Dr. Nichole R. O'Neill
	Research Plant Pathologist, USDA-ARS Soybean and Alfalfa Research Laboratory, Beltsville, MD 20705 Tel. 301-504-5723 FAX 301-504-5728 e-mail: noneill@asrr.arsusda.gov

	Project: Pathology of Medicago sativa and Related Species
	As a plant pathologist, I am studying the interaction between fungi and their host plants, and working to find or improve disease resistance to important fungal pathogens of alfalfa and forage legumes. In my laboratory we also evaluate newly discovered or highly virulent pathogenic species and races of fungi, and assess their potential destructiveness to crops. In addition to plant pathology, the research requires expertise in plant pathology, biochemistry, plant physiology, genetics, molecular biology, and mycology. One of the most serious problems in alfalfa is diseases caused by foliar pathogens. The diseases are caused by several genera of fungi, the most severe of which are Colletotrichum, Phoma, Leptosphaerulina, Stemphylium, and Stagonospora. There are at least five pathogenic Colletotrichum species: C. destructivum, C. dematium, C. gloeosporioides, C. trifolii, and C. graminicola. Progress in controlling disease losses by breeding for resistance is slow because small increments in improvement take years to achieve by traditional plant breeding programs, and there is little resistance in adapted germplasm. Progress is also hampered by the lack of basic knowledge of the biology and pathogenic potential of fungi causing many diseases of alfalfa. We are using several approaches to solve these problems. One is to determine the feasibility of using the phenomenon known as induced disease resistance to control foliar pathogens alfalfa. Induced resistance is a biochemical mechanism by which plants are rendered immune to attack by virulent fungi by prior inoculation with non-virulent strains of a fungus or by elicitors. The plant's own defenses are activated, and thus the plant protects itself from further attack! We have shown that this defense strategy works well with the alfalfa anthracnose disease caused by Colletotrichum trifolii. To determine whether induced resistance has broad-spectrum effectiveness, resistance induced by elicitors or by prior inoculation with C. trifolii is being assessed against other major alfalfa foliar and stem diseases caused by Phoma medicaginis, Leptophaerulina briosiana, and Stagonospora meliloti. If induced resistance is effective in reducing the severity of these diseases, the development of this strategy as a practical disease control method becomes more valuable. The feasibility of an induced resistance disease control strategy depends on an effective eliciting agent. Such agents could be biotic or abiotic, but abiotic agents are more easily handled and less likely to depend on environmental conditions for effectiveness. Isonicotinic acid (INA) and related compounds, yeast cell wall extracts, Colletotrichum cell wall fractions, and cyclic and non-cyclic glucan elicitors are being evaluated for effectiveness in activating, suppressing, or enhancing constitutive or inducible defense expression. Isonicotinic acid induces systemic acquired resistance (SAR) in several crops but is untested in alfalfa. We are investigating whether resistance can be induced by artificial elicitors, and whether induced resistance can be further enhanced by non-specific artificial elicitors. Elicitors are being evaluated for activities which condition the host to activate defense reactions in advance of subsequent pathogen invasion, as well as for activities which would enhance resistance in plants already in the induced resistant state. Knowledge of the mechanism of induced resistance will help us understand more about the biochemical events occurring during a host and parasite interaction. Recently, we discovered a new and unique component of induced resistance. Plants in an acquired immune state responded rapidly to challenge by potentially highly virulent pathogens by producing elevated levels of phytoalexins. This additional component, a response to challenge, rendered the plant more resistant to infection and disease. This enhancement of induced defense expression during challenge may play a very important role in disease resistance under field conditions, where plants are subject to variable disease pressure from numerous foliar pathogens. It appears that the acquired immune state in some way conditions the plant for subsequent rapid defense activation. Upon attack by virulent pathogens, the plant's defense response is then boosted, conferring the observed immunity. The basis for the response to challenge by plants in the acquired immune state is being investigated. Understanding pathogen population structure can contribute to improved disease management by allowing resistance genes and plant genotypes to be identified and characterized relative to the spectrum of the pathogen population. Information on genetic diversity within a pathogen population is also important for the design of strategies for producing resistant varieties. Molecular approaches have become quite important for analyzing systematic and evolutionary relationships in agriculturally significant pathogenic species. Recently we used a new technique, AFLP (amplified fragment length polymorphism, a PCR-based DNA fingerprinting method), to define genetic relationships in fungal populations. Using this technique, we characterized the genetic diversity in Colletotrichum races and species which are morphologically similar and are pathogens of alfalfa and other hosts. Earlier we characterized the pathogenicity, virulence, aggressiveness, and morphology of Colletotrichum isolates pathogenic to alfalfa, but little was known about the genetic or molecular basis for these traits. We are currently evaluating AFLP as a method to determine genotypic variation within (and between) species of pathogenic fungi in relation to morphological and pathotype criteria. In combination with other molecular approaches (MtDNA, rDNA, and RAPD analyses), this research will facilitate the identification of marker genes associated with pathogenicity, host and race specificity, and phylogeny, and provide a foundation for population structure analyses of other alfalfa foliar pathogens. In collaboration with Dr. Peter van Berkum, we are analyzing sequence homologies in fungal ribosomal DNA to provide estimates of genetic distances. This information us used to determine how closely pathogens or isolates are related, and whether the genomes evolved apart. Multilocus enzyme electrophoresis is another technique that we use to derive indices of association (linkage disequilibrium) as an estimate of genetic distance.
	Other projects:
	We are evaluating methods to control microbial populations during and after sprouting alfalfa seed. We constructed a working model to germinate, treat, and grow alfalfa sprouts, so that treatments can be evaluated during simulated sprout production. The efficacy of different anti-bacterial treatments is being assessed so that sprouts can be produced with minimal bacterial levels. In collaboration with Dr. Gary Bauchan, we are evaluating a wide diversity of germplasm for resistance to fungal diseases. Accessions in collections of perennial and annual species of Medicago may have resistance to important alfalfa diseases. These collections are a potential source of new or novel genes for resistance to major diseases, and these genes or gene products could be made available for incorporation into adapted germplasm. We found that annual Medicago species are a source of genes that produce novel or undescribed, fungitoxic compounds. The annual Medicago Core Subset, representing the genetic diversity inherent in species and accessions of annual Medicago, was screened for resistance to Colletotrichum trifolii and Phoma medicaginis. About 40 species and accessions out of several hundred exhibited high levels of resistance to Colletotrichum. We discovered new fungitoxic compounds in three species, M. turbinata from Lebanon, M. muricoleptis from Italy, and M. truncatula. Germplasm developed with high levels of disease resistance to major diseases would increase quality, yield, and adaptability of alfalfa.

	Relevant Publications:
	O'Neill, N.R., Elgin, J.H., Jr., and Baker, C.J. 1989. Characterization of induced resistance to anthracnose in alfalfa by races, isolates, and species of Colletotrichum. Phytopathology 79:750-756.
	Bauchan, G.R., Campbell, T.A., O'Neill, N.R., and Elgin, J.H., Jr. 1989. Self-incompatibility in two alfalfa populations. Crop Science 30(6):1205-1210.
	Elgin, J.H., Jr., and O'Neill, N.R. 1988. Comparison of genes controlling race 1 anthracnose resistance in Arc and Saranac AR alfalfa. Crop Science 28:657-659.
	O'Neill, N.R. 1991. Anthracnose Resistance. Section D-1. in: Standard Tests to Characterize Alfalfa Cultivars, third ed. C. C. Fox, R. Berberet, F. Gray, C. R. Grau, D. L. Jessen, and M. A. Peterson, eds. North American Alfalfa Improvement Conf., Beltsville, MD, publisher. 64 pp.
	Baker, C.J., O'Neill, N.R., Keppler, L.D., and Orlandi, E.W. 1991. Early responses during plant- bacteria interactions in tobacco cell suspensions. Phytopathology 81:1504-1507.
	Churchill, A.C.L., Baker, C.J., O'Neill, N.R., and Elgin, J.H., Jr. 1988. Development of Colletotrichum trifolii races 1 and 2 on alfalfa clones resistant and susceptible to anthracnose. Canadian Journal of Botany 66:75-82.
	Baker, C. Jacyn, O'Neill, Nichole R., and Tomerlin, J. Robert. 1989. Accumulation of phenolic compounds in incompatible clone/race interactions of Medicago sativa and Colletotrichum trifolii. Physiological and Molecular Plant Pathology 35:231-241.
	O'Neill, N.R., and Saunders, J.A. 1994. Compatible and incompatible responses in alfalfa cotyledons to races 1 and 2 of Colletotrichum trifolii. Phytopathology 84:283-287.
	O'Neill, N. R. 1996. Pathogenic variability and host resistance in the Colletotrichum trifolii/Medicago sativa pathosystem. Plant Dis. 80: 450-457.
	O'Neill, N.R. 1996. Defense expression in protected tissues of Medicago sativa is enhanced during compatible interactions with Colletotrichum trifoli i. Phytopathology 86:1045-1050.
	O'Neill, N. R., and Bauchan, G. R. 1998. Sources of resistance to anthracnose in the annual Medicago core collection. Plant Dis. 82: in press.
	Jianping Cheng, James A. Saunders, and Nichole R. O'Neill. 1998. Temporal Patterns of mRNA and Phytoalexin Accumulation from Isoflavonoid Metabolism during Compatible and Incompatible Interactions between Medicago sativa and Colletotrichum trifolii. In press.
	Gerald D. Baldridge, Nichole R. O'Neill, and Deborah A. Samac. 1998. Alfalfa (Medicago sativa L.) resistance to the root-lesion nematode, Pratylenchus penetrans: Defense-response gene mRNA and isoflavonoid phytoalexin levels in roots. Plant Molecular Biology. In Press.
	O'Neill, N.R, van Berkum, P.B., Lin, J.J., Kuo, J., Ude, G.N., Kenworthy, W., and Saunders, J.A. 1997. Application of Amplified Fragment Length Polymorphism (AFLP) for the genetic characterization of Colletotrichum pathogens of alfalfa (Medicago sativa). Phytopathology 87:745-750.

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