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United States Department of Agriculture

Agricultural Research Service

2010 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. 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 with emphasis on diseases of alfalfa.

Fungi pathogenic to important agricultural crops in the U.S. were obtained, cultured, and characterized. New strains from China and from California 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 Alternaria, Stemphylium, and Ulocladium, and three new species were described. A key to species and descriptions of species in the genus Ulocladum was produced. 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 genera Stemphylium and Ulocladium contributed to understanding the evolutionary basis for biological species, pathogenicity and host specificity.

The effects of four levels of elevated atmospheric carbon dioxide on the quantity and quality of spores produced by fungi growing on timothy hay were studied. Leaf carbon-to-nitrogen ratio was positively correlated with the quantity of A. alternata spores produced per gram of leaf, and spore production was increased nearly three-fold at the higher levels of carbon dioxide. Rising levels of carbon dioxide often increase the biomass and carbon-to-nitrogen ratio of plant leaves. This research demonstrates that leaf changes induced even at moderately increased carbon dioxide levels greatly enhance the sporulation of forage grass fungi, as well as the common allergenic species A. alternata.

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 Cevelopment); Component III: Forage Management (a) Forage Establishment and Persistance. Project Objectives are also relevant to National Program 303, Plant Diseases, Component I: Disease Diagnostics, 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. Elevated levels of carbon dioxide cause an increase in fungal spore production in the forage grass timothy. Rising environmental carbon dioxide levels have been shown to change the carbon-to-nitrogen ratio in plant leaves. In our research, we found that exposing the forage grass timothy to increasing carbon dioxide levels also resulted in an increase in the quantity of spores produced by fungi growing on the grass, a common component in hay. The fungi used in these experiments were Alternaria alternata, a common allergy-inducing species, and Cladosporium phlei, a plant pathogen of timothy. Alternaria alternata spore production increased nearly three-fold at the higher levels of carbon dioxide. This enhancement of spore production may be a contributing factor in the increasing prevalence of asthma. The research will be of interest to forage producers, plant scientists, health care providers and the general public.

Review Publications
Wolf, J.E., Oneill, N.R., Rogers, C.A., Muilenberg, M.L., Ziska, L.H. 2010. Elevated atmospheric carbon dioxide concentrations amplify Alternaria alternata sporulation and total antigen production. Environmental Health Perspectives. 118(9):1223-1228.

Wang, Y., Pei, Y., Oneill, N.R., Zhang, X. 2010. Ulocladium cantlous sp. nov. isolated from Northwest China: morphology and molecular phylogenetic position. Mycologia. 102(2):374-383.

Wang, Y., Fu, H., Oneill, N.R., Zhang, X. 2009. Two new species of Stemphylium from Northwest China. Mycological Progress. 8(4):301-304.

Last Modified: 10/7/2015
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