2003 Annual Report 1.What major problem or issue is being resolved and how are you resolving it? Emerging and re-emerging plant diseases due to walled bacteria and cell wall less microbes like phytoplasmas and spiroplasmas are causing economic losses to agricultural crops and damage to plants in the natural environment. To resolve these problems, we are investigating the detection, identification, genomics, and molecular biology of the pathogens and their interactions with plants. Our work contributes to reduction of pesticide use, aids efforts by APHIS to prevent introduction of exotic bacteria and phytoplasmas into the U.S., and enhances the export of U.S. plant products. 2.How serious is the problem? Why does it matter? Many agriculturally important crops are seriously affected by phytoplasma, spiroplasma, and bacterial diseases that cause economic losses to farmers in the U.S. and worldwide. The pathogens also impact U.S. exports of commodities and importation of useful germplasm. Our work will help to reduce these losses and will enhance movement of useful plant germplasm. 3.How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? National Program 303, Plant Diseases (100%). This research provides the basis for the design and development of new and improved tools and technologies for efficient, economical, and environmentally friendly management and control of plant diseases. Our research on the spiroplasma and phytoplasma genomes will (i) reveal the genetic basis for pathogenicity (ii) reveal targets for environmentally friendly molecular disease control, and (iii) lead to insights needed to understand specificity of their transmission by insects. 4.What were the most significant accomplishments this past year? A. Single Most Significant Accomplishment during FY 2003 (one per Research Project New knowledge gained from genome information is critical for developing novel strategies to combat Spiroplasma kunkelii, a wall-less bacterium that causes the devastating corn stunt disease of corn (maize) in the Americas. In the past year, the laboratory's Spiroplasma kunkelii genome project entered the annotation phase and significant progress has been made on two fronts: detailed annotation of large genome segments and assembly of major metabolic/functional pathways. Genes involved in different biochemical and physiological processes have been identified and categorized; several genes have been marked as candidates that might encode virulence factors. This accomplishment will aid scientists in the search for molecular targets and development of therapeutic agents against the disease. Other significant accomplishments (1) Genomic insertion sequences provide an avenue to investigate unknown mechanisms that control ability of phytoplasmas to induce varied symptoms in diseased plants. Insertion sequences (ISs) are small, mobile genetic elements that can insert into target DNA molecules with various degrees of site specificity, altering gene expression. Scientists from the Molecular Plant Pathology Laboratory at Beltsville, MD, identified putative IS elements in the genome of Aster Yellows (AY) phytoplasma that may account for chromosomal rearrangements, change of phenotype, and strain evolution of AY strains. The accomplishment will aid scientists to develop molecular tools for improved strain differentiation and for the study of pathogen-host interactions. (2) Phytoplasmas have a very small genome size and relatively few genes that approach the minimum number required for cellular life, parasitism, and pathogenicity in plants and insects. In a study of the evolution of parasitism and pathogenicity in phytoplasmas, scientists from the Molecular Plant Pathology Laboratory at Beltsville, MD, found that some genes undergo a process of degeneration leading to eventual elimination, and postulated that this process is part of an overall genome degradation and reduction of genome size still taking place as these microbes continue to evolve their host niche specialization. These findings begin to explain why phytoplasmas cannot be isolated in artificial culture, and point to new molecular targets for potential disease control. (3) More than a dozen phytoplasmas are associated with diseases of strawberry, causing deformation of fruit, leaf yellowing, stunting, and heavy fruit loss as well as plant death, but control of phytoplasmal diseases in strawberry has been very difficult because of the lack of curative methods and the lack of plant varieties resistant to phytoplasmas. Scientists from the Molecular Plant Pathology Laboratory at Beltsville, MD, in collaboration with Shandong Institute of Pomology, Shandong, China, established a new protocol for efficient genetic transformation and regeneration of strawberry plants. Using the protocol, phloem-specific expression of a reporter gene was achieved in genetically modified strawberry plants. This accomplishment opens an avenue for genetic engineering of disease-resistant strawberry plants, which will benefit growers and customers. (4) Aster yellows (AY) group (16SrI) phytoplasmas are associated worldwide with more than 100 diseases, several of which are quarantine regulated and economically important. Scientists from the Molecular Plant Pathology Laboratory at Beltsville, MD, found that combined use of 16S rRNA and ribosomal protein gene sequences enabled differentiation of 18 distinct subgroups in the AY group. A new species, 'Candidatus Phytoplasma asteri', was proposed to represent the AY phytoplasma group. This accomplishment will aid plant quarantine agencies in implementing new regulations to prevent these pathogens from being introduced into new regions. (5) Sweet cherries are susceptible to bacterial, phytoplasmal, and viral diseases that cause severe fruit loss. Scientists from the Molecular Plant Pathology Laboratory at Beltsville, MD, in collaboration with Shandong Institute of Pomology, Shandong, China, established a high-frequency cherry rootstock transformation and regeneration method for future targeted expression of antimicrobial genes in genetically modified cherry rootstock. The aim is to provide a broad-spectrum resistance to phytoplasmal and viral diseases while keeping the scion, the fruit-bearing part of the plant, genetically natural. This accomplishment provides plant biotechnologists a useful tool to overcome the bottleneck of genetic engineering of disease-resistant cherry trees. (6) In Lebanon a phytoplasma strain (AlmWB) belonging to pigeon pea witches¿-broom phytoplasma group (16SrIX) is devastating almond production. Scientists from the Molecular Plant Pathology Laboratory at Beltsville, MD, in collaboration with scientists from American University of Beirut, Beirut, Lebanon, found that this phytoplasma could also infect peach and nectarine, and designed a sensitive molecular probe to facilitate the detection of the phytoplasma pathogen. This accomplishment enhances the capability of regulatory agencies to detect and prevent the spread of pathogens in stone fruit germplasms. (7) Peach yellows in India is caused by a phytoplasma (subgroup rpV-M) that is unique in this geographical region and has spread widely in peach growing areas in the northwestern Himalayas. Scientists from the Molecular Plant Pathology Laboratory at Beltsville, MD, in collaboration with scientists at Dr. Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan, India, discovered that nectarine yellows disease is caused by a distinct phytoplasma strain (subgroup rpV-N) closely related to the peach yellows phytoplasma. This accomplishment will aid international regulatory agencies in implementing new regulations to prevent these pathogens from being introduced into new regions. (8) Phytoplasma taxonomy has adopted designation of a 16S rRNA sequence for delineation of 'Candidatus Phytoplasma species', although phytoplasma genomes each contain two, often heterogeneous, 16S rRNA genes, making mixed sequence errors and misidentifications possible. Scientists from the Molecular Plant Pathology Laboratory at Beltsville, MD, in collaboration with scientists from the Institute of Botany and the Institute of Biochemistry at Vilnius, Lithuania, completed a study of ribosomal interoperon heterogeneity in the genomes of phytoplasmas and the potential impacts of the sequence heterogeneities in phytoplasma detection, classification, and taxonomy. This work proposed new guidelines for describing new 'Candidatus Phytoplasma species' by including both operons in species descriptions or alternatively designating the operon source of signature sequence data. This accomplishment advances the emerging molecular-based taxonomy of phytoplasmas by demonstrating the need for precision in assignment of signature DNA sequences that define new species. 5.Describe the major accomplishments over the life of the project, including their predicted or actual impact. Advances from predecessor project include: (i) discovery, characterization, and naming of a new class of disease causing organism (spiroplasma); (ii) discovery of the bacterium that causes ratoon stunting disease of sugarcane, (iii) discovery of spiroplasma as the cause of corn stunt disease, (iv) establishment of the first taxonomic scheme for spiroplasmas and the first taxonomic scheme for phytoplasmas, (v) discovery of phytoplasma as the free branching agent in commercial poinsettia. 6.What do you expect to accomplish, year by year, over the next 3 years? 2004 - Complete ribosomal protein gene analyses of diverse phytoplasmas to clarify the status of subgroups as putative species. Complete the sequencing of the Spiroplasma kunkelii genome. Clone and characterize non-ribosomal gene sequences from phytoplasmas; construct phylogenetic relationship based on the new genes. Clone and characterize the genes in two spiroplasma plasmids. Construct a spiroplasma gene vector and shuttle vector based on spiroplasma plasmid nucleotide sequence. Continue to analyze and annotate the S. kunkelii genome sequence in preparation for functional genomics study, with the emphasis centered on the pathogen¿s secretion systems and surface proteins. Analyze the transgenic strawberry and cherry plants for phloem-specific expression of the engineered antimicrobial / antiapoptotic genes. 2005 - Construct physical maps of aster yellows phytoplasma strains that induce various distinct symptom types and develop molecular markers of pathogenicity. Discover the identities of unknown phytoplasmas causing emerging and economically significant plant diseases. Complete comparative genomics of the S. kunkelii genome with genomes of whole genome sequenced bacteria. Characterize selected spiroplasma and phytoplasma genes for their involvement in pathogenesis and pathogen-host interactions. Test and select antimicrobial peptides for their bactericidal or growth inhibitory effects on spiroplasmas and phytoplasmas. 2006 - Design gene constructs for the delivery and expression of pathogen-inhibiting protein genes in diseased plants by the use of virus-based gene vectors. Clone and characterize pathogenicity domains in the genome of aster yellows phytoplasma. Analyze the transgenic strawberry and cherry plants for phytoplasmal/viral disease resistance. Develop more transgenic lines based on the results of the virulence factor and antimicrobial peptide studies conducted in the previous years. 7.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Technology on phytoplasma detection and identification was transferred to scientists, from U.S. universities and commercial companies and from foreign universities and research institutes, who have spent periods of time in our laboratory to learn our technologies as part of this technology transfer. These technologies have already been made available to, and are being used by, commercial users and the scientific community. No constraints to the durability of the technology are known, although further improvements in the procedures are expected to become available from our work during the next two to three years. 8.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). None. Review Publications Abou-Jawdah, Y., Dakhil, E., El-Mehtar, Shoaa, and Lee, I.M. Almond witches'-broom phytoplasma: a potential threat to almond, peach, and nectarine. Canadian Journal of Plant Pathology. 2003. v. 25. p. 28-32. Alminaite, A., Davis, R.E., Valiunas, D., Jomantiene, R. First report of a group 16SrI, subgroup B, phytoplasma in diseased Epilobium hirsutum in the region of Tallin, Estonia. Plant Disease. 2002. v. 86. p. 1177. Davis, R.E., Jomantiene, R., Kalvelyte, A., Dally, E.L. Differential amplification of sequence heterogeneous ribosomal RNA genes and classification of the 'Fragaria multicipita' phytoplasma. Microbiological Research. 2003. v. 158. p. 229-236. Jacobs, K.A., Lee, I.-M., Griffiths, H.M., Miller, Jr., F.D. A new member of the clover proliferation phytoplasma group (16SrVI) associated with elm yellows in Illinois. Plant Disease. 2003. v. 87. p. 244-246. Lee, I.M., Zhao, Y., Bottner, K.D. Identification of a putative insertion sequence (IS) associated with members of the aster yellows (AY) phytoplasma group. Phytopathology. 2003. v. 93. (Supplement) Abstract p. 49. Lee, I.-M. Primers and probes for the detection of Ralstonia solanacearum race3/biovar2 by polymerase chain reaction. 2003. U.S. Patent Application S/N60/469,028. Liu, Q., Zhao, Y., Hammond, R.W., Zhao, H., and Davis, R.E. Agrobacterium-mediated transformation and plant regeneration from leaf segments of sweet cherry dwarf rootstock 'Gisela 6' (Prunus cerasus X P. canescens). Invitro. 2003. v. 39. (Supplement) Abstract. p. 43. Samuitiene, M., Navalinskiene, M., Davis, R.E., Jomantiene, R. Molecular detection and characterization of a phytoplasma infecting daisy (Bellis perennis L.) plants in Lithuania. Botanica Lithuanica. 2002. v. 8. p. 195-200. Zhao, Y., Hammond, R.W., Jomantiene, R., Dally, E.L., Lee, I.M., Jia, H., Wu, H., Lin, S., Zhang, P., Kenton, S., Najar, F.Z., Hua, A., Roe, B.A., Fletcher, J., Davis, R.E. Gene content and organizatioin of an 85-kb DNA segment from the genome of the phytopathogenic mollicute Spiroplasma kunkelii. Molecular Genetic Genomics. 2003. v. 269. p. 592-602. Zhao, Y., Wang, H., Dally, E.L., Liu, Q., Davis, R.E. The largest paralogous family of proteins in one of the smallest cellular pathogens: ATP-binding cassette proteins in Spiroplasma kunkelii. Phytopathology. 2003. v. 93. (Supplement) Abstract. p. 95.