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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR STRATEGIES FOR DETECTION AND IDENTIFICATION OF FORAGE LEGUME PATHOGENS AND DEVELOPMENT OF HOST RESISTANCE
2009 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
Rapid and accurate identification of disease-causing fungi is necessary for effective and appropriate disease control and is also critical for understanding the sources of epidemics and disease outbreaks. Knowledge of specific causal agents of diseases is necessary 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. Collections of fungi pathogenic to important agricultural crops in the U.S. were obtained and characterized. Strains from Europe, China, and India were characterized by morphology and DNA sequencing, and compared with domestic pathogens from forages, alfalfa, tree fruit, and vegetable crops. Fungi included strains and species in the genera Ulocladium, Alternaria, Pleospora, Stemphylium, and Phytophthora. Molecular markers were identified that enabled distinctions between strains within a species. Specific DNA analyses sequencing methods revealed new species, new lineages within families, and new host associations among these fungi. Further analyses at the sub-species level of strains in the genus Stemphylium contributed to understanding 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.


4.Accomplishments
1. Molecular genetic characterization of fungi causing leaf spot disease in alfalfa. Leaf spot diseases in alfalfa caused by fungi are devastating, causing losses in forage quality and yield. A high degree of variability exists within species and among strains of the fungi, which attack different crops world wide. Methods were developed to understand the molecular variation and the genetic structure of populations within species of fungi. Individual isolates were identified, and their relatedness to similar fungi that attack vegetable and fruit tree crops was determined. The research resulted in the identification and characterization of several new species of fungi. The research will enable scientists, breeders, and industry to identify the specific causes of Stemphylium diseases and lead to implementation of appropriate disease control strategies.


6.Technology Transfer

Number of Other Technology Transfer2

Review Publications
Sun, W.X., Jia, Y.J., Wu, Y.M., Zhang, X.G., Oneill, N.R. 2008. Genetic Diversity in Phytophthora capsici from Eastern China. Canadian Journal of Plant Pathology. 30(3):414-424.

Sun, W.X., Jia, Y.J., Feng, B.Z., Zhu, X.P., Xie, B., Oneill, N.R., Zhang, X.G. 2009. Functional analysis of Pcipg2 from the straminopilous plant Pathogen Phytophthora capsici. Genesis. 47(8):535-544. Available: http://dx.doi.org/10.1002/dvg.20530.

Last Modified: 10/22/2014
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