Restoring the American Chestnut with a Virus and Biotechnology

May 7, 2020

The legacy of the American chestnut (Castanea dentata) is inextricably linked to its losing battle against a fungal pathogen, chestnut blight (Cryphonectria parasitica). But, a virus and some genetic engineering may help us return this tree to the upper canopy of our forests.

In the Audubon Guide to North American Trees, the American chestnut has 3 descriptions. The first is a memory of this tree’s historical stature, which was once so common and useful that it would make both cribs and caskets. The second, more practical, entry describes how these trees look to the modern observer: small and enfeebled by the unrelenting chestnut blight fungus that prevents these trees from reaching their full potential. The third entry describes the etiology of chestnut blight, which killed an estimated 4 billion American chestnuts in the 50 years following its introduction to New York in the early 1900’s.

American Chestnut Foundation photo from the 19th century shows the monumental size of American chestnuts before chestnut blight spread throughout their natural range.
American Chestnut Foundation photo from the 19th century shows the monumental size of American chestnuts before chestnut blight spread throughout their natural range.

The loss of the American chestnut is often framed as a loss for American industry, but Native Americans in the Eastern United States have valued this tree for centuries. The tree’s abundant and edible nuts were easily storable provisions that could be stewed into porridge. This dish introduced chestnuts into Iroquois mythology, where a boy named Hodadenon foolishly uses up the remaining stored chestnuts and must brave great danger to collect more. This story ultimately places chestnuts as key to survival during hard times, and its moral is that chestnuts (and food generally) must be shared and planted widely. In this context, the damages done by chestnut blight are not just financial, but ecological and cultural.

How Chestnut Blight Kills American Chestnuts

Today, American chestnuts bear the scars of chestnut blight cankers. After C. parasitica enters the tree through breaks in the bark of chestnuts, it grows in the living layer beneath it called the cambium. As the fungus stretches its hyphae around this layer, the tree responds with thick growth to try to contain the pathogen. These outward symptoms of the struggle are called cankers. These become more obvious when bark is sloughed off where cambium has been killed by the fungus, revealing the wood underneath. The fungus kills the cambium by producing oxalic acid and enzymatically digesting this layer in order to grow. Throughout this battle, the fungus produces little orange, spore-bearing structures (perithecia) on the canker. These spores are spread by wind, water or other organisms to the next chestnut. Eventually, the hyphal threads of chestnut blight encircle the entire stem of the tree, strangling it in a process called girdling. Ultimately, these trees that used to live for centuries are now lucky to live a couple of decades.

Orange C. parasitica perithecia bearing stroma showing through bark as seen with the naked eye (left) or with a macro lens (right).
Orange C. parasitica perithecia bearing stroma showing through bark as seen with the naked eye (left) or with a macro lens (right).
Source: Matt T. Kasson

Since the fungus does not grow in the soil, buried roots are spared. If large enough, they will send up sprouts around the stump of old trees. These clones are susceptible to chestnut blight, just as the original tree was, so their fungal fate is spelled out before they even produce their first leaf. The lucky ones live long enough to produce some chestnuts, and may grow enough roots to support another generation of sprouts. This cycle is nearly ubiquitous, despite over a century of efforts to control the spread of chestnut blight since its initial sighting at the Bronx Zoo in New York. Even with humans as allies, chestnut trees have withered and the fungus has thus far prevailed.

The Virus of the American Chestnut’s Enemy Is Our Friend

There is one battle that C. parasitica is losing: its standoff with a naturally infecting hypovirus. Discovered in chestnut blight cankers in Italy by Antonio Biraghi in 1953, this virus lives in the fungal cytoplasm. When infected with the hypovirus, C. parasitica is weakened, preventing it from producing the devastating cankers that are a blight on American chestnuts. However, because these hypoviruses are limited to the cytoplasm, they are only passed on during the exchange of cytoplasm that takes place during anastomosis: when hyphae of 2 fungi join to form a single hyphal network. Fungi, perhaps to protect against the transmission of such viruses, have genes, called vegetative incompatibility genes (vic), that make this fusion incompatible in most cases.

To be 'compatible' for anastomosis, 2 fungi must have matching sets of vic genes. C. parasitica has 5 vic gene loci, each with 2 possible versions, or alleles. That means, for any given C. parasitica fungus, only 1 in 32 other C. parasitica strains will be compatible. This natural hurdle makes it difficult to introduce hypovirus to natural C. parasitica strains through hyphal fusion. When vic genes don't match,the hyphal cells that attempt anastomosis die, thus preventing the virus from being transmitted from one fungus to another.

Genetic Engineering Aids Virus Transmission

Genetic engineering is helping us overcome the vic gene hurdle and use hypoviruses as a means of controlling chestnut blight. By knocking out the vic gene loci one by one, engineered C. parasitica strains are able to fuse with an increasing proportion of natural strains. Engineered strains infected with hypovirus are referred to as “super donors,” because of their promiscuous ability to pass on the virus to wild fungi. Four vic genes can be removed from a single C. parasitica strain without dramatically affecting the fitness of the engineered fungus. The resulting strain can fuse hyphae with any C. parasitica matching its only remaining vic gene allele—now a 1 in 2 chance, instead of 1 in 32. In theory, a mixture of super donors with either version of the essential vic gene can deliver hypovirus to any wild C. parasitica strain through hyphal fusion.

Indeed, when applied to cankers directly in a forest setting, the super donor mixture passes hypoviruses on to nearly 95% of natural C. parasitica strains. To introduce the super donor mixture into the cambium, a hole is made on a canker using a leather punch and a super donor slurry is painted on. This slurry is composed of fungal growth medium, water and 2 super donor strains, each with 1 of 2 versions of the remaining vic gene. The small wound is taped over to heal while the natural C. parasitica and the super donor strains meet underneath the bark. Following this procedure, natural strains acquire the hypovirus, and become less able to kill trees and spread to new ones. Though historically chestnut blight has clearly gained the upper hand against our efforts to contain its spread, modern advances like genetic engineering are allowing us to leverage the full potential of these cytoplasmic viruses.

Other projects, such as genetically engineering chestnuts to be resistant or breeding American chestnuts with blight-resistant varieties like the Chinese chestnut, provide further promise in the battle against this merciless fungus. Engineered chestnuts will face considerable scrutiny before they can be applied, but with the state of American chestnuts today, it would be difficult to imagine an intervention that could make the situation worse. When integrated together, these tools will help us relegate C. parasitica to a footnote in the sordid history of the American chestnut.

Author: Brian Lovett, Ph.D.

Brian Lovett, Ph.D.
Brian Lovett is a postdoctoral researcher working on fungal biology and biotechnology at West Virginia University.