Progress 07/20/08 to 07/19/11
Outputs
OUTPUTS: Neonectria ditissima is a fungus that causes disfiguring cankers on a wide range of hardwood species, most significantly on black and yellow birch (Betula lenta and B. alleghaniensis), native tree species valued for their use in veneer production and, in the case of black birch, 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. In previous work, we developed the methods and tools necessary for molecular genetic analyses in the laboratory that allowed us to analyze natural populations of the fungus. Most importantly, we developed a set of 17 microsatellite markers that are polymorphic among sites across Connecticut and Massachusetts. Fifteen of these are polymorphic in the study site population, in West Rock Ridge State Park (WRRSP) in New Haven, Connecticut, that was the object of this study. We further divided the population, consisting of 38 cankers, into two subpopulations, WRA and WRB, separated by a geographic barrier. Each canker was dissected and sampled for at least two fruit bodies (perithecia), from which 8-16 sexual progeny were isolated and cultured. DNA extracted from each progeny isolate was then assayed at each of the 15 microsatellite loci, in order to determine (1) whether the perithecium was the product of selfing or outcrossing; and (2) if outcrossed, the extent of heterozygosity. In total, over 600 progeny isolates -23 WRA progeny arrays and 27 WRB progeny arrays were isolated, cultured, and genotyped at all microsatellite loci. Interest in maximizing efficiency and reducing costs by pooling progeny (cultures or DNA) resulted in the conclusion that pooling was not efficacious or reliable at any stage; therefore the project required that we culture and extract DNA from over 600 isolates. All microsatellite genotyping is complete, and data analysis is underway. Preliminary results of the analysis show that both subpopulations exhibit a high degree of genotypic diversity, and that there is a small degree of genetic differentiation between the two subpopulations. Therefore, it was unexpected to discover that self-fertilization, not outcrossing, predominates, leaving unanswered the question concerning the source of genotypic diversity, since recombination does not occur at a high rate. 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. Knowledge gained through experience in developing, using, and analyzing AFLP and microsatellite markers and data from them is 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 college students working as summer research assistants. 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 publication and presentation of this research to plant pathologists at the annual meeting of the Northeast Division of the American Phytopathological Society. 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 the development and use of AFLP and microsatellite markers will be shared with other researchers interested in developing genetic markers for population studies; such a collaboration has already been productive with Dr. Wade Elmer in the use of AFLPs to study populations of Fusarium palustre and other species implicated in decline of Spartina species in northeastern salt marshes. New knowledge from the WRRSP population, showing a high level of genetic variability and low rates of outcrossing, and therefore a mixed mating system, point to the need for additional research into the source of genetic variability and dispersal dynamics.
Publications
- No publications reported this period
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: Neonectria ditissima, a plant pathogenic fungus native to eastern N. America, is considered among the most widespread and important canker-causing species of northeastern forests. Extensive scarring and damage to yellow (Betula alleghaniensis) and black birch (B. lenta) leads to drastic reductions in log quality and value. To improve forest management strategies, a thorough understanding of the ecology, genetics, and reproductive biology of this pathogen is needed. The purpose of this project is to conduct a detailed genetic study of a population of N. ditissima in Connecticut, which will afford a better understanding of the roles of asexual and sexual reproduction, as well as dispersal, in the spread of this disease. Research over the past year has demonstrated that the microsatellite markers developed in the laboratory in the preceding year reveal a high degree of polymorphism in the current study site. Microsatellites are chains of repeating DNA motifs (e.g., acgacgacgacgacg) found throughout the genomes of most eukaryotes; variation in the number of motif repeats at a microsatellite locus underlies the length differences among alleles. In total, 17 microsatellite loci have been identified that are polymorphic and informative among numerous populations in Connecticut; fifteen of these loci have been demonstrated to be polymorphic within a single Connecticut population, West Rock Ridge State Park (WRRSP), consisting of 38 isolates. The number of alleles at each locus segregating among the WRRSP isolates ranged from two to 12. A more detailed study of the WRRSP population is underway: Two fruit bodies isolated from each canker were dissected to obtain cultures of between eight and 16 sexual progeny from each fruit body. DNA has now been extracted from an average of 12 progeny from each of 23 perithecia from the WRRSP "A" population and 27 perithecia from the WRRSP "B" population, for a total of over 600 DNA extractions. Because we are interested in determining the genotype of the composite progeny array, we tested methods of pooling sibling progeny (i.e., those from a single perithecium) that would streamline the process of genotyping without compromising the ability to detect alternate alleles at segregating loci. These pooling methods included: (a) bulk-culturing sibling progeny en masse; (b) extracting the DNA from pooled mycelium of sibling progeny; (c) genotyping the pooled DNA from sibling progeny. These pooling techniques were compared to data from individually genotyped progeny; unfortunately, none of the pooling methods reliably reflected data from the individually genotyped progeny. Therefore, we are proceeding with genotyping individual progeny for each of thirteen microsatellite loci. From these data we will determine the level of clonality and relatedness among isolates, and also determine whether sexual reproduction occurs by self-fertilization, outcrossing, or a mixture of both. PARTICIPANTS: The investigator on this project is Robert E. Marra, Assistant Scientist at The Connecticut Agricultural Experiment Station. Dr. Marra designs and conducts this study, performing and supervising all field and laboratory duties associated with this work. Knowledge gained through genotyping 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, as well as undergraduate students at St. Joseph's College and Northeastern University, and a high school student at Branford High School. Ms. Emily Arsenault, Ms. Danielle Marias, and Mr. William Ryan, provided technical assistance with DNA extractions and genotyping. TARGET AUDIENCES: The target audiences are as follows: state foresters, land managers, mycologists, plant pathologists, and evolutionary biologists. This topic is also important to stakeholders interested in state forests and parks and other open space venues. The primary method used to deliver science-based knowledge is through publication and presentation of this research to plant pathologists at the annual meeting of the Northeastern Division of the American Phytopathological Society. Information on this research is also communicated formally at the annual Northeast Forest Health Workshop, at the annual meeting of the Connecticut Urban Forest Council, and at lectures delivered to local chapters of the Audubon Society, as well as other lay groups interested in the ecology of the northeastern forests. PROJECT MODIFICATIONS: As a result of parallel testing of various methods of pooling sibling progeny, it became clear that none of the tested pooling techniques -- (a) bulk culturing of sibling progeny; (b) pooling of mycelium from individually cultured progeny prior to DNA extraction; (c) pooling of DNA extracted individually from each progeny's mycelium, prior to PCR -- could reliably produce data equivalent to that obtained from individually genotyped progeny. We are therefore proceeding with a strategy that involves genotyping each individual in the progeny array.
Impacts
Black birch is increasing in abundance in Connecticut, and is therefore a tree of growing importance and concern. According to USDA Forest Services estimates from 2006, black birch is the fifth most common tree species in Connecticut, with approximately 300 million cubic feet of live trees on Connecticut timberlands. By contrast, black and yellow birch account for only about 5% of the growing stock removed annually from Connecticut timberlands, demonstrating the extent of underutilization of birch wood, due in large part to the devaluation caused by N. ditissima perennial cankers, the most common and most damaging disease affecting birches. Although trees infected with N. ditissima perennial cankers can persist for decades, the extensive scarring caused by the cankers renders them of little value for lumber or veneer. This devaluation cannot be overemphasized: perennial cankers reduce the value of birch stems three- to four-fold, resulting in wood that can only be sold for dunnage and pallet wood. The extent of damage to black birch in records of tree mortality most recently available (2006), show that yellow and black birch have a mortality rate of approximately 1%, in stark contrast to 0.3% for red and white oaks. Most of this high mortality is due to cankering from N. ditissima. Currently, high quality uncankered black and yellow birch command prices as high or higher than white oak for veneer logs and milled lumber. It is worth nothing that the tree's value beyond its use in veneer may be underestimated; for example, early accounts praised the qualities of black birch as an excellent substitute for black walnut in furniture and flooring, applications for which black walnut, a tree of increasing rareness, was once prized. Our efforts to more fully understand the biology and natural history of N. ditissima will contribute to the identification and utilization of control strategies. Knowledge gained from the application of microsatellite markers to the WRRSP population will be shared with other researchers interested in the population dynamics of forest pathogens.
Publications
- Brazee, N. J. Marra, R. E. (corresponding author), Gocke, L. and van Wassenaer, P. 2010. Non-destructive assessment of internal decay in three hardwood species of northeastern North America using sonic and electrical impedance tomography. Forestry-An International Journal of Forest Research (in press)
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Neonectria ditissima, a plant pathogenic fungus native to eastern N. America, is considered among the most widespread and important canker-causing species of northeastern forests. Associated with cankers on a wide range of broadleaved trees, including fruit trees, particularly apple and pear, N. ditissima is particularly devastating to yellow (Betula alleghaniensis) and black birch (B. lenta). Extensive scarring and damage caused by N. ditissima leads to drastic reductions in log quality and value. To improve forest management strategies, a thorough understanding of the ecology, genetics, and reproductive biology of this pathogen is needed. The purpose of this project is to conduct a detailed genetic study of a population of N. ditissima in Connecticut, which will afford a better understanding of the roles of asexual and sexual reproduction, as well as dispersal, in the spread of this disease. Research over the past year has demonstrated that the microsatellite markers developed in the laboratory in the preceding year reveal a high degree of polymorphism in the current study site. Microsatellites are chains of repeating DNA motifs (e.g., acgacgacgacgacg) found throughout the genomes of most eukaryotes; variation in the number of motif repeats at a microsatellite locus underlies the length differences among alleles. In total, 17 microsatellite loci have been identified that are polymorphic and informative among numerous populations in Connecticut; fifteen of these loci have been demonstrated to be polymorphic within a single Connecticut population, West Rock Ridge State Park (WRRSP), consisting of 38 isolates. The number of alleles at each locus segregating among the WRRSP isolates ranged from two to 12. A more detailed study of the WRRSP population is underway. Two fruit bodies isolated from each canker were dissected to obtain cultures of between eight and 16 sexual progeny from each fruit body. We are in the process of extracting DNA from these samples, and have begun analyzing them using microsatellite molecular markers. From these data we will determine the level of clonality and relatedness among isolates, and also determine whether sexual reproduction occurs by self-fertilization, outcrossing, or a mixture of both. PARTICIPANTS: The investigator on this project is Robert E. Marra, Assistant Scientist at The Connecticut Agricultural Experiment Station. Dr. Marra designs and conducts this study, performing and supervising all field and laboratory duties associated with this work. Knowledge gained through genotyping 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, as well as undergraduate students at St. Joseph's College and Northeastern University, and a high school student at Branford High School. 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 publication and presentation of this research to plant pathologists at the annual meeting of the Northeast Division of the American Phytopathological Society. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Black birch is increasing in abundance in Connecticut, and is therefore a tree of growing importance and concern. According to USDA Forest Services estimates from 2006, black birch is the fifth most common tree species in Connecticut, with approximately 300 million cubic feet of live trees on Connecticut timberlands. By contrast, black and yellow birch account for only about 5% of the growing stock removed annually from Connecticut timberlands, demonstrating the extent of underutilization of birch wood, due in large part to the devaluation caused by N. ditissima perennial cankers, the most common and most damaging disease affecting birches. Although trees infected with N. ditissima perennial cankers can persist for decades, the extensive scarring caused by the cankers renders them of little value for lumber or veneer. This devaluation cannot be overemphasized: perennial cankers reduce the value of birch stems three- to four-fold, resulting in wood that can only be sold for dunnage and pallet wood. The extent of damage to black birch in records of tree mortality most recently available (2006), show that yellow and black birch have a mortality rate of approximately 1%, in stark contrast to 0.3% for red and white oaks. Most of this high mortality is due to cankering from N. ditissima. Currently, high quality uncankered black and yellow birch command prices as high or higher than white oak for veneer logs and milled lumber. It is worth nothing that the tree's value beyond its use in veneer may be underestimated; for example, early accounts praised the qualities of black birch as an excellent substitute for black walnut in furniture and flooring, applications for which black walnut, a tree of increasing rareness, was once prized. Our efforts to more fully understand the biology and natural history of N. ditissima will contribute to the identification and utilization of control strategies. Knowledge gained from the application of microsatellite markers to the WRRSP population will be shared with other researchers interested in the population dynamics of forest pathogens.
Publications
- Marra, Robert E. and Jason A. Corwin. 2009. Isolation and characterization of codominant markers for the perennial canker fungal pathogen Neonectria ditissima. Molecular Ecology Resources 9(3):906-909.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Neonectria ditissima, a plant pathogenic fungus native to eastern North America, is considered among the most widespread and important canker-causing species of northeastern forests. It is associated with cankers on a wide range of broadleaved trees, including fruit trees, particularly apple and pear. The fungus is also devastating to yellow and black birch (Betula lenta). Extensive scarring and damage caused by N. ditissima leads to drastic reductions in log quality and value. To improve forest management strategies, a thorough understanding of the ecology, genetics, and reproductive biology of this pathogen is needed. The purpose of this project is to conduct a detailed genetic study of a population of N. ditissima in Connecticut, which will afford a better understanding of the roles of asexual and sexual reproduction, as well as dispersal, in the spread of this disease. We have sampled the fungus and its fruit bodies from cankers on 40 trees in a single population in West Rock Ridge State Park (WRRSP) in New Haven, Connecticut. We are in the process of extracting DNA from these samples, which will be analyzed using microsatellite molecular markers. From these data, we will determine the level of clonality and relatedness among isolates, and also determine whether sexual reproduction occurs by self-fertilization, outcrossing, or a mixture of both. PARTICIPANTS: The 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 and laboratory studies associated with this work. Knowledge gained through genotyping 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. TARGET AUDIENCES: The target audiences are as follows: state foresters, mycologists, plant pathologists, and evolutionary biologists. The primary method used to deliver science-based knowledge is through publication and presentation of this research to plant pathologists at the annual meeting of the Northeast Division of the American Phytopathological Society. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Knowledge gained from the application of microsatellite markers to the WRRSP population will be shared with other researchers interested in the population dynamics of forest pathogens. A better understanding of pathogen dispersal and the dynamics of infection will be essential to changes in stand management practices that increase the frequency of canker-free boles.
Publications
- No publications reported this period
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