in potato phytoplasma strains belonging to five different phytoplasma groups; 16SrI, 16SrII, 16SrIII, 16SrVI, and 16SrXI Phytoplasmas belonging to groups 16SrIII and 16SrXII were most prevalent At least eight subgroups, including two new subgroups belonging to 16SrI and 16SrVI, were identified in various potato-growing regions of Russia. The accomplishment provides insights into the diversity of phytoplasmas involved in potato purple top and related diseases in Russia and will ai implementation of quarantine regulations, as well as providing new molecular biomarkers for extension workers and plant diagnosticians in Russia to identify the pathogens and to combat the diseases. 03 New phytoplasma species found in China. A new disease characterized by uncontrolled branching (witches�-broom disease) was observed in salt ced trees growing in their native habitat--China. We conducted extensive DNA fingerprint analysis on DNA samples extracted from stem and leaf tissues of symptomatic trees and found a direct connection between phytoplasma infection and the salt cedar witches�-broom (SCWB) disease. We found tha the 16S rRNA gene sequence of SCWB phytoplasma is distinctly different from that any previously described phytoplasmas. SCWB represents a new 16Sr phytoplasma group (16SrXXX) and is a novel phytoplasma species, whi we named �Candidatus Phytoplasma tamaricis�. 04 Automated phytoplasma classification. We launched a web-based research tool, iPhyClassifier, for online gene-based phytoplasma identification a classification. We implemented online the virtual RFLP analysis concept and automatic RFLP pattern comparison programs that we developed previously, and we integrated additional functions such as overall sequence comparison and similarity score calculation, intelligent trimmi of input sequences, and publication-ready virtual gel plotting. The new virtual gel plotting function is able to generate not only virtual gel images resulting from multiple enzyme analysis of a single 16S F2nR2 DNA sequence from any given strain, but also virtual gel images resulting fr a single enzyme digestion of multiple DNA sequences. iPhyClassifier also incorporates carefully curated databases of phytoplasma 16S rRNA gene sequences for up-to-date classification and comparative studies. A simpl operation of iPhyClassifier can quickly lead to identification of a phytoplasma strain under study, providing suggestions on its tentative 16Sr group/subgroup classification status and �Candidatus Phytoplasma species� assignment. We also expanded virtual RFLP analysis to multi-loc genotyping and finer differentiation of closely-related phytoplasma strains. 05 Genes for disease resistance in pears. We continued research on genetic engineering of fruit crops for resistance to phytoplasma and other vascular-inhabiting pathogen. We established protocols for high efficien transformation and regeneration of transgenic pear plants and demonstrat that foreign genes can be stably maintained and expressed in recipient pear plants even after several generations of propagation under laborato conditions. We also demonstrated that the introduced gene product was precisely delivered to places where the vascular tissue-inhabiting pathogens may live. The findings from our study suggest that this geneti engineering approach has high potential for targeted delivery of therapeutic agents to control vascular diseases in pear. 06 Grapevine disease and pathogen detection. We developed molecular markers and an assay for rapid detection and precise differentiation of three quarantine-regulated phytoplasmas responsible for yellows diseases in grapevine. Grapevine yellows (GY) is a destructive disease complex that affects a wide variety of grapevine cultivars in almost all major wine production countries. The causal agents of GY are phytoplasmas belonging to at least four distinct genetic groups and some have been listed as quarantine pests in the United States, European Union, China, and many other countries. The disease symptoms induced by different GY phytoplasm are very similar, making accurate visual diagnosis and quarantine inspection difficult. In the present study, we developed biomarkers and DNA fingerprinting-based procedure that can simultaneously detect and distinguish three major GY phytoplasmas in affected plants. The findings and the new technology resulted from this study will help phytoplasma researchers, plant doctors, and quarantine personnel to identify and distinguish GY phytoplasmas more efficiently. 07 Molecular characterization of a new phytoplasma causing disease in weste cedar. In this reporting period, we discovered a phytoplasma as the probable cause of a previously unreported disease of western cedar Juniperus occidentalis. The finding suggests that phytoplasma infection of Gymnosperms may be more common than previously thought. Using molecular methods for detection and identification, we found that the phytoplasma is related to a phytoplasma associated with a disease of almond trees reported in Lebanon and Iran, but the juniper witches�-broo phytoplasma represents a previously undescribed lineage. The discovery and characterization of this phytoplasma provided molecular markers for its detection and identification, expanded the known biodiversity of phytoplasmas discovered to infect conifers, and posed the question of whether western cedar could play a role in spread of phytoplasmal diseas in forest or landscape conifers. 08 We continued studying the genomes of phytoplasmas, unique plant disease- causing bacteria whose genomes contain �islands� called sequence variabl mosaics (SVMs), that were formed by viruses (phages). We initiated functional analysis of proteins encoded by the SVMs, to learn more about the interactions of phytoplasmas with their plant and insect hosts. Results so far indicate that some proteins encoded by these genomic islands possess activity that likely plays a role in those interactions. 09 Evolution of phytoplasma pathogens. While investigating phytoplasmas infecting onion, scientists from the Molecular Plant Pathology Laborator at Beltsville, MD, in collaboration with scientists from Lithuania, discovered a single nucleotide polymorphism (SNP) in the 16S rRNA gene that was a powerful molecular marker of evolutionary divergence of two major branches of phytoplasmas. In other work, we discovered, identified and classified two phytoplasma lineages associated with diseases in frui trees. The study opened new avenues for characterizing phytoplasmas in relation to single rRNA operons and interoperon sequence-heterogeneous rRNA genes, made recommendations for sequencing single-copy conserved genes, defined single nucleotide polymorphisms (SNPs) as markers for phytoplasma differentiation and predictions of affiliations, and propose a new convention for designating specific SNPs. The accomplishment provides information important for devising measures to mitigate the spread of phytoplasmal diseases of vegetables and fruit trees. 10 Phytoplasma classification presents some significant challenges. Sequenc heterogeneity of rrn operons and/or high level 16S rDNA sequence conservation can hinder differentiation of closely-related phytoplasmas, and then only an additional genetic marker can help to identify a new lineage. We proposed that a single copy gene, such gene (secA), which encodes SecA ATPase, should be incorporated into phytoplasma classification; one. In this reporting period, we expanded our work on t secA gene and confirmed its utility as a new molecular genetic marker fo detection and identification of phytoplasmas. The accomplishment provide elements important for eventual molecular descriptions of phytoplasmas, needed capability because phytoplasmas cannot be isolated in culture in the test tube.
Impacts
(N/A)
Publications
- Zhao, Y., Wei, W., Davis, R.E., Lee, I. 2010. Recent advances in 16S rRNA gene-based phytoplasma differentiation, classification and taxonomy. In: Weintraub, P., Jones, P., editors. Phytoplasmas: Genomes, Plant Hosts and Vector. Wallingford, UK: CABI Publishing. p. 64-92.
- Lee, I., Zhao, Y., Davis, R.E. 2010. Prospect of multiple gene-based systems for differentiation and classification of phytoplasmas. In: Weintraub, P., Jones, P., editors. Phytoplasmas: Genomes, Plant Hosts and Vectors. Wallingford, UK: CABI Publishing. p. 51-63.
- Lee, I., Bottner-Parker, K.D., Zhao, Y., Davis, R.E., Harrison, N. 2010. Phylogenetic analysis and delineation of phytoplasmas based on the secY gene. International Journal of Systematic and Evolutionary Microbiology. 60:2887-2897.
- Davis, R.E., Dally, E.L., Zhao, Y., Lee, I., Jomantiene, R., Detweiler, A. J., Putnam, M.L. 2010. First report of a new subgroup 16SrIX-E, 'Candidatus Phytoplasma phoenicium'-related, phytoplasma associated with juniper witches' broom disease in Oregon. Plant Pathology. 20:35.
- Zhao, Y., Wei, W., Lee, I., Shao, J.Y., Suo, X., Davis, R.E. 2009. Construction of an interactive online phytoplasma classification tool, iPhyClassifier, and its application in analysis of the peach X-disease phytoplasma group (16SrIII). International Journal of Systematic and Evolutionary Microbiology. 59(10):2582-2593.
- Secor, G.A., Rivera, V.V., Abad, J.A., Lee, I., Clover, G.R., Liefting, L. W., Li, X., De Boer, S.H. 2009. Association of "Candidatus Liberibacter solanacearum" with zebra chip disease of potato established by graft and psyllid transmission, electron microscopy and PCR. Plant Disease. 93:574- 583.
- Jomantiene, R., Davis, R.E., Lee, I., Zhao, Y., Bottner-Parker, K.D., Valiunas, D., Petkauskaite, R. 2010. Onion is a host for two phytoplasma lineages, subgroups 16SrI-A and 16SrI-L, in Lithuania: a HinfI site revealed a SNP marking divergent branches of evolution. Journal of Plant Pathology. 92:461-470.
- Valiunas, D., Davis, R.E., Jomantiene, R. 2009. Establishment of a new Phytoplasma subgroup, 16SRI-Q, to accommodate a previously undescribed Phytoplasma found in diseased cherry in Lithuania. Journal of Plant Pathology. 91:71-75.
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Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) Establish molecular biomarkers of evolutionary divergence for spiroplasma and phytoplasma detection and identification. Expand the molecular scheme for phytoplasma classification through genome-based selection of "constellations of identifier genes". Identify and characterize plant pathogenic mollicute genes involved in pathogenesis and symptom induction for eventual establishment of identifiers of pathotypes. Determine the correlation of chromosomally-integrated repetitive sequences and extrachromosomal DNAs in genome plasticity and biological diversity of phytoplasmas. Develop new practical strategies for the enhancement of plant defenses against diseases caused by phytoplasma and spiroplasma. Approach (from AD-416) Genome-based information will form the basis for improving detection, identification, and classification of plant pathogenic, wall-less prokaryotes (class Mollicutes). Gene sequencing, bioinformatics methods, recombinant DNA technology, phylogenetic and gene evolution analyses, enzyme and bacterial growth assays, gene function analyses, and biochemical methods will be employed with inoculations of susceptible plants, tissue culture and plant transformation, to investigate the structure and function of DNA sequences and gene products as molecular markers for detection and identification of genus-, species-, and strain- level evolutionary divergence. Phytoplasmas associated with newly emerging diseases will be identified, and molecular criteria for improving descriptions of 'Candidatus Phytoplasma' species and for developing a formal taxonomy will be sought. DNA sequences enabling genome plasticity and biological diversity will be investigated as potential molecular biomarkers for making finer distinctions at species, strain, and population levels. Agrobacterium-mediated stable transformation and plant viral vector-mediated transient expression technologies will be used to introduce antimicrobial peptide genes and other defense-related genes into plants, enhancing natural plant defense and mitigating phytoplasmal and spiroplasmal diseases. Significant Activities that Support Special Target Populations Plant diseases caused by bacteria called phytoplasmas and spiroplasmas are responsible for economic losses in U.S. and world agriculture. Knowledge from genomics will make it possible to devise improved and novel technologies for pathogen detection and classification to aid efforts to reduce crop losses due to plant diseases through improved crop management, production of certified disease-free germplasm, and implementation of plant quarantine regulations. This reporting year, our research has progressed on several fronts. We completed characterization of a new �Cadidatus Phytoplasma� species associated with emerging diseases in soybean, sweet pepper, and passion fruit in Costa Rica, and identified the phytoplasma strains associated with potato purple top diseases in Montana as a new subgroup. We designed technology for amplification of ribosomal protein L15 (rp10), adenylate kinase (adk), methionine aminopeptidase (map), and protein translocase (secY) gene sequences from 31 phytoplasma strains representing 11 phytoplasma groups and complete phylogenetic analysis based on secY gene sequence. Use of secY gene as biomarkers permits finer differentiation of closely related phytoplasma strains. We discovered a new phytoplasma associated with salt cedar witch�s-broom disease occurred in northwestern China. The phytoplasma represents a novel �Ca. Phytoplasma� species and a new 16Sr group. We completed a genetic variability study on phytoplasmas that cause Bois noir (BN) disease in grapevine, and identified new and distinct subgroup and single nucleotide polymorphism lineages in BN phytoplasma populations in Italy. We identified and characterized novel phytoplasma strains associated with cherry decline disease in Lithuania and sunshine tree stem fasciation disease in China. Distinct molecular markers were found that can be used to identify these new phytoplasma strains. We also devised an interactive online tool, iPhyClassifier, for rapid phytoplasma identification, classification and taxonomic assignment; bringing these capabilities within reach of scientists and students worldwide. Built upon our previous development of the computer-simulated 16S rDNA RFLP analysis system, the new online tool provides a user- friendly platform for multiple applications pertaining to phytoplasma classification and taxonomy including delineation of new phytoplasma groups/subgroups and discovery of new �Ca. Phytoplasma� species. In addition, we made substantial progress in piecing together the complex genome sequence of Spiroplasma kunkelii, cause of corn stunt disease - a major factor in corn production in the Americas. Following our discovery of a unique phytoplasma genome architecture (Sequence-Variable Mosaics, SVMs), we have determined SVMs were formed by recurrent and targeted virus (phage) attacks. We postulated that phage-mediated gene exchange enabled phytoplasmal transkingdom parasitism and pathogenicity, and we are examining genes responsible for the unique biology of phytoplasmas. Technology Transfer Number of Web Sites managed: 1
Impacts
(N/A)
Publications
- Wei, W., Lee, I., Davis, R.E., Suo, X., Zhao, Y. 2008. Automated RFLP pattern comparison and similarity coefficient calculation for rapid delineation of new and distinct phytoplasma 16S rDNA subgroup lineages. International Journal of Systematic and Evolutionary Microbiology. 58:2368- 2377.
- Lee, I., Bottner, K.D., Sun, M. 2009. An emerging potato purple top disease caused by a new 16SrIII group phytoplasma in Montana. Plant Disease. 93:574-583.
- Villalobos, W., Moreira, L., Rivera, C., Lee, I. 2009. First report of new phytoplasma diseases associated with soybean, sweet pepper, and passion fruit in Costa Rica. Plant Disease. 93:201.
- Wei, W., Davis, R.E., Jomantiene, R., Zhao, Y. 2008. Ancient, recurrent phage attacks and recombination events shaped dynamic sequence-variable mosaic structures at the root of phytoplasma genome evolution. Proceedings of the National Academy of Sciences. 105:11827-11832.
- Li, W.N., Abad, J.A., French-Monar, R.D., Rascoe, J., Wen, A., Gudmestad, G., Secor, G.A., Lee, I., Levy, L. 2009. Multiplex real-time PCR for detection, identification and quantification of "Candidatus Liberibacter solanacearum" in potato plants with zebra chip. Phytopathology. 78:59-65.
- Quaglino, F., Zhao, Y., Bianco, P.A., Wei, W., Casati, P., Durante, G., Davis, R.E. 2009. New 16Sr subgroups and distinct SNP lineages among grapevine Bois noir phytoplasma populations. Annals of Applied Biology. 154:279-289.
- Valiunas, D., Jomantiene, R., Ivanauskas, A., Abraitis, R., Staniene, G., Zhao, Y., Davis, R.E. 2009. First Report of a New Phytoplasma Subgroup, 16SrIII-S, Associated with Decline Disease Affecting Sweet and Sour Cherry Trees. Plant Disease. 93:500.
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) Establish molecular biomarkers of evolutionary divergence for spiroplasma and phytoplasma detection and identification. Expand the molecular scheme for phytoplasma classification through genome-based selection of "constellations of identifier genes". Identify and characterize plant pathogenic mollicute genes involved in pathogenesis and symptom induction for eventual establishment of identifiers of pathotypes. Determine the correlation of chromosomally-integrated repetitive sequences and extrachromosomal DNAs in genome plasticity and biological diversity of phytoplasmas. Develop new practical strategies for the enhancement of plant defenses against diseases caused by phytoplasma and spiroplasma. Approach (from AD-416) Genome-based information will form the basis for improving detection, identification, and classification of plant pathogenic, wall-less prokaryotes (class Mollicutes). Gene sequencing, bioinformatics methods, recombinant DNA technology, phylogenetic and gene evolution analyses, enzyme and bacterial growth assays, gene function analyses, and biochemical methods will be employed with inoculations of susceptible plants, tissue culture and plant transformation, to investigate the structure and function of DNA sequences and gene products as molecular markers for detection and identification of genus-, species-, and strain- level evolutionary divergence. Phytoplasmas associated with newly emerging diseases will be identified, and molecular criteria for improving descriptions of 'Candidatus Phytoplasma' species and for developing a formal taxonomy will be sought. DNA sequences enabling genome plasticity and biological diversity will be investigated as potential molecular biomarkers for making finer distinctions at species, strain, and population levels. Agrobacterium-mediated stable transformation and plant viral vector-mediated transient expression technologies will be used to introduce antimicrobial peptide genes and other defense-related genes into plants, enhancing natural plant defense and mitigating phytoplasmal and spiroplasmal diseases. Significant Activities that Support Special Target Populations This progress contributes to NP303 Plant Diseases; Component I, Disease Diagnosis: Detection, Identification, Characterization of Plant Pathogens. Plant diseases caused by bacteria called phytoplasmas and spiroplasmas are responsible for economic losses in U.S. and world agriculture. Progress is based on the hypothesis that knowledge from genomics will make it possible to devise improved and novel technologies for pathogen detection and classification to aid efforts to reduce crop losses due to plant diseases through improved crop management, production of certified disease-free germplasm, and implementation of plant quarantine regulations. This reporting year, our research has progressed on several fronts. We improved classification of phytoplasmas, and identified and classified phytoplasmas associated with diseases whose causes had been unsolved. We identified and characterized a new phytoplasma associated with emerging diseases associated with soybean, sweet pepper, and passion fruit in Costa Rica.; identified a new phytoplasma strain associated with potato purple top diseases in Montana and Alaska, the first group 16SrIII phytoplasma associated with a potato disease in the U.S.; designed technology for amplification of ribosomal protein L15 (rp10), adenylate kinase (adk), methionine aminopeptidase (map), and protein translocase (secY) gene sequences as biomarkers for finer differentiation of strains; and completed research on genetic diversity of phytoplasmas affecting cactus plants, gaining new insight into relationships between ecosystem distribution and emergence of new phytoplasma lineages. We also devised, and made available online, automated computer-assisted programs for phytoplasma identification and classification; bringing these capabilities within reach of scientists and students worldwide. Our development of the computer-simulated 16S rDNA RFLP analysis system led to our delineation of 10 new putative phytoplasma species. In addition, we made substantial progress in piecing together the complex genome sequence of Spiroplasma kunkelii, cause of corn stunt disease - a major factor in corn production in the Americas. We determined gene sequences of previously unknown regions of the chromosome and gained insight into how the spiroplasma genome is organized. This progress is leading to a complete genetic blueprint of this pathogen, information critical for identifying and targeting molecules for safe and effective disease control. Fifth, following our discovery of a unique genome architecture (Sequence-Variable Mosaics, SVMs), we postulated that these structures formed, by mobile genetic elements, early in phytoplasma evolution and are at the root of the evolutionary branch giving rise to phytoplasmas. In a major advance, we determined that ancient, targeted attacks by phage (bacterial virus) was responsible for SVM formation, and we documented the distribution, composition, and physical characteristics of the phage genomes integrated within SVMs in phytoplasma chromosomes. This advance points to genes responsible for the unique biology of phytoplasmas and explains the emergence of new life forms, including pathogens.
Impacts
(N/A)
Publications
- Cai, H., Wei, W., Davis, R.E., Chen, H., Zhao, Y. 2008. Genetic diversity among phytoplasmas infecting Opuntia: virtual RFLP analysis identifies new subgroups in the peanut witches'-broom phytoplasma group. International Journal of Systematic and Evolutionary Microbiology. 58:1448-1457.
- Lee, I., Zhao, Y., Davis, R.E., Wei, W., Martini, M. 2007. Prospects of DNA-based Systems for Differentiation and Classification of Phytoplasmas. Bulletin of Insectology. 60:239-244.
- Munyaneza, J.E., Crosslin, J., Lee, I. 2007. Phytoplasmas Diseases and Insect Vectors in Potatoes of the Pacific Northwest of the United States. Bulletin of Insectology 60:181-182
- Wei, W., Davis, R.E., Lee, I., Zhao, Y. 2007. Computer-simulated RFLP analysis of 16S rRNA genes: Identification of 11 new phytoplasma groups. International Journal of Systematic and Evolutionary Microbiology. 57:1855- 1867.
- Wei, W., Hua, J., Yang, Y., Youfu, W., Davis, R.E., Zhao, Y. 2007. Molecular identification of a new phytoplasma strain associated with the first observation of jujube witches'-broom disease in northeastern China. Plant Disease. 91:1364.
- Zhao, Y., Sun, Q., Davis, R.E., Lee, I., Liu, Q. 2007. First report of witches�-broom disease in a Cannabis species and its association with a phytoplasma of elm yellows group (16SrV). Plant Disease. 91:227.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) Establish molecular biomarkers of evolutionary divergence for spiroplasma and phytoplasma detection and identification. Expand the molecular scheme for phytoplasma classification through genome-based selection of "constellations of identifier genes". Identify and characterize plant pathogenic mollicute genes involved in pathogenesis and symptom induction for eventual establishment of identifiers of pathotypes. Determine the correlation of chromosomally-integrated repetitive sequences and extrachromosomal DNAs in genome plasticity and biological diversity of phytoplasmas. Develop new practical strategies for the enhancement of plant defenses against diseases caused by phytoplasma and spiroplasma. Approach (from AD-416) Genome-based information will form the basis for improving detection, identification, and classification of plant pathogenic, wall-less prokaryotes (class Mollicutes). Gene sequencing, bioinformatics methods, recombinant DNA technology, phylogenetic and gene evolution analyses, enzyme and bacterial growth assays, gene function analyses, and biochemical methods will be employed with inoculations of susceptible plants, tissue culture and plant transformation, to investigate the structure and function of DNA sequences and gene products as molecular markers for detection and identification of genus-, species-, and strain- level evolutionary divergence. Phytoplasmas associated with newly emerging diseases will be identified, and molecular criteria for improving descriptions of 'Candidatus Phytoplasma' species and for developing a formal taxonomy will be sought. DNA sequences enabling genome plasticity and biological diversity will be investigated as potential molecular biomarkers for making finer distinctions at species, strain, and population levels. Accomplishments Developed a computer-simulated restriction fragment length polymorphism (RFLP) analysis method to facilitate 16S rDNA sequence-based phytoplasma classification: Phytoplasmas are a large group of small bacteria that infect several hundred plant species, causing numerous diseases in economically important vegetable, cereal, fruit, ornamental, and forest crops worldwide. Since phytoplasmas cannot be cultivated in the laboratory, DNA fingerprinting is the best way to distinguish them from one another. Conventional DNA fingerprinting involves several complicated and expensive laboratory procedures. Scientists from the Molecular Plant Pathology Laboratory at Beltsville, MD, in collaboration with a visiting scientist from China, exploited computer programs to mimic the laboratory DNA fingerprinting procedures and rapidly generated digital DNA fingerprint patterns. This accomplishment determined the classification status of numerous previously unclassified phytoplasmas, delineated 10 new phytoplasma groups, identified seven potential new phytoplasma species, and provided research community an updated set of standard fingerprint patterns for phytoplasma identification. The method and findings of this accomplishment will help phytoplasma researchers, plant doctors, and quarantine personnel to identify and classify phytoplasmas more quickly. NP303 Plant Diseases; Component 1, Disease Diagnosis: Detection, Identification, and Characterization of Plant Pathogens; Problem Statement: 1A New Diagnostic Methods and Tools. Developed a computer program for automated RFLP pattern comparison and similarity coefficient calculation: Differentiation and classification of unculturable phytoplasmas are mainly relied on DNA fingerprinting of conserved genes. Visual comparison of DNA fingerprint patterns of numerous and diverse phytoplasmas has become increasingly difficult. Scientists from the Molecular Plant Pathology Laboratory at Beltsville, MD have developed a computer program that can distinguish subtle differences among multiple phytoplasma DNA fingerprints and calculate the degree of their similarities. This accomplishment greatly eases the task of fingerprint pattern comparison and it helped identify a large number of new phytoplasma fingerprint patterns and therefore potential new and distinct phytoplasma strains. NP303 Plant Diseases; Component 1, Disease Diagnosis: Detection, Identification, and Characterization of Plant Pathogens; Problem Statement: 1A New Diagnostic Methods and Tools. Identified phytoplasmas associated with symptomatic potato plants in Russia: Many commercial potato plants grown in various regions in Russia have exhibited symptoms resembling those caused by phytoplasmas. Phytoplasmal etiology of the disease has been long suspected but has never been proved by means of molecular-based procedures. In collaboration with scientists from Russia, we employed PCR-RFLP assay procedures developed in our laboratory for the detection and identification of the phytoplasmas that may be associated with these symptomatic potato plants. The results indicated the majority of the symptomatic plants were associated with phytoplasmas. At least three distinct phytoplasma strains belonging to groups 16SrXII and 16SrI were identified. The accomplishment will aid implementation of quarantine regulation and it will help Russian researchers, extension workers and plant diagnosticians to identify the pathogens and to combat this disease. NP303 Plant Diseases; Component 1, Disease Diagnosis: Detection, Identification, and Characterization of Plant Pathogens; Problem Statement: 1B Detection, Identification, Characterization, and Classification of Pathogens. Confirmed that phytoplasma plays a role in a newly emerging disease complex, potato zebra-chip, in Texas: A newly emerging potato disease (potato zebra chip), first identified in commercial potato fields in Texas and Mexico in 1995, has become widespread since 2000, resulting in serious economic losses in both fresh and processing markets. The disease has since been found in several potato producing areas in the US including Texas and Nebraska. Infected potato plants exhibit a wide range of symptoms similar to the purple top wilt syndrome, including leaf curl, mild chlorosis, bud breaking, swollen nodes, aerial tubers, vascular discoloration, and wilting. The infected tubers exhibit extensive brown discoloration of the medullary rays throughout the entire tuber, and death of eyes which do not sprout, or rarely produce hair sprouts. Chips made from diseased potatoes develop dark brown banding patterns, termed 'Zebra chip'. The severity of ZC symptoms varies with sources of infected potato plants. ZC infected potato plants in Dalhart, TX usually exhibit mild symptoms with less brown discoloration on tubers. Extensive testing over several years has failed to find the cause(s) of the disease. In cooperation with scientists from North Dakota State University, we have initially identified a new phytoplasma (�Candidatus Phytoplasma americanum�) that may play a significant role in disease development in some, but not in all, potato growing areas. In this accomplishment we demonstrated that by graft-inoculation of infected potato tissues (collected from Dalhart, TX) onto healthy plants in the greenhouse, we were able to transmit the associated phytoplasma to both tomato and potato. The inoculated potato plants with scions prepared from either infected potato or tomato plants exhibited similar symptoms as those infected with ZC in the fields. PCR assays confirmed that the new phytoplasma was consistently associated with symptomatic plants. The results confirmed the hypothesis that the new phytoplasma play a role in the development of ZC symptoms. The accomplishment will aid implementation of quarantine regulation and it will help Russian researchers, extension workers and plant diagnosticians to identify the pathogens and to combat this disease. NP303 Plant Diseases; Component 1, Disease Diagnosis: Detection, Identification, and Characterization of Plant Pathogens; Problem Statement: 1B Detection, Identification, Characterization, and Classification of Pathogens. Identified and characterized a new disease, sesame witches�-broom, in Oman: Sesame is cultivated in the Sultanate of Oman mainly in Dakhliya region for seed and animal feed. Recently sesame crops have been found associated with witches broom disease. During a field survey in November � December 2005, symptoms of phyllody (leaf-like floral structure) and virescence (green discoloration of flowers) that are usually associated with phytoplasmal infection have been observed in sesame fields. The causal agent of sesame witches�- broom disease and its identity have never been clarified. In collaboration with scientists from Oman, we detected by PCR assay a phytoplasma that was associated with symptomatic, but not asymptomatic sesame plants. Based on RFLP analysis and sequencing of PCR-amplified 16S rDNA sequences, the phytoplasma was identified as a new member of phytoplasma group 16SrII, subgroup 16SrII-D. The phytoplasma is most closely related to another Omani phytoplasma that is the causal agent of Alfalfa witches�-broom. The accomplishment will aid implementation of quarantine regulation and it will help local extension workers and plant diagnosticians in Oman to identify the pathogens and to combat this disease. NP303 Plant Diseases; Component 1, Disease Diagnosis: Detection, Identification, and Characterization of Plant Pathogens; Problem Statement: 1B Detection, Identification, Characterization, and Classification of Pathogens. Phytoplasma genomes: We have analyzed genome architectures of diverse phytoplasma strains and explored structural motifs, including sequence- variable mosaics and conserved sequence repeats as, potential biomarkers for phytoplasma detection, differentiation, and classification. This progress will advance work by epidemiologists and other scientists interested in strain identification and characterization of phytoplasma populations in relation to host-pathogen interactions. NP303 Plant Diseases; Component 2, Biology, Ecology, Epidemiology, and Spread of Plant Pathogens and their Relationships with Hosts; Problem Statement: Pathogen Biology, Virulence Determinants, and Genetics of the Pathogen; and Component 1, Disease Diagnosis: Detection, Identification, and Characterization of Plant Pathogens; Problem Statement: 1B Detection, Identification, Characterization, and Classification of Pathogens. New phytoplasmas and phytoplasma hosts in Europe: Previously unknown plant hosts of phytoplasmas and new phytoplasmas were identified in Europe. This research will be of interest to scientists involved in plant disease diagnostics centers, implementation of quarantines, production of disease-free plant materials, and disease control. NP303 Plant Diseases; Component 1, Disease Diagnosis: Detection, Identification, and Characterization of Plant Pathogens; Problem Statement: 1B Detection, Identification, Characterization, and Classification of Pathogens. Technology Transfer Number of Web Sites managed: 3 Number of Non-Peer Reviewed Presentations and Proceedings: 3
Impacts
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Publications
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