The American Chestnut Story
Sam Cox, 1997
Table of Contents
HISTORY OF THE AMERICAN CHESTNUT
HISTORICAL ACCOUNT OF THE CHESTNUT BLIGHT
BIOLOGY OF THE FUNGUS SPECIFICITY OF CHESTNUT BLIGHT
EARLY EFFORTS AT BREEDING RESISTANCE
RECENT BREEDING EFFORTS - THE BACKCROSS METHOD
The Backcross Method Diagram
HYPOVIRULENCE IN CHESTNUT BLIGHT
Once beautiful and abundant, the American chestnut tree covered huge tracts of land across the eastern United States for thousands of years until a fungus from Asia decimated virtually every tree standing on North American soil. Will future generations ever have the opportunity to see forests of this gracious tree again? Only through diligent efforts by man can the tree make a comeback in the forseeable future. This paper examines the possibility of just such a comeback.
HISTORY OF THE AMERICAN CHESTNUT TREE
The American chestnut tree, Castanea dentata, is a member of the family Fagaceae, closely allied to oaks and beeches. Its natural range before 1900 stretched from the coasts of Maine and Ontario to the coasts of Georgia, west through the mountains and highlands to Alabama, and north to the plains of Indiana and Illinois (8). The chestnut tree of America comes from a small genus of only four North American trees, including the chinquapin tree. It grew to be a very large tree, up to a hundred feet in height and four feet in diameter. The leaves are very distinctive, long and narrow with parallel veins leading to large serrations on the leaf margin. The hallmark of the American chestnut was, of course, the edible nut. Suitable for roasting over an open fire, street corner vending, or stuffing a Thanksgiving turkey, the American chestnut was in high demand. Despite being smaller than its European counterpart, the nut enjoyed the benefit of superior taste. It was therefore a fairly important crop species in the U.S., especially to the farmers in the Appalachian Mountains where the chestnut grew to its most impressive dimensions. In addition to the commercial value from the nuts it produced, the American chestnut tree also was the primary provider of tannin, a compound used to treat and cure leather. Without question it was one of the more desirable hardwood timber species. Its trunk grew straight and thick. Lighter than oak, and yet just as strong, the wood split easily down the grain. This, combined with its terrific rot resistance, made it ideal for telephone poles, fencing and building materials. Many a lumber man's fortune was made at the cost of the chestnut tree. The leaves of this tree are also said to have had medicinal values. But overshadowing all the economic values of the tree was its simple beauty. The chestnut tree made a grand and graceful tree in maturity, and was used throughout the east as a welcome landscaping addition. It lined the avenues of the famous Bronx Zoo. Thomas Jefferson planted these trees on his Monticello estate (4). The founder of the famous Du Pont company grew chestnut trees on his estate and gave them to friends as well (4). The list goes on. In the forest, the chestnut tree was prominent and abundant, making up 25% of the forest in many areas (2). In the hills of the central Appalachians, the chestnut tree grew solid, and covered miles and miles of rolling hills with its distinct leaves and long, white catkins. The wildlife enjoyed the benefits of the trees' nuts as well. Bear, deer, squirrels, wild turkeys and even the once-tremendous flocks of Passenger Pigeons all benefited from the heavy nuts. Since its sudden disappearance from eastern forests, no other tree has filled its niche, and the ecosystem has teetered on instability ever since (2).
HISTORICAL ACCOUNT OF THE CHESTNUT BLIGHT
Shortly after the turn of the century, in 1904, curators of the Bronx Zoo in New York noticed an unusual malady afflicting the magnificent chestnut trees lining the avenues of the zoo. Symptoms of this malady included wilting leaves, large cankers with rupturing bark, sprouts below the cankers and shortly thereafter, death of the tree's trunk and upper limbs (11). In 1907 and 1908, trees in the New York Botanical Garden exhibited the same symptoms. Before anyone knew what was happening, the mysterious infection spread by unknown means to chestnuts throughout New England, decimating entire forests in a few short years. By 1906, the blight was reported to be in New Jersey, Virginia and Maryland. Spreading as far as 50 miles a year, the blight worked its way across the east, killing virtually every chestnut tree in its path (9). By the time the blight reached the forests of Pennsylvania, the federal government, along with the state of Pennsylvania, were entrenched and determined to stop the blight. Quarantine lines were set up in attempt to halt the march of the blight (5). All chemical control options were explored, but in vain. The blight swept through Pennsylvania, outpacing quarantine lines, and continued on. By 1950, even the remote stands of the tree in southern Illinois were brought down by the blight. Because the situation looked so bleak, lumber men scrambled to cut down the remaining chestnuts for their timber before they were infected and began to rot (10). This may or may not have ultimately impacted the results of the blight, but one can imagine a possible scenario where blight resistance was erased from the gene pool by eager and worried businessmen.
At the hands of the forces acting upon it, less than 50 years after its peak of commercial value, the tree was essentially gone. It has been estimated that 3.5 billion trees were lost in the 40 year span from 1910 to 1950 (10). Millions of acres (3.6 million hectares) of land that had once been shaded by the lofty, full boughs of the chestnut tree now stood empty, shadowed only by leafless, dead remnants (1). Estimates of financial loss for the year of 1912 from three states, Pennsylvania, South Carolina and West Virginia, totaled $82.5 million (1).
Although the blight does not kill the roots of the tree, it does not allow the tree's suckers to attain an appreciable height before reinfecting the trunks and killing them back to the ground. These suckers grow into nothing more than a shrub; a minor understory species outcompeted by other trees. The suckers certainly never attain a maturity required to bear fruit, and so, the species would gradually slip into extinction if left alone. In 1950, the situation looked bleak indeed. All of the once vast chestnut forests were gone, and no hybrid had yet been produced that combined resistance to the blight with the quality of the American chestnut fruit. The only chestnut trees remaining were in scattered isolated groves planted out west by early settlers (10), beyond the range of the blight, and a few groves back east kept alive through diligent applications of hypovirulence (a virus of the fungus). All of this devastation was attributed to a minute creature of another kingdom.
BIOLOGY OF THE FUNGUS
The blight that decimated the chestnut tree is a fungus, Cryphonectria parasitica, a member of the fungal group including such villains as Dutch Elm Disease, Peach Leaf Wilt, and other hardwood pathogens. The fungal spores are borne either by wind or on birds and insects and enter the tree through cracks in the bark or through wounds caused by beetles or other animals. The spores germinate into a parasitic fungus that grows beneath the bark.
The fungus mycelia grow in the cambium layer of the tree, the layer responsible for active growth and nutrient transport. In response to the invasion, the tree forms callus tissue around the infected area in a maneuver designed at isolating the infection. The cells of the tree that are isolated are essentially dead at that point, as the tree can never again use them. The fungus defeats this response by growing faster than the tree can form callus tissue around it. The barrier cannot be erected fast enough. This callus tissue formed by the tree under the bark causes the outer bark to swell in a characteristic canker. As the infection spreads through the cambium layer, so too spreads the canker spread. Wherever the canker rises, that part of the tree is dead, and all parts above it are losing nutrients. This canker eventually encircles the trunk and destroys the cambium layer completely, cutting off nutrients to the upper reaches of the tree. In effect, the tree is girdled from the inside. All tissue above this point of infection is choked off, and death occurs in about four days - frighteningly efficient (9). The tree responds to this by sending out shoots directly below the canker. These shoots never live long due to the close proximity to the fungus. They are quickly infected, and killed. By this time, the fungus has entered its teleomorph, or fruiting body stage, and bright yellowish-orange fruiting bodies about the size of a pin head emerge from cracks in the rotting bark (10). The spores are then passed along on the slightest breeze to other trees.
Despite the destruction to the crown of the tree, the roots are never affected. This allows the chestnut tree to send up suckers that may attain a height of ten to twelve feet before being reinfected by the fungus (10). This is a very fortunate condition of the tree. Many trees do not have the ability to send up suckers. The fact that the roots survive and are able to reproduce tissue above ground serves both to ensure that the tree will not face immediate extinction, and that researchers are provided with a reliable supply of germplasm on which to do their research (1).
The fungus not only affects Castanea dentata, but other members of the Castanea genus as well, including the chinquapins in the eastern US, and even post oaks which are in the same family. The infection of these trees does not usually kill them, but does serve to spread the disease to other chestnut trees.
SPECIFICITY OF CHESTNUT BLIGHT
Why doesn't chestnut blight attack other trees as well? The blight attacks only members of the Castanea genus, and select members of the Fagaceae family. Why? As explained by Dr. Jakobi of Colorado State University, it is dependent on the genetics of the tree itself.
The spores of Cryphonectria parasitica land on the wounds of many trees and germinate into the cambium layer. The toxins produced by most trees are too powerful for the fungus to survive long. The reason the fungus thrives in chestnut trees is that this tree is lacking the toxicity in a certain protein that other trees have. The chestnut tree is genetically predisposed to the blight just as humans are genetically predisposed to AIDS, for example. It is not known exactly which protein(s) or compound(s) is lacking in chestnut trees to cause this vulnerability to the blight.
EARLY EFFORTS AT BREEDING RESISTANCE
As the ultimate cause of the blight's effects on the American chestnut, man may also be the ultimate salvation of the American chestnut. Shortly after the isolation of the fungus, United States Department of Agriculture plant explorer Frank Meyer confirmed that the fungus existed in both Japan and China, and further evidence emerged of importations and mail order sales of Japanese and Chinese chestnut nursery stock into the U.S. by horticulturists from 1876 on (4). It seems likely that the blight had been accidentally imported by horticulturists. This information led directly to the passage of the Plant Quarantine Act in 1912 which stopped the indiscriminate and hasty introduction of germplasm into the United States (1). Significant efforts are now made to ensure that such a disaster never happens again.
As scientists began to understand the nature of the fungus and its mode of infection, they increasingly resigned with great frustration to the conclusion that there was no stopping the blight. Efforts shifted to the only option capable of saving the chestnut tree, and that was breeding a new, blight-resistant tree. The obvious method was to cross American chestnuts with Asian chestnuts and evaluate progeny. The blight will infect Chinese chestnuts, but is not lethal in them, causing only cosmetic damage (2). All chestnut trees can be crossed with ease (2). The difficulty arose in the physical differences between the trees. The American chestnut is significantly larger than the Asian species, which are more of an orchard type tree (2). The ideal progeny had to have blight resistance coupled with the favorable traits that marked the American species. The goal was to find at least one tree that could then be propagated clonally (4). These early breeding efforts were done by a multitude of groups, both public and private, and by amateurs as well. Although hordes of cultivars were produced by dozens of breeders, none rivaled the stature and size of the American chestnut (4). The method ultimately proved too simple to be effective.
The United States Department of Agriculture funded an extensive breeding project that crossed American chestnuts with Asian chestnuts and then crossed the progeny in a myriad of ways and combinations in a hope of finding just the right cross (2). Again, success was limited and the genetics of the genes controlling resistance explain why.
Resistance has been found to be associated with at least two genes, possibly more (3). These two genes exist on different locci. Also, the trait is incompletely dominant (2), meaning that the genome must be homozygous for the resistance allele at both locci for the tree to be effectively immune. Trees that are 3/4 dominant show slight resistance, but are not able to thrive in association with the virus. Therefore, the crosses must ensure that the progeny receive ALL of the resistance genes from the Asian parent, and a significant amount of other genes from the American parent.
Knowing this, early breeders attempted to ensure full resistance by flooding the progeny with Chinese genes (2). The progeny from the initial cross were then crossed again to the Chinese parent, and again, and again and so on. The resulting progeny were very blight resistant, but of course very Chinese-like as well (3).
In 1960, after many decades of fruitless research, the USDA pulled out of the search for a blight resistant chestnut tree. Interest in chestnut breeding seemed to founder at this point, no doubt due to lack of funding.
RECENT BREEDING EFFORTS - THE BACKCROSS METHOD
After the USDA discontinued its program in 1960 (2), the Connecticut program, begun by plant breeder Dr. Arthur Graves on his own land in Hamden in 1930, became the leader in research on this front. Today it is the longest running continuous breeding program for chestnut trees in the US (4). In 1983, under renewed interest of the subject, the American Chestnut Foundation was founded in Virginia to attempt to produce a blight-resistant tree. The current trend in breeding is the backcross method (6), and it is actually quite simple in theory. In fact, it is curious that this method was not tried decades ago.
The backcross method begins by crossing a Chinese parent with an American parent to produce an F1 that is 1/2 Chinese and 1/2 American. The progeny are grown out for a couple of years and then inoculated with the blight to assay resistance. Only those trees which show strong resistance to the blight are used for the next cross. The second cross is actually the first backcross. This involves backcrossing the surviving progeny to the American parent. In effect, the crosses are aimed at flooding the genome with American genes. The progeny from this cross, called BC1 (backcross 1) are again assayed for resistance. The BC1 generation has a genome composed of 3/4 American and 1/4 Chinese genes. The survivors are backcrossed again to the American parent and produce a BC2 generation which has a genome of 7/8 American and 1/8 Chinese genes. Resistance is assayed and the survivors are backcrossed a third time to the American parent to result in a BC3 generation which is 15/16 American and only 1/16 Chinese. However, at each backcross that introduces new American genes, non-resistant genes are reintroduced into the system. Continuously selecting only for the resistant types keeps the number of non-resistant genes low, but it is very difficult to distinguish between fully resistant trees and 3/4 resistant trees. Therefore, after the three backcrosses, the progeny are selfed, or intercrossed. The progeny of this cross are called BC3F2 (backcross 3 generation 2). The intercross serves to expose the parents that are not fully resistant. A cross between parents that are only 3/4 resistant will yield progeny that are only 1/2 resistant and very susceptible to the blight. On the other hand, a cross between parents that are fully resistant will yield fully resistant progeny. These progeny that are fully resistant are intercrossed again to ensure that all American type resistance genes have been eliminated from the gene pool. This final generation is called the BC3F3. So, after 6 crosses, a tree is produced that is 15/16 American, yet retains full resistance to the blight. Since it is homozygous for resistance, all of its progeny will retain the resistance.
The issue of diversity is an important one, after all, lack of diversity of resistance genes was what led to the destruction of the chestnut in the first place. To ensure that the resulting gene pool is sufficiently diverse, the breeders in this program have used numerous trees from different locations and diversity (2). Active breeding is occuring in Connecticut, Indiana, Pennsylvania and Virginia incorporating local trees that will presumably be better adapted to those regions (2). Numerous Chinese parents have been, and will be, used as well to ensure diversity in the resistance genes.
Each generation of backcrossed trees requires six years to mature and produce nuts for planting. Intercross trees can be evaluated earlier and require only 5 years to planting. With six crosses in the project, the entire program should take 33 years to produce the first blight resistant chestnut ready for planting. The initial crosses were made in 1977. This means that a fully blight resistant chestnut tree that is 15/16 American should be ready for planting in 2010 (2). That's extremely promising news for a tree that has been conspicuously absent from the landscape for over 60 years.
Since the 15/16 American chestnut tree has yet to be produced, no one knows exactly what it will look like. It is logical to conceive of a tree that is virtually identical to the original American chestnut tree, but no one knows for sure. There are no standards across the breeding world for what qualifies as the same species after being crossed. A beefalo is the name of a cow with at least 1/8 buffalo genes. A soybean must contain at least 31/32 soybean genes or else it is not a soybean. Scientists predict that a 15/16ths American tree will be indistinguishable from the original (2).
THE BACKCROSS METHOD (3)
American ----This produces an F1.
F1 x American ----This is the first backcross to the American and produces a BC1.
BC1 x American ----This is the second backcross to the
| American and produces the BC2.
BC2 x American ---- This is the third backcross and produces a BC3.
BC3 x BC3 ---- This is the first intercross which produces a BC3F2.
BC3F2 x BC3F2 ---- This is the second intercross
which produces a BC3F3.
BC3F3 ---- This is the final product; a 15/16ths
American chestnut with resistance equal
to that of the Chinese parent.
American gene content at each generation:
F1 = 1/2
BC1 = 3/4
BC2 = 7/8
BC3 = 15/16
BC3F2 = 15/16
BC3F3 = 15/16
If a blight resistant chestnut tree were to
be crossed with a wild tree, the resistance to the blight would be lost
in the progeny. If the blight resistant tree is introduced into the
wild, this is a possibility. American chestnut trees could cross
with the blight resistant trees. However, since no effective control
for the blight has yet been designed (11), pure American chestnuts almost
never attain a maturity to be in a position to reproduce. Therefore,
unless the blight is eradicated, making the blight resistance a mute point,
there is no worry of the resistance being diluted by sexual crossing in
most areas of the wild. What little crossing does occur will not
impact the system much because the progeny will not survive (being only
1/2 resistant to the blight).
An important question one may consider: How are American chestnut trees kept alive on farms in order to be a part of this breeding program in places like Connecticut and Pennsylvania where wild chestnuts still fall victim to the blight? The trees on the farm are kept alive by routine inoculations of "hypovirulence", a strain of the pathogen that is not lethal, and tempers the blight to a point of causing only cosmetic damage (7). This is the subject of the next section.
HYPOVIRULENCE IN CHESTNUT BLIGHT
Roughly twenty five years after the blight infected the trees in North America, it was introduced into Europe, and quickly began infecting European chestnuts (Castanea sativa) with the same deadly results (1). However, shortly after the epidemic began, a promising new discovery was made when Italian pathologist Antonio Biraghi found chestnut trees living with blight infection. His observations aroused the curiosity of Jean Grente, a French mycologist, who cultured out the isolates from these non-lethal strains and tested them on chestnut trees (1). The lighter pigmented strain almost never proved lethal to its host. Jean Grente called the strain "hypovirulent". An important biological control for chestnut blight had been discovered.
Hypovirulence, although not a breeding technique, is highly significant in the efforts at breeding a blight resistant tree. Without hypovirulence, healthy chestnut trees in their native areas would never stay alive long enough to be crossed with Chinese trees and produce resistant hybrids. Therefore, it is important to the breeding effort.
Hypoviruelence is a condition any pathogen may have when it has somehow become less virulent than the expected normal level (7). In the case of the blight, the hypovirulence is caused by a virus that attacks the fungus cells (1). Infected fungus cells are not as virulent, exhibit different growth morphology, sporulate with less vigor and exhibit marked pigment reduction. The tree infected with the hypovirulent strain can fight it off to a point that the tree will very rarely die. The tree ends up fighting a "flu" rather than "pneumonia". What's more, the hypovirulence is a cytoplasmic trait that is easily transmissible from one fungus cell to another through hyphal fusion, where cytoplasmic material is exchanged through a tube between two cells (1). Therefore, a virulent strain can quickly be made hypovirulent when put in an environment near hypovirulent cells (7). This is why a tree infected with the hypovirulent strain is usually not killed, even if the virulent strain of the fungus infects it as well.
The exact mechanism of the hypovirulence is not entirely understood. Scientists have found a strong correlation between hypovirulence and dsRNA (double stranded ribonucleic acid) in the cytoplasm (7). dsRNA is characteristic of viruses. It is dsRNA that a virus injects into the cell to infect it. The hypovirulent strain seems to almost always contain this dsRNA in the cytoplasm while the virulent strain does not. Passage of this dsRNA through hyphal fusion has been confirmed, as has the subsequent effect at decreasing the virulence of the blight (7). The virus is passed from cell to cell much like a human disease is transmitted sexually. Furthermore, when the dsRNA from a hypovirulent strain is eliminated from the cell by complex techniques involving cyclohexamide, the strain becomes virulent (7).
Some trees in Pennsylvania and in Michigan are actively growing despite blight infection (9,6). Cultured isolates of this strain show the same bright coloration of the virulent strain, yet behave like a hypovirulent strain, being less destructive and able to pass this quality on to other fungus cells (1). Today, over 30 chestnut stands, although severely damaged by the blight, continue to thrive. The stands consist of mature trees, seedlings and saplings. In a few Michigan stands, evidence of the blight has all but disappeared. The location of these stands is outside the tree's natural range, yet the significance of this is not clear (9).
The dsRNA of the American hypovirulent strain was sequenced and compared to the European hypovirulence dsRNA (7). The sequences were genetically different, but had some terminal sequences in common. dsRNA appears to be the reason for hypovirulence; however, hypovirulent strains have been isolated that do not contain the dsRNA. The significance of this discovery has not been explored (7).
Theoretically, the effects of the blight could be nullified by widespread application of the hypovirulent strain. This is exactly what saved the European chestnut. Scientists applied hypovirulence to trees all over Europe and within 25 years, the threat was neutralized (7). The hypovirulent strain spread its depressed virulence by hyphal fusion to all the virulent fungi. Nature had provided a seemingly perfect defense; however, the same technique failed to work as well in North America (10). Today, so successful was the European chestnut recovery that the U.S. imports chestnuts from Europe (7).
Why doesn't the hypovirulence strain eliminate virulence in the U.S.? The answer hinges on the fact that many different strains of the blight had evolved in the 80 years before the tests with hypovirulence in the US. (6). Europe had only a few strains. It appears that vegetative incompatibility between the different strains that have evolved halts the spread of the hypovirulence dsRNA (1). Without active hyphal fusion, the dsRNA is not passed along. It is estimated that as many as 250 different vegetatively incompatible strains of the blight exist in the U.S. (9). To overcome this, scientists would have to isolate each and every strain, infect those strains with the hypovirulence virus, and then return those strains to the wild in order to spread the hypovirluence to all populations. So far, only 70 strains are known (7).
The procedure of inoculating trees with the hypovirulence was further reduced in effectiveness due to the extremely depressed sporulating qualities of the hypovirulent strain (7). Hypovirulent spores are produced in very small quantities from asexual production. Since the hypovirulence is only a cytoplasmic condition, any sexual reproduction between hypovirulent strains and virulent strains produces all virulent progeny. The virulent strains out-compete the hypovirulent strains, thus causing the hypovirulent strains to die off. The low persistence of the hypovirulent strains requires that the trees be inoculated on a regular basis to avoid infection. This is exactly what is done on research farms within the tree's natural range (4). The simple approach used in Europe clearly has failed in the US, yet the possibility of manipulating hypovirulence for effective blight control is very real, and an exciting field of active research.
Molecular biology techniques have been employed in an effort to incorporate the hypovirus into the nucleus of C parasitica (6). The goal is to transfer the dsRNA responsible for hypovirulence into the DNA strands of the fungus genome inside the nucleus. This would be a tremendous step in the dissemination of hypovirus because it would allow hypovirulence to be transmitted through sexual reproduction. Since vegetative incompatibility barriers are not present in sexual fusion, the hypovirus would be free to spread to all strains (6). The progeny of a sexual cross between hypoviruent strains and virulent strains would be about 1/2 hypovirulent (6).
The story of the American chestnut is an unfortunate tragedy, yet one that still retains hope for a happy ending. Manipulations of hypovirulence could render the blight weakened and non-lethal. Because chestnut blight does not kill the roots of the tree, sprouts that develop from the roots could grow into mature trees to once again fill the forests with chestnuts. On the breeding front, the backcross method is proceeding smoothly and seems capable of producing a blight resistant chestnut tree that is nearly identical to the original American chestnut. This would be an exciting breakthrough; however, introduction of this tree into the wild and re-establishment of the tree as a forest species would prove difficult. The answer to the chestnut problem may ultimately entail both of these methods. The field of chestnut restoration is an exciting and fast-paced area of research. After nearly 70 years of absence, the American chestnut is poised to make a comeback.
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