1a.Objectives (from AD-416)
Objective 1: Characterize genetic differences in isolates of plant pathogenic streptomycetesand Phytophthora using targeted PCR, markers, regional genome sequencing and biological assays. Sub-objectives: a. Characterize populations of Streptomyces causing common scab in the U.S., using PCR and regional genome sequencing of 16s ribosomal RNA genes and genes from the pathogenicity island. b. Identify and characterize genes and corresponding gene product proteins, peptides and other compounds produced by Streptomyces and Phytophthora that may contribute to pathogenicity. c. Identify critical components of pathogen genetic variability contributing to variation in severity of common scab and late blight. d. Identify critical components in the genetic variability of Phytophthora infestans which contributes to variation in disease severity.

Objective 2: Characterize genetic differences in disease resistance of solanaceous hosts in response to Phytophthora and Streptomyces scabies. Sub-objectives: a. Characterize differences in gene expression in both hosts and pathogens to identify pathogenicity determinants, applying RT-PCR, microarray analysis, and functional genomics. b. Map genes for resistance to common scab onto potato chromosomes, and develop molecular markers for common scab resistance.

Objective 3: Identify plant defense responses in tomato induced by the biocontrol agent F. oxysporum strain CS-20 and determine their contribution to observed biocontrol of Fusarium wilt. Other biocontrol-host-pathogen systems and biocontrol mechanisms will be included as time and resources permit, and as indicated by results. Objective 4: Identify and characterize genetic traits and gene expression related to biocontrol ability in the biocontrol fungus F. oxysporum strain CS-20. Objective 5: Characterize interactions between the biocontrol fungus F. oxysporum strain CS-20 and other components of the tomato rhizosphere community.

1b.Approach (from AD-416)
Characterization of genetic differences in isolates of plant pathogenic streptomycetes and Phytophthora will be performed using targeted PCR, markers, regional genome sequencing and biological assays. Populations of Streptomyces causing common scab in the U.S. will be characterized, using PCR and regional genome sequencing of 16S ribosomal RNA genes and genes from the pathogenicity island. Genes and corresponding gene product proteins, peptides and other compounds produced by Streptomyces and Phytophthora that may contribute to pathogenicity will be identified and characterized. Critical components of pathogen genetic variability contributing to variation in severity of common scab and late blight will also be identified. Additionally, genetic differences in disease resistance of solanaceous hosts in response to Phytophthora and Streptomyces scabies are to be characterized, utilizing differences in gene expression in both hosts and pathogens to identify pathogenicity determinants, applying RT-PCR, microarray analysis, and functional genomics. Genes for resistance to common scab will be mapped onto potato chromosomes, and molecular markers for common scab resistance developed.

3.Progress Report
Hydrolase (cellulose-modifying) genes from Phytophthora were found in multiple copies with moderate differences between individual genes. The function of each individual gene is being assessed to determine if discrete enzymatic properties exist.

Sequences for XIPs (xyloglucanase inhibitor proteins) have been determined for a spectrum of Solanaceous plants including potato, tomato, eggplant, night shade, tamarillo, datura, and physalis. The ability of each XIP to inhibit hydrolases from Phytophthora is being investigated to determine their use in plant disease protection.

Disease severity is associated with different isolate groups as differences were found in the seriousness of common scab disease caused by distinct Streptomyces species and strains on four potato varieties that have contrasting susceptibility to common scab. Recognition of specific interactions between pathogenic Streptomyces species and individual potato cultivars is important in testing common scab tolerance in potato varieties and will be used to recommend the most suitable potato cultivars for specific growing regions. Isolates of P. infestans have been collected from multiple state locations. The ability of the pathogen to expand its range is being investigated relative to molecular fingerprinting characterization. This allows for tracking of pathogen outbreaks and aids in determining the original source of the pathogen.

4.Accomplishments
1. Identifying, characterizing and preserving pathogen strains causing potato common scab in North America. There are no reliable methods for controlling common scab disease, which causes quality losses in root and tuber crops, especially potatoes, and has become increasingly troublesome for northeastern and Midwest potato growers in recent years. With the cooperation of potato growers and research personnel, a research collection of the pathogenic bacteria associated with potato common scab in North America was assembled during the last 7 years. Characterization of the collection revealed a number of species (in the genus Streptomyces) associated with the disease. Pathogenic strains showed unexpectedly dynamic variation in gene content associated with pathogenicity, which may provide an explanation for differences in disease pressure in different potato-growing regions. Pathogenic variation poses challenges in developing strategies to control the disease. The survey is the most extensive conducted anywhere in the world to date. This molecularly characterized and preserved Streptomyces research collection is available to other researchers interested in population biology studies and tracing the evolutionary history and spread of common scab-associated Streptomyces. There are also potential applications in identification of new antimicrobial products. Identifying and characterizing the diversity of pathogens causing the disease provides essential information for developing new disease control methods, selecting appropriate potato cultivars to plant in a location, and breeding or testing for common scab disease resistance.

2. Genetic diversity and field dynamics of an international pathogen. In 2008, scientists in Taiwan in cooperation with ARS scientists in Beltsville, Maryland reported the isolation and characterization of Phytophthora infestans strains from tomato hosts, growing in heavily blighted fields in several locations in Taiwan. P. infestans, the microbe that causes this blight, is a highly variable pathogen of these hosts. The disease, late blight, has suddenly reemerged as a major concern in most potato and tomato producing areas. This disease organism shows broad genetic variability in Taiwan which has created the opportunity to screen North American breeding clones at this location. For several years, breeding materials derived from highly diverse populations have been tested in successive years. More aggressive, fungicide-resistant and host-specialized isolates have appeared attacking potato and tomato crops. Characterization of pathogens with molecular markers with information on aggressiveness and virulence, mating type (thus likely frequency of sexual reproduction) and fungicide resistance was the fundamental aim of a multiyear study. This information would indicate the rate of genetic change within late blight populations and possibly the mechanisms involved. This information will benefit other scientists, plant breeders and various producers.

Review Publications
Chen, C., Sheu, Z., Wang, T., Black, L.L., Perez, F.G., Deahl, K.L. 2009. Phenotypic and Genotypic Changes in the Phytophthora infestans Population in Taiwan - 1991 to 2006. Journal of Phytopathology. 157:248-255.