Editing American chestnut (section)

==Attempts at restoration==
[[File:castanea dentata-field trial2009.jpg|thumb|upright|American chestnut field trial sapling from the American Chestnut Cooperators Foundation]]
[[File:Tower Hill Botanic Garden - American chestnuts.jpg|thumb|right|250px|Experimental trials by [[The American Chestnut Foundation]] at [[Tower Hill Botanic Garden]] in [[Massachusetts]]]]

===Transgenic blight-resistant American chestnut===
Researchers at the [[State University of New York College of Environmental Science and Forestry]] (SUNY ESF) have developed the [[Darling 58]] chestnut cultivar. This cultivar expresses the gene for [[wheat]] [[oxalate oxidase]] enzyme, which breaks down the [[oxalic acid]] produced by the blight fungus, preventing the death of the tree. When pollen of transgenic fathers fertilizes an ovule of a native mother in the field, those resulting seedlings that express the oxalate oxidase enzyme show growth similar to non-transgenic full siblings, indicating that the [[transgene]] does not impede growth under field conditions.<ref>{{cite journal |last1=Riendeau |first1=Tyler |title=Advancing American Chestnut (Castanea Dentata) Restoration Through Science, GIS And Partnerships |url=https://dune.une.edu/theses/444/ |website=All Theses And Dissertations |date=1 July 2023}}</ref> The modified chestnut does not affect survival, pollen use, or reproduction of [[Bombus impatiens|bumble bees]].<ref>{{cite journal |last1=Newhouse |first1=Andrew E. |last2=Allwine |first2=Anastasia E. |last3=Oakes |first3=Allison D. |last4=Matthews |first4=Dakota F. |last5=McArt |first5=Scott H. |last6=Powell |first6=William A. |title=Bumble bee (Bombus impatiens) survival, pollen usage, and reproduction are not affected by oxalate oxidase at realistic concentrations in American chestnut (Castanea dentata) pollen |journal=Transgenic Research |date=10 June 2021 |volume=30 |issue=6 |pages=751–764 |doi=10.1007/s11248-021-00263-w|pmid=34110572 |pmc=8580921 |doi-access=free }}</ref> A deregulation petition for the Darling 58 variant has been submitted.<ref>[https://www.aphis.usda.gov/brs/aphisdocs/19-309-01p.pdf "Petition for Determination of Nonregulated Status for Blight-Resistant Darling 58 American Chestnut"]. https://www.aphis.usda.gov, Retrieved August 28, 2020.</ref><ref>[https://www.federalregister.gov/documents/2020/08/19/2020-18135/state-university-of-new-york-college-of-environmental-science-and-forestry-petition-for "State University of New York College of Environmental Science and Forestry; Petition for Determination of Nonregulated Status for Blight-Resistant Darling 58 American Chestnut"]. https://www.federalregister.gov, August 19, 2020. Retrieved August 28, 2020.</ref> If approved, these trees could be the first genetically modified forest trees released in the wild in the United States.<ref>{{cite journal |last1=Thompson |first1=Helen |year=2012 |title=Plant science: The chestnut resurrection |journal=[[Nature (journal)|Nature]] |volume=490 |issue=748|pages=22–23 |doi=10.1038/490022a |pmid=23038446 |bibcode=2012Natur.490...22T |doi-access=free }}</ref><ref>{{cite news|last=Wines|first=Michael|title=Like-Minded Rivals Race to Bring Back an American Icon|url=https://www.nytimes.com/2013/07/14/us/like-minded-rivals-race-to-bring-back-an-american-icon.html?ref=earth&_r=0|date=July 13, 2013|newspaper=New York Times|access-date=July 14, 2013}}</ref>

Unlike American chestnut, Japanese chestnut exhibits resistance to ''Phytophthora cinnamomi'', the fungal pathogen that causes ink disease. The mechanism of resistance of ''C.&nbsp;crenata'' to ''Phytophthora cinnamomi'' may derive from its expression of the Cast_[[ginkbilobin|Gnk2]]-like gene (99.6% identical with {{UniProt|A0A8J4V9V8}}).<ref>{{cite journal |vauthors=Santos C, Nelson CD, Zhebentyayeva T, Machado H, Gomes-Laranjo J, Costa RL |title=First interspecific genetic linkage map for Castanea sativa x Castanea crenata revealed QTLs for resistance to Phytophthora cinnamomi |journal=PLOS ONE |volume=12 |issue=9 |pages=e0184381 |date=2017 |pmid=28880954 |pmc=5589223 |doi=10.1371/journal.pone.0184381 |bibcode=2017PLoSO..1284381S |doi-access=free }}</ref> Transgenic modification of ''C.&nbsp;dentata'' with the Cast_Gnk2-like gene may provide a mechanism for developing American chestnut trees resistant to ''Phytophthora cinnamomi''.<ref name=McGuigan20/> Stacking of the Cast_Gnk2-like gene and the oxalate oxidase gene may provide a means of developing genetically modified American chestnut trees resistant to both the chestnut blight and to ink disease.<ref name=McGuigan20>{{cite journal |last1=McGuigan |first1=Linda |last2=Fernandes |first2=Patrícia |last3=Oakes |first3=Allison |last4=Stewart |first4=Kristen |last5=Powell |first5=William |title=Transformation of American Chestnut (Castanea dentata (Marsh.) Borkh) Using RITA Temporary Immersion Bioreactors and We Vitro Containers |journal=Forests |date=2020-11-13 |volume=11 |issue=11 |page=1196 |doi=10.3390/f11111196|doi-access=free }}</ref>

The American Chestnut Research and Restoration Project at SUNY-ESF is not pursuing [[intellectual property]] protection through patents. [[William Powell (biologist)|Dr William Powell]], the project's co-director, states that the decision to not pursue a patent on the project's transgenic lines was to allow the plant to be more accessible for conservationists and members of the public.<ref name="Barnes-Delborne-2021" /> Powell posits that a patent would constrain the spread of the oxalate oxidase transgene into American chestnut populations by limiting the ability to freely plant transgenic trees and cross the trees with surviving American chestnuts or the hybrids produced in the backcross program. Powell states that patents would be a barrier to chestnut restoration and in direct opposition to the program's goals of collaboration.<ref name="Barnes-Delborne-2021" /> While patent protection is not sought, the non-profit [[The American Chestnut Foundation|American Chestnut Foundation]] (TACF) maintains control through a [[Germplasm]] Agreement, ensuring authorized use of chestnut germplasm. The agreement safeguards TACF's rights and aligns with the organization's restoration goals.

A laboratory error resulted in mistaken use of the Darling 54 cultivar instead of the Darling 58 cultivar in some field trials.<ref>{{cite web |last1=Todd |first1=Roxy |title=After GMO program hits snag, what's the future of restoring American chestnuts? |url=https://www.alleghenyfront.org/american-chestnut-blight-genetically-modified/ |website=The Allegheny Front |date=8 March 2024}}</ref> TACF and colleagues have also reported decreased growth rates, and poor heritability of resistance of the Darling 58 cultivar.<ref>{{cite web |title=TACF Chestnut Chat |url=https://www.youtube.com/watch?v=9w_ehgYyxGY |website=YouTube | date=September 15, 2023 |publisher=The American Chestnut Foundation |access-date=8 June 2024}}</ref> In response, the American Chestnut Foundation withdrew its support of development of the Darling 58 cultivar in December 2023.<ref>{{Cite web |last=Brindley |first=Hal |date=2023-12-08 |title=Press Release: TACF Discontinues Development of Darling 58 |url=https://tacf.org/tacf-discontinues-development-of-darling-58/ |access-date=2024-06-05 |website=The American Chestnut Foundation |language=en-US}}</ref> The American Chestnut Research & Restoration Program at SUNY ESF is continuing to pursue deregulation.<ref>{{Cite news |last=Grandoni |first=Dino |date=2023-12-27 |title=Genetic engineering was meant to save chestnut trees. Then there was a mistake. |url=https://www.washingtonpost.com/climate-environment/2023/12/24/chestnut-tree-genetic-engineering-mistake/ |access-date=2024-02-05 |newspaper=Washington Post |language=en-US |issn=0190-8286}}</ref><ref>{{Cite web |title=Progress Update 2023 |url=https://www.esf.edu/chestnut/progress-report/2023.php |access-date=2024-02-05 |website=www.esf.edu |language=en}}</ref><ref>{{Cite web |title=The American Chestnut Research & Restoration Project at ESF |url=https://www.esf.edu/chestnut/index.php |access-date=2024-06-05 |website=www.esf.edu |language=en}}</ref><ref>{{Cite web |last=Brindley |first=Hal |date=2023-12-08 |title=Press Release: TACF Discontinues Development of Darling 58 |url=https://tacf.org/tacf-discontinues-development-of-darling-58/ |access-date=2024-02-05 |website=The American Chestnut Foundation |language=en-US}}</ref><ref>{{Cite web |title=Darling 58 |url=https://tacf.org/darling-58/ |access-date=2024-02-05 |website=The American Chestnut Foundation |language=en-US}}</ref>

In 2022, the SUNY ESF group developed transgenic American chestnut trees incorporating both the oxalate oxidase transgene from wheat and the win3.12 promoter transgene from the [[Populus deltoides|necklace poplar]]. Unlike the CAMV 35S promoter which acts at all times, this poplar promoter drives OxO expression at a low level under basal conditions, but elevates to high levels under conditions of wounding or tissue infection. In laboratory bioassays, win3.12-OxO lines showed elevated disease tolerance similar to that exhibited by blight-resistant Chinese chestnut.<ref>{{cite journal |last1=Carlson |first1=Erik |last2=Stewart |first2=Kristen |last3=Baier |first3=Kathleen |last4=McGuigan |first4=Linda |last5=Culpepper |first5=Tobi |last6=Powell |first6=William |title=Pathogen-induced expression of a blight tolerance transgene in American chestnut |journal=Molecular Plant Pathology |date=March 2022 |volume=23 |issue=3 |pages=370–382 |doi=10.1111/mpp.13165|pmid=34841616 |pmc=8828690 |bibcode=2022MolPP..23..370C }}</ref> Limiting expression of the OxO gene to blight infected tissues is expected to reduce the metabolic cost of gene expression, resulting in increased competitiveness of these new cultivars. As of January 2025, the win3.12 OxO chestnut is still in the research and development phase and has not been deployed for restoration purposes.<ref>{{cite web |title=Darling 58 /54 {{!}} The American Chestnut Foundation |url=https://tacf.org/darling-58/}}</ref>

===Intercrossing surviving American chestnuts===
{{main|American Chestnut Cooperators' Foundation}}

[[File:Floyda.jpg|thumb|upright|Large surviving blight-resistant American chestnut in its natural range. Surviving trees passing resistance tests are used in ACCF's All American Breeding program.]]
[[American Chestnut Cooperators' Foundation]] (ACCF) is not using crosses with Asian species for blight resistance, but [[Hybrid (biology)|intercrossing]] among American chestnuts selected for native resistance to the blight, a breeding strategy described by the ACCF as "All-American intercrosses". John Rush Elkins, a research chemist and [[professor emeritus]] of chemistry at [[Concord University]], and Gary Griffin, professor of plant pathology at [[Virginia Tech]], think there may be several different characteristics which favor blight resistance. Both Elkins and Griffin have written extensively about the American chestnut.<ref name=griffin>[http://www.accf-online.org/biblio.html "Bibliography."] {{Webarchive|url=https://web.archive.org/web/20160305080130/http://www.accf-online.org/biblio.html |date=March 5, 2016 }} ''www.accf-online.org.'' Retrieved January 11, 2016.</ref> They believe that by making intercrosses among resistant American chestnuts from many locations, they will continue to improve upon the levels of blight resistance to make an American chestnut that can compete in the forest. Griffin, who has been involved with American chestnut restoration for many years,<ref name=griffin/> developed a scale for assessing levels of blight resistance, which made it possible to make selections scientifically. He inoculated five-year-old chestnuts with a standard lethal strain of the blight fungus and measured growth of the cankers. Chestnuts with no resistance to blight make rapid-growing, sunken cankers that are deep and kill tissue right to the wood. Resistant chestnuts make slow-growing, swollen cankers that are superficial: live tissue can be recovered under these cankers. The level of blight resistance is judged by periodic measurement of cankers. Grafts from large survivors of the blight epidemic were evaluated following inoculations, and controlled crosses among resistant American chestnut trees were made beginning in 1980. The first "All-American intercrosses" were planted in Virginia Tech's Martin American Chestnut Planting in [[Giles County, Virginia]], and in [[Beckley, West Virginia]]. They were inoculated in 1990 and evaluated in 1991 and 1992. Nine of the trees showed resistance equal to their parents, and four of these had resistance comparable to hybrids in the same test.<ref name=griffin/><ref name="elkins">Griffin, G. J., J. R. Elkins, D. McCurdy, and S. L. Griffin. [http://ecosystems.psu.edu/research/chestnut/information/conference-2004/conference/griffin "Integrated use of resistance, hypovirulence, and forest management to control blight on American chestnut."] ''www.ecosystems.psu.edu'', 2005.</ref><ref name=integrated>[https://archive.org/stream/restorationofame00stei/restorationofame00stei_djvu.txt "Restoration of American Chestnut to Forest Lands: Proceedings of a Conference and Workshop Held May 4-6, 2004 at The North Carolina Arboretum."] ''www.archive.org.'' Retrieved January 22, 2016.</ref><ref name="resist">[http://www.accf-online.org/breed.html "Breeding for Blight Resistance."] {{Webarchive|url=https://web.archive.org/web/20160304215224/http://www.accf-online.org/breed.html |date=March 4, 2016 }} ''www.accf-online.org.'' Retrieved January 11, 2016.</ref> Many ACCF chestnuts have expressed blight resistance equal to or greater than an original blight survivor but so far, only a handful have demonstrated superior, durable blight control. Time will tell if the progeny of these best chestnuts exhibit durable blight resistance in different stress environments.<ref>[http://www.accf-online.org/news.html "American Chestnut Cooperators' Foundation 2015 Newsletter: Grower Reports."] {{Webarchive|url=https://web.archive.org/web/20160122062941/http://www.accf-online.org/news.html |date=January 22, 2016 }} ''www.accf-online.org.'' Retrieved January 11, 2015.</ref>

===Backcrossing===
Backcrossing as a treatment for blight was first proposed by [[Charles Burnham (geneticist)|Charles Burnham]] of the [[University of Minnesota]] in the 1970s.<ref name=gary/><ref name=hebard/><ref name=doctor>[http://www.charliechestnut.org/Phase1/Scrapbook/Conservation3AGenetic.html "Conservation - Genetic Research"] {{Webarchive|url=https://web.archive.org/web/20160427063734/http://www.charliechestnut.org/Phase1/Scrapbook/Conservation3AGenetic.html |date=April 27, 2016 }}. ''www.charliechestnut.org.'' Retrieved January 12, 2016.</ref> Burnham, a professor emeritus in [[agronomy]] and [[plant genetics]] who was considered one of the pioneers of [[maize]] genetics,<ref name=stories>Galloway, Paul R. [http://www.tacf.org/pdfs/chapters/VTNH/My%20Chestnut%20Story%20-%20Paul%20Galloway.pdf "My Chestnut Story"] {{Webarchive|url=https://web.archive.org/web/20161027081123/http://www.tacf.org/pdfs/chapters/VTNH/My%20Chestnut%20Story%20-%20Paul%20Galloway.pdf |date=October 27, 2016 }}. ''www.tacf.org.'' Retrieved October 5, 2015.</ref> realized that experiments conducted by the [[USDA]] to cross-breed American chestnuts with European and Asian chestnuts erroneously assumed that a large number of genes were responsible for blight resistance, while it is currently believed the number of responsible genes is low. The USDA abandoned their cross-breeding program and destroyed local plantings around 1960 after failing to produce a blight-resistant hybrid.<ref name=failure>{{cite journal |last1=Burnham |first1=Charles R |year=1986 |title=Chestnut Hybrids from the USDA-Connecticut Breeding Programs |url=http://ecosystems.psu.edu/research/chestnut/information/journal/vol1-issue2 |journal=The Journal of the American Chestnut Foundation |volume=1 |issue=2|pages=8–13 }}</ref> Burnham's recognition of the USDA's error led to him joining with others to create [[The American Chestnut Foundation]] in 1983, with the sole purpose of breeding a blight-resistant American chestnut.<ref name=doctor/> [[The American Chestnut Foundation]] is [[backcrossing]] blight-resistant Chinese chestnut into American chestnut trees, to recover the American growth characteristics and genetic makeup, and then finally intercrossing the advanced backcross generations to eliminate genes for susceptibility to blight.<ref name=lori>Valigra, Lori. [https://web.archive.org/web/20060104013902/http://news.nationalgeographic.com/news/2005/12/1229_051229_chestnut.html "Back-Breeding Could Restore Chestnut Trees Ravaged by Blight"]. ''[[National Geographic News]]'', December 29, 2005. Retrieved September 26, 2015.</ref> The first backcrossed American chestnut tree, called "Clapper", survived blight for 25 years, and [[grafting|graft]]s of the tree have been used by The American Chestnut Foundation since 1983.<ref name=failure/> The Pennsylvania chapter of The American Chestnut Foundation, which seeks to restore the American chestnut to the forests of the [[Mid-Atlantic states]], has planted over 22,000 trees.<ref name=penn>[http://www.tacf.org/pdfs/resources/planting_manual.pdf "Planting and growing chestnut trees"] {{Webarchive|url=https://web.archive.org/web/20160304074428/http://www.tacf.org/pdfs/resources/planting_manual.pdf |date=March 4, 2016 }}. ''www.tacf.org.'' Retrieved January 15, 2016.</ref>

The [[Surface Mining Control and Reclamation Act of 1977]] requires owners of abandoned coal mines to cover at least 80 percent of their land with vegetation. While many companies planted invasive grasses, others began funding research on planting trees, because they can be more cost-effective, and yield better results.<ref name=cadiz>Barnes, Philip. [https://www.ohio.edu/research/communications/gilland.cfm "Return of the Native: Biologists revive the chestnut tree at former coal mine sites"] {{Webarchive|url=https://web.archive.org/web/20151001195133/https://www.ohio.edu/research/communications/gilland.cfm |date=October 1, 2015 }}. ''www.ohio.edu.'' Retrieved September 30, 2015.</ref> Keith Gilland began planting American chestnut trees in old [[strip mine]]s in 2008 as a student at [[Miami University]], and to date has planted over 5,000 trees.<ref name=cadiz/> In 2005, a hybrid tree with mostly American genes was planted on the lawn of the [[White House]].<ref>{{cite news
 |title=Trying to Light A Fire Under Chestnut Revival
 |newspaper=[[The Washington Post]]
 |date=December 29, 2005
 |url=https://www.washingtonpost.com/wp-dyn/content/article/2005/12/28/AR2005122801235.html
 |access-date=October 29, 2010 }}</ref> A tree planted in 2005 in the tree library outside the [[USDA]] building was still very healthy seven years later; it contains 98% American chestnut DNA and 2% Chinese chestnut DNA. This tree contains enough Chinese chestnut DNA that encodes for systemic resistance genes to resist the blight. This is essential for restoring the American chestnut trees into the Northeast.<ref name=GreenXC>[https://web.archive.org/web/20110703095726/http://greenxc.com/2011/american-chestnut-restoration-breakthrough-tale-tree/ "American Chestnut Restoration Breakthrough: The Tale of a Tree"]. [[Wayback Machine]]. ''www.greenxc.com'', June 28, 2011. Retrieved September 23, 2014.</ref> The Northern Nut Growers Association (NNGA) has also been active in pursuing viable hybrids.<ref name=nuts>[http://www.nutgrowing.org/ng-american-chestnut.htm "Nut Grower's Guide--Chestnut: American Chestnut"] {{Webarchive|url=https://web.archive.org/web/20150923060209/http://www.nutgrowing.org/ng-american-chestnut.htm |date=September 23, 2015 }}. ''Northern Nut Growers Association, Inc.'' www.nutgrowing.org. Retrieved September 22, 2015.</ref> From 1962 to 1990, [[Alfred Szego]] and other members of the NNGA developed hybrids with Chinese varieties which showed limited resistance. Initially the backcrossing method would breed a hybrid from an American chestnut nut and a Chinese chestnut, the hybrid would then be bred with a normal American chestnut, subsequent breeding would involve a hybrid and an American chestnut or two hybrids, which would increase the genetic makeup of the hybrids primarily American chestnut but still retain the blight resistance of the Chinese chestnut.<ref>{{Cite journal|last=Burnham|first=Charles R.|date=1988|title=The Restoration of the American Chestnut: Mendelian genetics may solve a problem that has resisted other approaches|journal=American Scientist|volume=76|issue=5|pages=478–487|jstor=27855387|issn=0003-0996}}</ref>

A backcross breeding program has integrated desirable American chestnut traits with traits from the [[Castanea mollissima|Chinese chestnut]], achieving intermediate resistance to ''Cryphonectria parasitica'' and ''Phytophthora cinnamomi'' in the hybrid genome.<ref>{{cite journal |last1=Clark |first1=Stacy L. |last2=Schlarbaum |first2=Scott E. |last3=Saxton |first3=Arnold M. |last4=Jeffers |first4=Steven N. |last5=Baird |first5=Richard E. |title=Eight-year field performance of backcross American chestnut (Castanea dentata) seedlings planted in the southern Appalachians, USA |journal=Forest Ecology and Management |date=March 2023 |volume=532 |page=120820 |doi=10.1016/j.foreco.2023.120820|s2cid=256572816 |doi-access=free |bibcode=2023ForEM.53220820C }}</ref>

The B3F3 strain, a product of backcrossing and intercrossing with selection for blight resistance, is approximately 94% American chestnut and 6% Chinese chestnut<ref>{{cite magazine|title=Breeding for Resistance: TACF and the Burnham Hypothesis|last=Smith|first=Anna Huckabee|date=March–April 2012|volume=26|issue=2|pages=11–15|magazine=The Journal of the American Chestnut Foundation|url=https://tacf.org/wp-content/uploads/2016/08/Blight-Resistance.pdf|access-date=10 May 2024}}</ref><ref>{{cite web|url=https://www.missouribotanicalgarden.org/PlantFinder/PlantFinderDetails.aspx?taxonid=444846&isprofile=0&hf=1|title=''Castanea'' hybrid (15/16 B3F3)|website=Plant Finder|publisher=Missouri Botanical Garden|access-date=10 May 2024}}</ref> and has been planted experimentally in Maryland in an orchard.<ref>{{Cite web |url=https://www.fws.gov/endangered/about/ep_46_2015.html |title=Featured Species {{!}} American chestnut (Episode 46)|website=fws.gov |date=8 October 2015|archive-date=29 October 2020 |archive-url=https://web.archive.org/web/20201029082941/https://www.fws.gov/endangered/about/ep_46_2015.html }}</ref>

===Hypovirulence===
[[Hypovirus]] is the only [[genus]] in the family [[Hypoviridae]]. Members of this genus infect fungal pathogens and reduce their ability to cause disease (hypovirulence).<ref name=fungus>[http://www.dpvweb.net/notes/showgenus.php?genus=Hypovirus "Notes on Genus: Hypovirus"] {{Webarchive|url=https://web.archive.org/web/20160304093422/http://www.dpvweb.net/notes/showgenus.php?genus=Hypovirus |date=March 4, 2016 }}. ''www.dpvweb.net.'' Retrieved October 14, 2015.</ref> In particular, the virus infects ''Cryphonectria parasitica'', the fungus that causes chestnut blight, which has enabled infected trees to recover from the blight. The use of hypovirulence to control blight originated in [[Europe]] where the fungal virus spread naturally through populations of [[European chestnuts]]. The reduced ability of the fungus to cause disease allowed the European chestnut to regenerate, creating large stands of trees. Hypovirulence has also been found in North America, but has not spread effectively.<ref name=spread>[http://www.tacf.org/FAQ.php "Frequently Asked Questions"] {{Webarchive|url=https://web.archive.org/web/20151020065031/http://www.tacf.org/FAQ.php |date=October 20, 2015 }}. ''www.tacf.org.'' Retrieved November 1, 2015.</ref> The "Arner Tree" of [[Southern Ontario]] is one of the best examples of naturally occurring hypovirulence. It is a mature American chestnut that has recovered from severe infections of chestnut blight. The cankers have healed over and the tree continues to grow vigorously. Scientists have discovered that the chestnut blight remaining on the tree is hypovirulent, although [[Genetic isolate|isolates]] taken from the tree do not have the fungal viruses found in other isolates.<ref name=healed>[http://www.canadianchestnutcouncil.ca/index.cfm?page=hypovirulence "Hypovirulence"]. ''www.canadianchestnutcouncil.ca.'' Retrieved October 14, 2015.</ref> Trees inoculated with isolates taken from the Arner tree have shown moderate canker control.<ref name=control>[http://ecosystems.psu.edu/research/chestnut/meetings/crees-ne-projects/minutes-pdfs/minutes-2001 "NE-140 Technical Committee Meeting Biological Improvement of Chestnut (''Castanea'' spp.) and Management of Pests"]. ''www.ecosystem.psu.edu'', October 20, 2001. Retrieved October 14, 2015.</ref> The cankers of hypovirulent American chestnut trees occur on the outermost tissues of the tree but the cankers do not spread into the growth tissues of the American chestnut tree, thereby providing it with a resistance.<ref>{{Cite journal|last=Griffin|first=Gary |title=Blight Control and Restoration of the American Chestnut |journal=Journal of Forestry |volume=98 |issue=2 |date=February 2000 |pages=22–27 |doi=10.1093/jof/98.2.22 |doi-access=free|url=https://academic.oup.com/jof/article/98/2/22/4614209}}</ref>