Progress 10/01/03 to 09/30/08
Outputs
OUTPUTS: Findings have been reported to diverse audiences via presentations that include: West Virginia Invasive Species Forum; Presentations to National and State Chapters of The American Chestnut Foundation; The Society of American Foresters; The Audubon Society; West Virginia Division of Forestry and Natural Resources; Canadian Chestnut Council; USDI-National Park Service; A feature on WV-National Public Radio on chestnut restoration; Seminar to the biology departments at Hood College (MD) and West Virginia University; Theses and research publications have been disseminated on-line. PARTICIPANTS: Individuals: William L. MacDonald, PI; Mark L. Double; Students: Gordon Tyler Wright; Miranda King; Matthew Malone; Andrew Naymick; Rebecca Rush; Shawn Kenaley----Partner Organizations: Wisconsin Department of Natural Resources (Jane Cummings Carlson); Michigan State University (Dennis Fulbright and Andrew Jarosz); University of Maryland Biotechnology Institute-Shady Grove (Donald Nuss); The American Chestnut Foundation (Fred Hebard and Robert Paris) TARGET AUDIENCES: Efforts included formal classroom instruction, laboratory instruction and experience for undergraduate students in the SURE Program (Summer Undergraduate Research Experience). Target audience includes researchers at other institutions, The American Chestnut Foundation (the national organization and state chapters) and chestnut growers. PROJECT MODIFICATIONS: There were no major changes made in 2008.
Impacts
Outcomes of this project have focused on three field-oriented studies. The first is a long-term experiment designed to initiate biological control of chestnut blight at an American chestnut stand near West Salem, Wisconsin. The study has been in place for 20 years and involves the release of hypoviruses (viruses that reduce the virulence of the chestnut blight fungus) by introducing them into the resident population of Cryphonectria parasitica, the fungus that causes chestnut blight. Annual evaluations of the disease have demonstrated that biological control is possible especially on trees that have been treated with strains that contain hypoviruses. Although some trees in the stand have died, others now are in remission from the disease and should survive. The transition from high levels of disease to acceptable levels of biological control appears to require significant time; in this case, more than 20 years. As biological control is achieved, seed production should be restored so that the American chestnut component of the stand can be perpetuated. A second research topic has involved field testing transgenic (genetically modified) strains of Cryphonectria parasitica to assess whether they can enhance the biological control potential of hypoviruses. The advantage the modified strains bring is their ability to transmit hypoviruses at high levels to asexual spores and also to sexual spores that result when fungal mating occurs. To date, the experimentation has demonstrated that the transgenic strains can function in the forest by increasing the production of hypovirus-infected inoculum. While this condition should allow for better biological control, additional time will be required to confirm their effectiveness. A third series of studies is designed to evaluate whether increased resistance to chestnut blight that has resulted from The American Chestnut Foundation's breeding program can be combined with biological control afforded by utilizing hypoviruses. To this end, American, Chinese, European chestnuts and a variety of Chinese X American backcrosses have been planted in an orchard setting and will be used to test whether the two approaches to disease control can be combined effectively to manage chestnut blight. Impact Statement: The natural biological control of the chestnut blight fungus is known in several locations in North America and Europe. Field and laboratory tests are designed to understand the mechanisms by which hypoviruses become established and spread. The ultimate goal is to employ them as biological control agents in eastern North American forests.
Publications
- Jones, William E. 2008. Comparing virulence of Cryphonectria parasitica isolates removed from portions of cultures or cankers established before versus those after hypovirus inoculations. M.S. Thesis, West Virginia University, Morgantown, WV.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Assessment of disease progress, the spread of hypoviruses and canker evaluation continues at a West Salem, WI American chestnut stand. Canker treatments with hypovirus-laden inoculum were made from 1992-1997and were resumed in 2002-2007 after analyses demonstrated that hypovirus spread occurred principally on trees that received treatment inoculum. In 2007, 712 cankers were sampled. Yearly trends indicate that hypoviruses persist very well on trees where they are introduced by canker treatment but spread at a reduced rate to cankers on adjacent, untreated trees. Hypovirus treatment has prolonged the life of treated trees when compared to non-treated trees. A second study in WV to evaluate the biological control potential of transgenic C. parasitica strains containing an infectious cDNA copy of the CHV1-Euro7 hypovirus was designed to evaluate whether transgenic strains show greater potential to biologically control chestnut blight than their cytoplasmically infected
counterparts. Treatments employed compared transgenic hypovirulent strains (TG), cytoplasmic hypovirulent strains (CH) and virulent strains (V). To produce ascospore inoculum, cankers were spermatized by painting cankers with a conidial mixture that contained both mating types of the appropriate treatment strain (TG, CH or V). To produce conidial inoculum, cankers were initiated (SI) on separate trees each June (2004-07) by scratching the surface of the bark and painting the wounded area with a mycelial-agar slurry of the treatment strain (TG, CH, or V). Non-treated trees were left to monitor natural canker development and hypovirus spread. As of November 2007, 118, 62 and 55 cankers existed in the TG, CH and V plots, respectively. Many treated cankers in TG and CH plots acquired hypovirus from treatment. Cankers on SI trees also acquired hypovirus. Ascospore production has been assessed annually since 2004 by serially diluting ascospore contents from individual perithecia collected.
Colonies that resulted from culture were scored for pigmentation and morphology. In 2006, 24,097 individual ascospore colonies were examined from 988 perithecia. Hypovirulent ascospore (HVA) isolates were recovered from 70% of the spermatized cankers in the TG plots. Significant findings from 2007 include the increased detection of hypoviruses in the canker thallus and greater outcrossing to transgenic strains than in the previous years. Hypoviruses also were detected for the first time from cankers on trap trees. Transgenic strains also were detected in CH and V plots where they had not been introduced. A third study was established that included six plots of 150 trees each for the purpose of assessing the interaction of host resistance with virulent and hypovirulent strains of C. parasitica. Backcross lines along with pure American, Chinese and European chestnuts were included. As of August 2007, 48% of the seedlings were growing.
PARTICIPANTS: Individuals: William L. MacDonald, PI; Mark L. Double; Gordon Tyler Wright; Miranda L. King; Rebecca Rush; Brieann Mauser; Shawn Kenaley----Partner Organizations: Wisconsin Department of Natural Resources (Jane Cummings Carlson); Michigan State University (Dennis Fulbright and Andrew Jarosz); University of Maryland Biotechnology Institute-Shady Grove (Donald Nuss); American Chestnut Foundation (Fred Hebard and Robert Paris)
TARGET AUDIENCES: Efforts included formal classroom instruction, laboratory instruction and experience for one undergraduate student in the SURE Program (Summer Undergraduate Research Experience). Target audience includes researchers at other institutions, The American Chestnut Foundation state chapters and chestnut growers.
PROJECT MODIFICATIONS: There were no major changes made in 2007.
Impacts
The natural biological control of the chestnut blight fungus is known in several locations in North America and Europe. Our field and laboratory tests are designed to understand the mechanisms by which hypoviruses become established and spread. The ultimate goal is to employ them as biological control agents in eastern North America.
Publications
- Hebard, F.V., M.L. Double and W.L. MacDonald. 2007. A pathogen without rival. Pages 171-177 in: Mighty Giants: An American Chestnut Anthology. Chris Bolgiano, ed., The American Chestnut Foundation, Bennington, VT.
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Progress 01/01/06 to 12/31/06
Outputs
The spread of two hypoviruses and evaluation of canker condition continues at a West Salem, WI American chestnut stand. Canker treatments with hypovirus-laden inoculum were resumed after analyses demonstrated that hypovirus spread was best on trees that received treatment inoculum. In 2006, 635 cankers were sampled in 12 permanent plots. Yearly trends indicate that hypoviruses persist best on trees where they are introduced by canker treatment but spread poorly to cankers on adjacent, untreated trees. Hypovirus treatment has prolonged the life of treated trees. In a second WV study initiated in 2004, the biological control potrential of C. parastica strains containing an infectious cDNA copy of the CHV1-Euro7 hypovirus (transgenic) is being evaluated. This study compares inoculum production, sexual reproduction and dissemination of cytoplasmic (CH), transgenic (TG) and virulent (V) isolates and is designed to evaluate whether TG strains containing a cDNA transgene
encoding the viral genome of CHV1-Euro7 show greater potential to biologically control chestnut blight than their CH infected counterparts. To introduce inoculum, naturally occurring and artificially established cankers were spermatized by painting cankers in May, June and July with a conidial mixture that contained both mating types (MAT-1 and MAT-2) of either TG, CM, or V inoculum. To produce conidial inoculum, cankers on separate trees were created each June (2004-06) by scratching the surface of the bark and painting the wounded area with a mycelial-agar slurry of the appropriate treatment strain (TG, CH, or V). Non-treated trees were left to monitor natural canker development as well as hypovirus spread. Each October (2004-2006), tree condition and natural canker establishment were assessed. By October 2006, there were 79 natural cankers in TG plots, 35 in CH plots, and 42 in V plots. These cankers were sampled to determine the hypovirus infection status of the thallus. Many
treated cankers acquired hypovirus from the treatment inoculum. Cankers occurring below scratch initiated cankers also acquired hypovirus. However, no transmission of introduced inoculum has been detected on trap trees in the plots. Ascospore production was assessed by collecting bark discs in the fall of 2004, 2005 and 2006 and serially diluting ascospore contents from individual perithecia. Pigmentation and morphology were recorded for ascospores from all TG, CH and V plots. From 2005 bark collections, 13,455 individual ascospore colonies were examined from 565 individual perithecia. Hypovirulent ascospore isolates were collected from 75% of the spermatized cankers in TG plots. From those cankers, 68% of the perithecia yielded trangenic ascospores. Distribution of hypovirus to many different vegetative compatibility types should increase the biological control potential of the hypoviruses. Monitoring of canker development in 2007 will provide further information on the fate of the
transgenic inoculum being produced and whether this approach results in improved biological control.
Impacts
The natural biological control of the chestnut blight fungus is know in several locations in North America and Europe. Our field and laboratory tests are designed to understand the mechanisms by which these hypoviruses become established and spread. The ultimate goal is to employ them as biological control agents in eastern North America.
Publications
- MacDonald, W.L. and M.L. Double. 2006. Hypovirulence: Use and limitations as a chestnut blight biological control. Pages 87-95 in: Steiner, K.C. and J.E. Carlson eds. Restoration of American Chestnut To Forest Lands-Proceedings of a Conference and Workshop. May 4-6, 2004, The North Carolina Arboretum. Natural Resources Report NPS/NCR/CUE/NRR-2006/001, National Park Service, Washington, DC.
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Progress 01/01/05 to 12/31/05
Outputs
Disease progress, spread of two hypoviruses and evaluation of host responses continues at a West Salem, WI American chestnut stand. Canker treatments with hypovirus-laden inoculum were made from 1992-1997 and were resumed in 2002-2005 after analyses demonstrated that hypovirus spread occurred principally on trees that received treatment inoculum. In 2005, 628 cankers were sampled in 12 permanent plots. Year-to-year trends indicate that hypoviruses persist very well on trees where they are introduced by canker treatment but spread poorly to cankers on adjacent, untreated trees. Hypovirus treatment has prolonged the life of treated trees when compared to non-treated trees. A second study in WV was initiated in 2004 to evaluate the biological control potential of C. parasitica strains containing an infectious cDNA copy of the CHV1-Euro7 hypovirus (transgenic). This study compares inoculum production, sexual reproduction and dissemination of cytoplasmic and transgenic
isolates and is designed to evaluate whether transgenic C. parasitica strains containing a cDNA transgene encoding the viral genome of CHV1-Euro7 show greater potential to biologically control blight than their cytoplasmically infected counterparts. Three treatments were employed to compare transgenic hypovirulent strains (TG), cytoplasmic hypovirulent strains (CH), and virulent strains (V). To produce ascospore inoculum, naturally occurring and artificially established cankers were spermatized by painting cankers in June, July, and September with a conidial mixture that contained both mating types (MAT-1 and MAT-2) of the appropriate treatment strain (TG, CH, or V). To produce conidial inoculum, cankers were surface-initiated (SI) on separate trees in June 2004 by scratching the bark and painting the wounded area with a slurry of the appropriate treatment strain (TG, CH, or V). Non-treated trees also were left to monitor natural canker formation. Most trees were asymptomatic after
the first treatment season and the incidence of natural infection remained relatively low. In the fall of 2004 and 2005, cankers were sampled to determine the hypovirus infection status of the thallus. Although the purpose of the spermatization treatment was to produce ascospores, many treated cankers also acquired hypovirus from the treatment inoculum. Ascospore production was assessed by collecting bark discs in October 2004 and serially diluting ascospore contents from perithecia. Hypovirulent ascospore (HVA) isolates were only collected from TG plots and at less than expected Mendelian ratios. Pigmentation segregated as expected in V and CH plots. The transgenic MAT-1 treatment strain effectively spermatized and produced HVAs on both initiated cankers and treated natural infections. To assess the increased conversion capability of HVAs, 18 HVA isolates were paired with 17 vegetative compatibility (v-c) types isolated from the study site; pairings were examined for hypovirus
transmission. Collectively, HVA isolates were able to convert 15 of the 17 v-c types. Overall, the production of HVAs with different conversion capabilities increased the biological control potential of transgenic strains.
Impacts
The natural biological control of the chestnut blight fungus is known in several locations in North America and Europe. Our field and laboratory tests are designed to understand the mechanisms by which these hypoviruses become established and spread. The ultimate goal is to employ them as biological control agents in eastern North America
Publications
- Root, C., C.J. Balbalian, R. Bierman, L.M. Geletka, S.L. Anagnostakis, W.L. MacDonald, M.L. Double and D.L. Nuss. 2005. Multiseasonal field release and spermatization trials of transgenic hypovirulent strains of Cryphonectria parasitica containing cDNA copies of hypovirus CHV1-EP713. For. Path. 35:277-297.
- Bauman, J. 2005. A copmarison of the growth and asexual reproduction by Cryphonectria parasitica isolates infected with hypoviruses via anastomosis and transfection. M.S. Thesis, West Virginia University, Morgantown. 91 pp.
- McGuire, I.C., J.E. Davis, M.L. Double, W.L. MacDonald, T. Raushcer, S. McCawley and M.G. Milgroom. 2005. Heterokaryon formation and parasexual recombination between vegetatively incompatible lineages in a population of the chestnut blight fungus, Cryphonectria parasitica. Mol. Ecol. 14:3657-3669.
- Rittenour, W.R. 2005. The biological control potential of Cryphonectria parasitica strains containing an infectious cDNA copy of the hypovirus CHV1-Euro7. M.S. Thesis, West Virginia University, Morgantown. 78 pp.
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Progress 01/01/04 to 12/31/04
Outputs
Assessment of disease progress, spread of two hypoviruses and evaluation of host responses continues at a West Salem, WI American chestnut stand. Canker treatments with hypovirus-laden inoculum were made from 1992-1997. In 2003 and 2004, hypovirus introductions were resumed after analyses demonstrated that hypovirus spread was restricted principally to trees that had received treatment inoculum. In 2004, approximately 850 cankers were sampled in 12 permanent plots. Year-to-year trends indicate that hypoviruses persist very well on trees where they are introduced by canker treatment but become poorly established on adjacent, untreated trees. Hypovirus treatment has prolonged the life of treated trees when compared to non-treated ones. The number of vegetative compatibility types of Cryphonectria parasitica, while increasing, is still dominated by founder types. A second study in WV has focused on procedures to introduce hypovirulent inoculum to virulent cankers.
Treatment procedures involved a variety of wounding protocols and canker coverings. Success of treatment was evaluated by assessing hypovirus transmission to the canker inciting strain. The study concluded that wounding was an important treatment prerequisite and covering made little difference except in the survival of the treatment inoculum. Another WV study compared the performance of Cryphonectria parasitica isolates that had been infected with several different hypoviruses by transfection or anastomosis. Their growth and sporulation was compared in laboratory and field tests. In general, the manner of hypovirus infection did not significantly alter their ability to form cankers or produce asexual hypovirulent inoculum. However, different hypoviruses and host backgrounds often influenced their performance. An additional field study was initiated in 2004 to evaluate the biological control potential of C. parasitica strains containing an infectious cDNA copy of the CHV1-Euro7
hypovirus. This study will compare inoculum production, sexual reproduction and dissemination of cytoplasmic and trangenic isolates.
Impacts
The natural biological control of the chestnut blight fungus is known in several locations in North America and Europe. Our field and laboratory tests are designed to understand the mechanisms by which these hypoviruses (the agents responsible for biologcial control) become established and spread. The ultimate goal is to employ hypoviruses as control agents in eastern hardwood forests.
Publications
- Bell, B.C. 2004. Comparison of chestnut canker treatment procedures for hypovirus introduction. M.S. Thesis, West Virginia University, Morgantown.
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