2008 Annual Report 1a.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. 1b.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. 3.Progress Report 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. 4.Accomplishments 1. Developed a computer-simulated 16SrDNA RFLP analysis system for identification and classification of phytoplasmas. Over the last few years, numerous emerging plant diseases worldwide have been reported to be associated with diverse phytoplasmas. Because phytoplasmas cannot grow outside their hosts, they cannot be differentiated and classified by traditional methods that are applied to culturable prokaryotes. Over the past decade, establishment of a phytoplasma classification scheme based on 16S rDNA restriction fragment length polymorphism (RFLP) patterns made possible the accurate and reliable identification and classification of a wide range of phytoplasmas. In the present study, we expanded this classification scheme through the use of computer-simulated RFLP analysis, achieving rapid differentiation and classification of phytoplasmas. This study classified more than 250 previously unclassified phytoplasmas, delineated 10 new phytoplasma groups. This accomplishment provides a new practical tool for rapid identification of phytoplasma strains and for discovery of novel phytoplasma species. The publication reporting this study was selected by the International Journal of Systematic and Evolutionary Microbiology as a “hot off the press” article and was highlighted by Microbiology Today. NP303 Plant Diseases; Component 1, Disease Diagnosis: Detection, Identification, and Characterization of Plant Pathogens; Problem Statement: 1B Detection, Identification, Characterization, and Classification of Pathogens. 2. Identified potential biomarkers for finer differentiation of phytoplasma strains. We have designed PCR primers for amplification of ribosomal protein L15 (rp10), adenylate kinase (adk), methionine aminopeptidase (map), and protein translocase (secY) gene sequences. These conserved house keeping genes are more variable than 16S rRNA gene and represent potential biomarkers that will facilitate differentiation of closely related phytoplasma strains. This progress will advance work by epidemiologists and other scientists interested in identification of biologically or ecologically distinct phytoplasma strains involved in various phytoplasmal diseases. NP303 Plant Diseases; component 1, Disease Diagnosis: Detection, Identification, and Characterization of Plant Pathogens; Problem Statement: 1B Detection, Identification, Characterization, and Classification of pathogens. NP303 Plant Diseases; Component 1, Disease Diagnosis: Detection, Identification, and Characterization of Plant Pathogens; Problem Statement: 1B Detection, Identification, Characterization, and Classification of Pathogens. 3. Phytoplasma genome architecture and foreign gene acquisition. We have continued our analysis of the unique phytoplasmal genome architecture that is characterized by structures that we have named sequence-variable mosaics (SVMs). We gained evidence that these structures were formed early in phytoplasma evolution -- after divergence of phytoplasmas from their acholeplasma-like ancestors but before diverse phytoplasmas evolved. Thus, we found evidence for SVMs in phytoplasmas representing diverse branches of the phytoplasma evolutionary tree, placing SVM formation at the root of phytoplasma evolution. We postulated that the size and nonrandom chromosomal distribution of SVMs could be explained by recurrent, mobile element attack targeted to specific regions in the phytoplasma chromosome, and that hyper-variable regions within the SVMs remained active as sites for horizontal acquisition of foreign genes. This scientific advance provided a fundamental basis for our subsequent discovery of the nature of mobile element(s) responsible for forming SVM and launching evolution of the phytoplasma clade. 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, and the evolutionary emergence of new kinds of pathogens. 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. 4. Phage attacks explain SVMs and evolutionary emergence of phytoplasma pathogens. Mobile genetic elements have impacted biological evolution across all studied organisms, but evidence for a role in launching evolutionary emergence of an entire phylogenetic clade has not been forthcoming. Our previous studies discovered a unique feature of phytoplasmal genome architecture, genes clustered in sequence variable mosaics (SVMs), and suggested that such structures formed through recurrent, targeted attacks by mobile elements. In the present study, we discovered that cryptic prophage remnants, originating from phages in the order Caudovirales, formed the SVMs and comprised exceptionally large percentages of the chromosomes of ‘Candidatus Phytoplasma asteris’-related strains OYM and AYWB, occupying nearly all major non-syntenic sections, and accounting for most of the overall size difference between the two genomes. The clustered phage remnants formed genomic islands exhibiting distinct DNA physical signatures. Numerous phytoplasma strain-specific genes were identified as phage-carried, laterally-transferred genes, indicating that prophage remnants played important roles in generating phytoplasma genetic diversity. Since no SVM-like structures could be identified in genomes of ancestral relatives including Acholeplasma spp., we hypothesize that ancient phage attacks leading to SVM formation occurred after divergence of phytoplasmas from acholeplasmas, triggering evolution of the phytoplasma clade. NP303 Plant Diseases; Component 1, Disease Diagnosis: Detection, Identification, and Characterization of Plant Pathogens; Problem Statement: 1B Detection, Identification, Characterization, and Classification of Pathogens 5. Completed research on genetic diversity of phytoplasmas affecting cactus plants. Discovered that: i) cacti can be infected by two distinctly different groups of phytoplasmas, ii) significant genetic diversity exists among phytoplasmas even belonging to the same group, and iii) the extent of phytoplasma genetic diversity differs, depending on geographical and ecological characteristics. As a result of this research, several new phytoplasma subgroup lineages were identified. NP303 Plant Diseases; Component 1, Disease Diagnosis: Detection, Identification, and Characterization of Plant Pathogens; Problem Statement: 1B Detection, Identification, Characterization, and Classification of Pathogens 6. Identified and characterized new phytoplasma strains and a new phytoplasma host in China. Phytoplasma infection in jujube causes witches’-broom disease, which severely reduces the quantity and quality of jujube production. The disease has spread rapidly in jujube-growing regions in Asia recently. The present study identified a new phytoplasma strain affecting jujube in northern China and a closely related phytoplasma strain that infects hemp fiber. Hemp fiber is a new host of phytoplasmas in the elm yellows group (16SrV) and a potential reservoir for jujube witches’-broom disease. The findings point to genetic diversity and ecological complexity of phytoplasmas causing jujube witches’-broom disease. This accomplishment provides new insights for management of jujube witches’-broom 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. 7. 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. 8. Phytoplasma identified in disease of potato. Identified a new phytoplasma strain associated with potato purple top diseases in Montana and Alaska: Potato purple top (PPT)-related diseases have caused tremendous damage to potato tuber production in South America, Mexico, the US, and elsewhere in the world. Many of these diseases are caused by phytoplasmas. In 2007, sporadic diseased potato plants exhibiting symptoms including rosette and stunting of plants with purple discoloration of top leaves and shoot proliferation were found in one field in Montana and some fields in western Alaska close to Canadian border. In collaboration with scientists from Montana and Alaska, we identified that the phytoplasma strains associated with symptomatic plants belong to members of 16SrIII group and are most closely related to subgroup 16SrIII-F (milkweed yellows and related phytoplasma strains). This phytoplasma can be transmitted through tubers. This is the first report that 16SrIII group phytoplasmas are associated with potato diseases in the U.S. This accomplishment will aid implementation of quarantine regulations and will help extension workers and plant diagnosticians to identify and combat these newly emerging diseases. This progress will be of interest to scientists involved in production of disease-free seed potatoes. NP303 Plant Diseases; component 1, Disease Diagnosis: Detection, Identification, and Characterization of Plant Pathogens; Problem Statement: 1B Detection, Identification, Characterization, and Classification of pathogens. 9. Identified potential biomarkers for finer differentiation of phytoplasma strains: We have designed PCR primers for amplification of ribosomal protein L15 (rp10), adenylate kinase (adk), methionine aminopeptidase (map), and protein translocase (secY) gene sequences. These conserved house keeping genes are more variable than 16S rRNA gene and represent potential biomarkers that will facilitate differentiation of closely related phytoplasma strains. This progress will advance work by epidemiologists and other scientists interested in identification of biologically or ecologically distinct phytoplasma strains involved in various phytoplasmal diseases. NP303 Plant Diseases; component 1, Disease Diagnosis: Detection, Identification, and Characterization of Plant Pathogens; Problem Statement: 1B Detection, Identification, Characterization, and Classification of pathogens. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of New Commercial Licenses Executed 9 Review 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.