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United States Department of Agriculture

Agricultural Research Service

2008 Annual Report

1a.Objectives (from AD-416)
Objective 1: Establish basic, fundamental genetic structures to identify and characterize fungal populations pathogenic to alfalfa and other forage legume crops. Develop diagnostic tools and phylogenies to aid in the identification of fungal pathogens of alfalfa. Describe new species and lineages based on this information. Apply existing and new methodologies to identify molecular evolutionary genetic relationships within fungal species that integrate morphology, biogeography, and host specificity.

Objective 2: Identify novel sources of resistance to fungi. Evaluate Medicago truncatula and Medicago lines and accessions for disease resistance in order to identify molecular markers associated with resistance. Determine host specificities of closely related pathogenic Stemphylium strains to M. sativa and M. truncatula lines relative to their host of origin.

1b.Approach (from AD-416)
A species and strain phylogeny for fungi causing diseases of alfalfa, specifically Stemphylim and Pleospora, will be developed based on strains from diverse hosts based sequences of multiple loci. The molecular phylogenies will be integrated with data that distinguish morphological characters to enable the production of keys to species. New and existing strains or pathotypes from alfalfa or its associated rotation crops and weed hosts will be characterized and integrated into updated phylogenetic frameworks. Once the molecular and morphological descriptions of new collections are complete, keys to groups of species will be prepared that can be used for the identification of species of Stemphylum and Pleospora. Closely related strains identified as single species groups will be analyzed for further molecular diversity using two approaches: DNA fingerprinting (AFLP), and a new multi-gene approach which will type specific sequences from multiple loci (MLST).

Research will be developed to utilize resistant and susceptible M. truncatula lines models for functional genomic studies including research to map and identify resistance genes, determine mechanisms of defense, and investigate host-pathogen relationships. Cultivar selection will be based on the presence and range of highly desirable agronomic traits, including fall dormancy, genetic diversity, winter hardiness, yield, and a variety of growth habits. To determine the vulnerability of alfalfa to Stemphylium strains and species that cause disease in other crops, pathogens of alfalfa will be evaluated for virulence on M. sativa and M. truncatula cultivars selected for high and low susceptibility to leaf spot disease.

3.Progress Report
Effective and appropriate disease control depends on rapid and accurate identification of the pathogens causing the disease. Accurate identification of pathogens is also critical for understanding the sources of epidemics and disease outbreaks, and for making sound decisions regarding quarantine of imported and exported plant materials and commodities. Knowing how pathogens are related to each other is also essential for developing appropriately targeted disease control strategies.

New collections of fungi pathogenic to important agricultural crops in the U.S. and world wide were obtained from Europe, India, and the Republic of China. These strains were characterized by morphology and DNA sequencing, and compared with domestic pathogens from forage especially alfalfa as well as tree fruit, and vegetable crops. Fungi included strains and species in the genera Colletotrichum, Phoma, Phytophthora, Pleospora, and Stemphylium. Molecular analyses revealed a high level of diversity within these populations and molecular markers were identified that enabled distinctions between pathogenic and non-pathogenic strains within a species. DNA analyses revealed new species, new lineages within families, and new host associations.

Research plans were developed and a new project initiated to expand these collections with emphasis on alfalfa and to further analyze the population and sub-species structure of fungi in these genera in order to understand the evolutionary basis for biological species, pathogenicity, and host specificity.

Breeders, extension agents, and programs aimed at producing healthy, disease resistant forages will be provided with basic information so that the specific causal agent of a disease can be determined. The project’s overall goal is to provide molecular tools and integrated phylogenies to facilitate the identification and classification of fungi.

The research addresses National Program 205 Rangeland, Pasture and Forages, Component II: Plant Resources a) Lack of Available Germplasm and c) Overcoming Limitations to Plant Growth and Development); Component III: Forage Management (a) Forage Establishment and Persistence. Project objectives are also relevant to National Program 303, Plant Diseases, Component I: Disease Diagnosis, Detection, Identification and Characterization of Plant Pathogens a) New Diagnostic Methods and Tools and b) Detection, Characterization, and Classification of Pathogens; and Component III Plant Disease Resistance b) Disease Resistance in New Germplasm and Varieties.

1. Molecular variability of Phoma medicaginis, cause of spring black stem and leaf spot of alfalfa.

Spring black stem and leaf spot of alfalfa caused by Phoma medicaginis is one of the most common diseases on alfalfa in the U.S. Within strains of this pathogen a high degree of variability exists; however, little is known about the molecular variation or the genetic structure of populations within this species. Techniques for identifying individual isolates and determining their relatedness called DNA fingerprinting methods were developed. The amount of molecular variation found in the pathogen population suggests that alfalfa breeding programs for enhancing resistance to disease should use specific genetically diverse isolates to assess alfalfa lines. In addition, it was determined that two additional Phoma species cause similar symptoms on alfalfa. This research will be used by plant breeders to develop lines of alfalfa that are resistant to spring black stem and leaf spot. This accomplishment addresses National Program 205 Rangeland, Pasture and Forages, Component II: Plant Resources a) Lack of Available Germplasm and c) Overcoming Limitations to Plant Growth and Development); Component III: Forage Management (a) Forage Establishment and Persistence.

5.Significant Activities that Support Special Target Populations

6.Technology Transfer

Number of Other Technology Transfer2

Review Publications
Castell-Miller, C., Szabo, L.J., Gale, L.R., O'Neill, N.R., Samac, D.A. 2008. Molecular variability of a Minnesota population of Phoma medicaginis var. medicaginis, the causal agent of spring black stem and leaf spot of alfalfa. Canadian Journal of Plant Pathology. 30:85-96.

Davidson, C.R., Evans, T.A., Mulrooney, R.P., Gregory, N.F., Carroll, R.B., Oneill, N.R. 2008. Lima Bean Downy Mildew Epiphytotics Caused by New Physiological Races of Phytophthora phaseoli. Plant Disease. 92:670-674.

Last Modified: 11/25/2015
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