Progress 07/20/04 to 07/19/08
Outputs
OUTPUTS: Neonectria ditissima is a fungus that causes disfiguring cankers on a wide range of hardwood species, most significantly on black birch (Betula lenta), a native tree valued for its use in veneer production and of increasing representation in northeastern forests. Optimal forest management strategies will require fundamental knowledge of the pathogen's natural history, including population biology, genetics, and ecology. It was, therefore, necessary to develop methods and tools for classical and molecular genetic analyses in the laboratory that can be applied to field studies. We developed a method for isolating and culturing both maternal and progeny genotypes from cankers and perithecia (the fruiting structures). We developed a long-term storage method that allows us to efficiently store viable cultures for several years. We have developed an efficient and inexpensive method for reliably extracting DNA from cultures that is suitable for all molecular manipulations used in the laboratory. We tested several methods for observing matings in the laboratory, and found only one method, using sterile birch stems embedded in a specialized nutrient agar, that produced perithecia, albeit unreliably. We tested six different media for observing vegetative incompatibility reactions, and found one method that, while it produced barrage zones, did not do so reliably in replication. We have created a large collection of geographically disparate isolates that are grouped into two screening populations. These have been used to screen two categories of genetic markers for polymorphisms, Amplified Fragment Length Polymorphisms (AFLP) and Microsatellites. The first screening population comprises 22 isolates from across eastern North America, including Canada, New York, Massachusetts, Tennessee, Virginia, West Virginia, and Connecticut. The second population comprises 38 isolates from a single site in West Rock Ridge State Park (WRRSP) in New Haven, Connecticut. We used three sets of selective AFLP primers and observed 79 dominant (presence/absence) loci that segregated among isolates in both screening populations. Microsatellite markers are superior to AFLP markers because they are non-anonymous codominant single-locus markers that can be assayed and studied in aggregate or independently. Fifteen microsatellite markers were identified that are polymorphic in the broader screening population; twelve of these are polymorphic in the WRRSP population. This information was disseminated to colleagues at the annual meeting of the Northeastern Division of the American Phytopathological Society in Cape May, New Jersey, 12 October 2007; to stakeholders at the Forest Health Monitoring Workshop, 21 February 2008; and to colleagues at a conference and workshop titled "Analogies in the Evolution of Gender Expression and Sexual Strategies in Animals and Plants" in Stuttgart, Germany, on 12 September 2008. PARTICIPANTS: The main investigator on this project is Robert E. Marra, Assistant Scientist at The Connecticut Agricultural Experiment Station. Dr. Marra designs and directs the experiments, and conducts the field work. Mr. Jason Corwin, a Technician at The Connecticut Agricultural Experiment Station, gives technical assistance in the laboratory, focusing principally on the cloning and screening of the microsatellite markers. Knowledge gained through experience in developing AFLP and microsatellite markers is being shared with graduate students and postdocs at Yale University, the University of Connecticut, the University of Southern Alabama, and the University of Massachusetts at Amherst. Additionally, this project has served as training for the project's technician, Mr. Jason Corwin. TARGET AUDIENCES: The target audiences are as follows: state foresters, mycologists, plant pathologists, and evolutionary biologists. The primary methods used to deliver science-based knowledge are through publication, as well as presentations of this research to plant pathologists at the annual meeting of the Northeast Division of the American Phytopathological Society and to arborists and foresters at the annual Forest Health Monitoring Workshop held at The Connecticut Agricultural Experiment Station. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Knowledge of the availability of genetic markers benefits other researchers studying Neonectria species; in particular, PCR primers for microsatellite markers developed for N. ditissima will be applicable, with some modification, to other Neonectria species, especially N. faginata, which causes beech bark disease on American beech (Fagus grandifolia) in eastern North America and in Europe. Expertise gained during this project has resulted in a collaboration with four other researchers on an NSF grant proposal to use beech bark disease in studying the impact of host-pathogen dynamics on forest community structure. Knowledge gained in learning how to develop these markers, particularly microsatellite markers, will be shared with other researchers interested in developing genetic markers for population studies; such a collaboration is already planned with Dr. Wade Elmer in the study of populations of Fusarium species implicated in decline of Spartina species in northeastern salt marshes. New knowledge about the high level of genetic variability showed that variability among isolates within the state of Connecticut is as high as that among isolates in the larger screening population, which includes isolates from Virginia, West Virginia, Tennessee, and Massachusetts. Knowledge gained through application of these markers to progeny sets from West Rock Ridge State Park in New Haven, CT, showed that N. ditissima has a mixed mating system (i.e., both selfing and outcrossing occur in the same population), a significant finding that warrants further study. In concert with data from current ongoing research on the population dynamics of this pathogen, these findings will inform recommendations for stand management practices that reduce the probability of new infections.
Publications
- Marra, R. E., and Corwin, J. A. 2009. Isolation and characterization of codominant markers for the perennial canker fungal pathogen, Neonectria ditissima. Molecular Ecology Resources 2008, in press.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Neonectria ditissima is a fungus that causes disfiguring cankers on a wide range of hardwood species, most significantly on black birch (Betula lenta), a native tree valued for its use in veneer production and of increasing representation in northeastern forests. Optimal forest management strategies will require fundamental knowledge of the pathogen's natural history, including population biology, genetics, and ecology. It was, therefore, necessary to develop methods and tools for classical and molecular genetic analyses in the laboratory that can be applied to field studies. A screening population of 22 geographically disparate isolates was used to screen two categories of genetic markers for polymorphisms, Amplified Fragment Length Polymorphisms (AFLP) and Microsatellites. Using three sets of selective primers, 79 dominant (presence/absence) AFLP loci were identified that segregated among isolates in the screening population. Microsatellite markers are superior to AFLP
markers because they are non-anonymous codominant single-locus markers that can be assayed and studied in aggregate or independently. Ten polymorphic microsatellite markers have been identified to date. Because these markers are codominant, in contrast to AFLP loci, all alleles are uniquely scored; hence, in the screening population, three loci have two alleles, four loci have three alleles, one locus has four alleles, one locus has five alleles, and one locus has nine alleles. This information was disseminated to colleagues at the annual meeting of the Northeastern Division of the American Phytopathological Society in Cape May, New Jersey, 12 October 2007.
PARTICIPANTS: The main investigator on this project is Robert E. Marra, Assistant Scientist at The Connecticut Agricultural Experiment Station. Dr. Marra designs and directs the experiments, and conducts the field work. Mr. Jason Corwin, a Technician at The Connecticut Agricultural Experiment Station, gives technical assistance in the laboratory, focusing principally on the cloning and screening of the microsatellite markers. Knowledge gained through experience in developing AFLP and microsatellite markers is being shared with graduate students and postdocs at Yale University, the University of Connecticut, and the University of Massachusetts at Amherst. Additionally, this project has served as training for the project's technician, Mr. Jason Corwin.
TARGET AUDIENCES: The target audiences are as follows: state foresters, mycologists, plant pathologists, evolutionary biologists. The primary method used to deliver science-based knowledge is through presentation of this research to plant pathologists at the annual meeting of the Northeast Division of the American Phytopathological Society.
Impacts
Knowledge of the availability of genetic markers benefits other researchers studying Neonectria species; in particular, PCR primers for microsatellite markers developed for N. ditissima will be applicable, with some modification, to other Neonectria species, especially N. faginata, which causes beech bark disease on American beech (Fagus grandifolia) in the Northeast. Knowledge gained in learning how to develop these markers, particularly microsatellite markers, will be shared with other researchers interested in developing genetic markers for population studies; such a collaboration is already planned with Dr. Wade Elmer in the study of populations of Fusarium species implicated in decline of Spartina species in northeastern salt marshes. New knowledge about the high level of genetic variability showed that variability among isolates within the state of Connecticut is as high as that among isolates in the larger screening population, which includes isolates from
Virginia, West Virginia, Tennessee and Massachusetts. Knowledge gained through application of these markers to progeny sets from West Rock Ridge State Park in New Haven, CT, showed that N. ditissima has a mixed mating system (i.e., both selfing and outcrossing occur in the same population), a significant finding that warrants further study.
Publications
- Marra, R. E. Amplified Fragment Length Polymorphic (AFLP) markers for the forest fungal pathogen, Neonectria ditissima. Abstract, Annual Meeting, Northeastern Division of the American Phytopathological Society, 10-12 October 2007, Cape May, NJ (in press).
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Progress 01/01/06 to 12/31/06
Outputs
Neonectria ditissima is a fungal pathogen of growing importance in northeastern hardwood forests, causing perennial cankers on a wide range of hardwood species. The greatest damage and death occur to black birch, Betula lenta, a native tree of increasing representation in the Northeast, prized in veneer production. Canker damage caused by N. ditissima drastically diminishes log value of black birch. An effective control strategy for perennial canker will require fundamental knowledge of the pathogen's natural history, including population biology, genetics, and ecology. To that end, we are improving and developing methods and tools for classical and molecular genetic analyses in the laboratory that can be applied to field studies. We have created a collection of N. ditissima isolates representative of the organism's genetic diversity in the Northeast, by sampling the fungus from black birch as well as yellow birch (B. alleghaniensis), gray birch (B. populifolia), and
other hosts. A broad distribution of isolates is critical to our goal of capturing the genetic diversity of N. ditissima found in Connecticut and the Northeast. We routinely isolate from cankers both maternal tissue as well as perithecia (fruiting bodies, containing sexual progeny). Based on published reports for other fungal species, we tested six types of culture media for laboratory matings, consisting of either birch pulp, elm pulp, or whole wheat grains mixed into either water agar or Neonectria Crossing Medium (NCM). We also tested embedding birch or beech stems in either water agar or NCM. While sparse perithecia were observed on some of the matings after six weeks, none produced abundant perithecia. However, the birch-stem/NCM combination produced sparse perithecia on the highest proportion (75%) of attempted matings. We have continued to test media and conditions for successful observation of the barrage zones that result from vegetative incompatibility, a self-nonself
recognition mechanism controlled by several unlinked genetic loci. Potato dextrose agar (PDA) supplemented with tannic acid and a pH-sensitive green dye appears to be the most successful method, but further testing is needed. We are using a screening population of twelve geographically disparate strains to identify informative polymorphic genetic markers. We have so far identified 55 Amplified Fragment Length Polymorphisms (AFLP) based on a single pair of selective PCR primers. The robustness of the procedure was confirmed by showing nearly 100% reproducibility of fragment patterns among 3 replicates of each of the 12 strains. Further testing with additional primer pairs should identify additional polymorphisms. Subsets of polymorphic markers will be used to test hypotheses on dispersal and mating, as well as other more fine-scale genetic studies, e.g. relatedness among cankers occurring on the same tree and on neighboring trees. In order to obtain a parallel set of single-locus
markers, we are currently constructing genetic libraries that have been enriched for microsatellites, chains of repeating DNA motifs (e.g., acgacgacgacgacg) that frequently exist as length polymorphisms within populations.
Impacts
Black birch is increasing in abundance in Connecticut, as well as other parts of the Northeast, and is therefore a tree of growing importance and concern to stakeholders. N. ditissima cankers scar and distort the tree, rendering a potentially valuable wood essentially worthless. Because of the impact that N. ditissima can impose on birch and therefore on forest dynamics, an understanding of the ecology and natural history of this fungus is critical to identifying and employing control strategies.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
Neonectria ditissima (formerly known as N. galligena) is a fungal pathogen of growing importance in northeastern hardwood forests, causing perennial target-like cankers on a wide range of hardwood species. The greatest amount of damage and death occurs to black birch, Betula lenta, a native tree of increasing representation in the Northeast, prized for its use in veneer production. Canker damage caused by N. ditissima drastically diminishes log value of black birch. An effective control strategy for perennial canker will require fundamental knowledge of the pathogen's natural history, including population biology, genetics, and ecology. To that end, we are improving and developing methods and tools for classical and molecular genetic analyses in the laboratory that can be applied to field studies. We have created a collection of N. ditissima isolates representative of the organism's genetic diversity in the Northeast, by sampling the fungus from black birch as well as
yellow (B. alleghaniensis) and gray (B. populifolia) birches. The broad distribution of isolates is critical to our goal of capturing the genetic diversity of N. ditissima found in Connecticut and the Northeast. In most cases, we have been able to isolate from cankers both the maternal tissue as well as perithecia (fruiting bodies, which contain the products of sexual reproduction). We continue to test a long-term storage method that involves fungal mycelium dried onto sterile glass fiber filter paper, and are finding no loss of viability. We are testing laboratory mating assays, using three different culture media (birch pulp, elm pulp, and whole grain), but have to date been unsuccessful in obtaining perithecia. We have tested six different media for observing vegetative incompatibility reactions, and have been successful with one; barrage zones--manifestations in media of vegetative incompatibility interactions--have been observed among a random collection of isolates from the
eastern United States. We have been amplifying by the PCR several important gene regions from a collection of isolates, and will be testing these for informative polymorphisms.
Impacts
Black birch is increasing in abundance in Connecticut, as well as other parts of the Northeast, and is therefore a tree of growing importance and concern to stakeholders. Because of the impact that N. ditissima can impose on birch and therefore on forest dynamics, an understanding of the ecology and natural history of this fungus is critical to identifying and employing control strategies.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
Neonectria galligena is a fungal pathogen of growing importance in northeastern hardwood forests, causing perennial target-like cankers on a wide range of hardwood species. The greatest amount of damage and death occurs to black birch, Betula lenta, a native tree of increasing representation in the Northeast, prized for its use in veneer production. Canker damage caused by N. galligena drastically diminishes log value of black birch. An effective control strategy for perennial canker will require fundamental knowledge of the pathogen's natural history, including population biology, genetics, and ecology. To that end, it is necessary to improve and develop methods and tools for classical and molecular genetic analyses in the laboratory that can be applied to field studies. To create a collection of N. galligena isolates representative of the organism's genetic diversity in the Northeast, state forests and parks throughout the state are being sampled for N. galligena on
black, as well as yellow (B. alleghaniensis) and gray (B. populifolia) birches. We have developed a method for isolating from cankers the maternal genotype, which gives rise to perithecia (fruiting bodies). Samples are dissected under the microscope to obtain both maternal fungal tissue as well as the sexual progeny contained in perithecia. Maternal fungal tissue is isolated from wood immediately surrounding the perithecia by plating onto cornmeal agar supplemented with antibiotics; taxonomic characters are used to distinguish N. galligena from other fungi. We have developed a method for long-term storage of isolates that obviates other more space- and time-consuming methods. DNA extraction protocols are being tested; the most successful results obtained so far have been with inexpensive and environmentally-friendly kits. The resulting DNA has been tractable for PCR and restriction enzyme digestions. Samples have so far been obtained from eighty cankered trees found at ten locations
across the state, with collections from more locations planned. Most of these have been collected from black birch, although several isolates have been obtained from yellow birch, gray birch, and sassafras, as well. The broad distribution of isolates is critical to our goal of capturing the genetic diversity of N. galligena found in Connecticut and the Northeast.
Impacts
Black birch is increasing in abundance in Connecticut, as well as other parts of the Northeast, and is therefore a tree of growing importance and concern to stakeholders. Because of the impact that N. galligena can impose on birch and therefore on forest dynamics, an understanding of the ecology and natural history of this fungus is critical to identifying and employing control strategies.
Publications
- No publications reported this period
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