American chestnuts used to be a vital part of the eastern forest, but they were nearly wiped out around the turn of the century when a deadly fungus, chestnut blight, was accidentally introduced into the United States from China. Various researchers are conducting projects aimed at returning these great trees to the Appalachian forest. I spent this summer carrying out research that may help in the efforts to reestablish the American chestnuts. Half of my research time was spent working in the Lesesne State Forest; surveying American-Chinese chestnut crosses for their level of resistance to chestnut blight. The other half of my time was spent comparing the growth of chestnut blight with and without tannins to the growth of other fungi with and without tannins.

NATURAL HISTORY OF THE AMERICAN CHESTNUT

Before the turn of the twentieth century, American chestnuts made up 25% of the Appalachian Forest. They would grow to be 100 feet tall and live for hundreds of years. Many people called them the redwoods of the East. The American chestnut provided wildlife and people with a large yearly supply of nuts. The chestnut was also an excellent timber source and supported a booming tanning industry.

Around 1907 chestnut blight was accidentally introduced into the United States from either Chinese chestnut nursery trees or timber. The introduction was first noticed in New York City when unusual cankers began to form on the American chestnut trees in a park. The American chestnut has very little resistance to the blight and is easily killed by it within a few years. By the middle of the twentieth century, nearly all standing chestnuts throughout the Appalachian Mountains had been killed. Once the spread of destruction seemed inevitable, many people cut down large numbers of chestnuts in order to salvage the lumber and try stopping the spread of the blight. However, the fungus spreads easily by wind and lives on oak trees without killing them. Today, chestnut trees survive only as small understory trees. The chestnut blight does not affect the roots of a chestnut because it cannot compete with other soil fungi. The American chestnut can resprout many times from its roots before completely dying. Occasionally, a resprouted tree will live long enough to flower and produce nuts.

NATURAL HISTORY OF CRYPHONECTRIA PARASITICA

Cryphonectria parasitica, or chestnut blight, is a fungus that lives between the bark and the wood of the tree it is using as a host. When a spore lands on a tree, it grows a germ tube into a wound in the bark. Once under the bark, it spreads in a round, fan-like shape, excreting poisons ahead of it that kill the cambium of the tree. The cambium is the living part of a tree that becomes xylem and phloem, which are the tree's water and nutrient transportation tissues. Once the fungus has completely girdled a tree the tree's food and water transportation to locations above the infection is cut off. Consequently, the tree starves to death. Chestnut blight can produce both sexual and asexual spores. The sexual spores travel on the wind. The asexual spores are released in a sticky, stringy matrix that becomes stuck to the feet of birds and insects or is splashed off a tree by rain.

REACTION OF THE TREE TO A BLIGHT INFECTION

When a tree is under attack from chestnut blight, it forms a canker to try to wall off and/or push out the fungus. There are four different types of cankers. The worst is a sunken canker. It indicates that the chestnut blight has reached the cambium and killed it, causing the bark to collapse and appear sunken. A tree with many sunken cankers would be considered to have very little resistance to blight. The best type of canker is a swollen canker. It usually indicates that a tree has good resistance and is able to push out the fungus before it reaches the cambium.

Chinese chestnuts are resistant to chestnut blight. They can become infected but will not be killed by the fungus. European chestnut trees show some resistance to chestnut blight, but not as much as the Chinese chestnut does. Most infected trees in Europe are now surviving because the blight there has become infected with a virus that makes it hypovirulent and unable to kill the tree it attacks. American chestnuts have the least amount of resistance to the fungus. Once infected, most American chestnuts will eventually die. There are, however, a few large American chestnuts still living in the Appalachian forests. This small number of trees is considered either to have some naturally occurring resistance to chestnut blight or to have become infected with hypovirulent blight. Hypovirulent chestnut blight exists in the U.S. in low levels but does not spread as well here as it does in Europe.

RESEARCH TO PRESERVE THE AMERICAN CHESTNUT

Researchers are conducting a number of programs aimed at returning the American chestnut to the eastern deciduous forest. Two such programs are breeding programs. One, run by the American Chestnut Cooperators' Foundation, is an all-American breeding program. In this program American trees that show resistance to chestnut blight are bred with each other in the hopes of developing a line of pure American chestnut trees that can survive blight infections. The American Chestnut Foundation runs a backcross-breeding program. Their hopes are to breed the Chinese chestnut resistance into American trees by creating a tree that is 1/16 Chinese chestnut and 15/16 American chestnut. Such a tree should be resistant to blight and still have the form of an American chestnut.

Other researchers are looking into the mechanisms behind resistance. We are able to identify resistance by a tree's ability to survive an attack of chestnut blight, but we do not know what causes a tree to have resistance. One idea is that the type of tannins in a tree contributes to resistance against blight. Tannins are organic molecules produced by many plants in order to prevent invasions from microorganisms; however, chestnut blight uses tannins as a food source. Differences in the concentrations and types of tannins have been found between Chinese and American chestnuts, but it is still unclear as to whether these differences affect a tree's level of resistance.

FIELD RESEARCH: STUDYING RESISTANCE IN HYBRID FAMILIES

My field research took place at the Lesesne State Forest in Nelson County. At this location the Virginia Department of Forestry is working on the backcross-breeding program. To produce a tree with 1/16 Chinese chestnut genes and 15/16 American chestnut genes, a Chinese and an American chestnut first have to be crossed to create a hybrid tree. This hybrid tree, 1/2 Chinese and 1/2 American chestnut, is then bred ("backcrossed") to an American tree, creating a tree that is 1/4 Chinese chestnut and 3/4 American chestnut. Breeding this tree to an American tree will create a tree that is 1/8 Chinese chestnut and 7/8 American chestnut. This tree will be bred with another pure American tree, creating the final product. At each step of the breeding program, the trees are surveyed to determine which ones have the most resistance. The trees with the most resistance are picked as parents for the next backcross.

For my research I carried out a survey of the first backcrossed trees (1/4 Chinese and 3/4 American chestnut). The backcrossed trees were planted between 1990 and 1993. For each tree in the plot, I measured its diameter, noted whether it was alive, dead, or resprouted, and recorded data on each of its cankers: location, size, and canker type. I then created a ranking system (1-5) for the cankers and averaged the canker ranks on each tree (1 being worst, 5 being best) in order to give each tree a number that would represent its level of resistance. I used these rankings to determine which individual trees had the most resistance.

I organized the data by full-sibling family in order to determine which families had the most resistance. A full-sibling family of trees has the same two trees as parents. When organizing the data, I only looked at the families that had five or more individuals; smaller sets of siblings would not give an accurate picture of the resistance in a family. All of this information will help the Virginia Department of Forestry to select the trees to use for the next backcross.

Seven full-sibling families had a survival rate greater than 75%. Out of the seven families with a survival rate greater than 75%, five had the same hybrid parent. This suggests that a few hybrid trees transmitted resistance to their offspring better than the others did. Seventeen families had a survival rate between 50% and 75%, and 26 families had a survival rate of less than 50%. When looking at individual trees instead of families, 299 trees (63%) are alive and 477 trees are dead or resprouted. Of the living trees, 231 had a canker average of 4.0 or greater, indicating a high level of resistance.

MY GREAT DISCOVERY

American chestnuts produce both male and female flowering parts but cannot self-fertilize. The pollen, which contains sperm, must come from a different tree than the female ovules. Chestnut breeders hand-pollinate their trees, and they often take advantage of the need for a different fertilizing tree to transport pollen from American trees (usually located in rural forests) to their orchard of hybrid and backcrossed trees. There was, however, a small group of trees in which the opposite was true: hybrid pollen was taken from the orchards to the American trees in the forests. When surveying the trees, I noticed that the survival rate of the trees with American mothers was much lower than that of the trees with hybrid mothers. The sample size of American mother trees was small, but there was statistical significance to which tree (the hybrid or the pure American) was the mother tree. If this finding were to hold true in a larger sample size, then it would appear that resistance in chestnuts may be transmitted to offspring primarily or solely from the female parent.

LAB RESEARCH: WHY CHESTNUT BLIGHT IS UNIQUE

For my lab research I wanted to investigate further how chestnut blight, in comparison to other fungi, reacts to tannins. To accomplish this task I compared the growth of various fungi on artificial media mixed with different concentrations of tannins. The fungi used were as follows: eight different samples of chestnut blight, one non-blight fungus from a chestnut tree, four fungi from a dogwood (Cornus florida), and one fungus from a sassafras tree (Sassafras albidum). Of the eight samples of blight, I was given one by Dr. Gary Griffin from Virginia Tech, collected two from the Lesesne State forest, and gathered the remaining samples from living pure American Chestnuts in Amherst County. The tannin concentrations used in the media were as follows: no tannins, 0.5 mg tannins per mL of media, 1.0 mg/mL, 2.0 mg/mL, and 4.0 mg/mL. I expected the chestnut blight samples to have an increased growth as the concentration of tannins increased. I expected the growth of the other fungi to decrease as the tannin concentration increased.

The results I obtained from the dogwood fungi samples were as expected. When the tannin concentration increased, the growth rate of the each fungus sample decreased. For example, the average diameter of a pink fungus after 7 days without tannins was 67.1 mm. The same pink fungus after 7 days with a tannin concentration of 1.0 mg/mL had a diameter of 54.3 mm. When the tannin concentration was increased to 4.0 mg/mL, the diameter of the fungi was 40.3 mm.

Not all of my lab results for the chestnut blight samples were what I had expected. Only three of the chestnut blight samples had an increased growth when grown with tannins rather than without tannins. Two blight samples had no significant growth rate difference between being grown with and without tannins. I was surprised to find that there were also three blight samples that grew more slowly with than without tannins. One of the samples that grew better with tannins was a known virulent strain. I believe that the strains that grew more slowly with tannins could be hypovirulent strains of blight. Further testing of the samples would need to be done to determine whether this hypothesis is true. The differences in growth amongst the blight samples demonstrate that there is naturally occurring variation in the fungus. These data helps to show that the characteristics of the fungus that infects a tree, and not just the tree's resistant characteristics, contributes to whether a tree will survive an infection.

FURTHER RESEARCH

The tree survey work done in the field can give the Virginia State Forestry Department a good idea as to which trees and which families are the most resistant. However, it was not a perfect survey because the trees were infected out in the wild, and the strains of blight that infected the trees did not all attack at the same time. A tree that appears to have resistance may only be surviving because it has just recently been infected with the blight or because it is infected with a hypovirulent strain of blight. One way to be sure that each tree has equal exposure to blight is to infect the trees within the same time period by injecting them with a known virulent strain of blight. After three months the cankers that develop from the inoculation can be looked at to determine the trees' levels of resistance.

The biggest question raised by my lab research is why different samples of chestnut blight react differently to tannins. I would first like to determine whether the samples I collected are virulent or hypovirulent strains of blight. To do this I would need to find some young, uninfected American chestnuts and inoculate them with my blight strains. After three months I would look at the cankers that formed. Swollen cankers would indicate a hypovirulent strain of blight; sunken cankers would indicate a virulent strain.

Other research that should be done to follow up on my lab work includes trying to identify the non-blight fungi I collected, and gathering fungi from trees other than chestnuts that have a high tannin concentration. The other fungi collected would be grown on the same tannin concentrations used in the current experiment. I would also like to compare the tannase activity in hypovirulent and virulent strains of blight. Tannase is the enzyme that breaks down tannins inside of fungi. I would expect tannase to be less active in hypovirulent strains than in virulent strains. Finally, I want to use higher concentrations of tannins in the media; I found out partway through my experiment that the concentrations I was using were much lower than the concentrations of tannins found in chestnut trees.

ACKNOWLEDGMENTS

I would like to thank John Scrivani of the Virginia Tech State Forestry Department, Dr. Gary Griffin of Virginia Tech, and Dr. Robin Davies and Dr. Linda Fink of Sweet Briar College for all the help they have given me in carrying out my research.

SOURCES

Griffin, Gary J. "Chestnut Blight and its Control" Horticultural Reviews 1986 Vol. 8, pp. 292-336.

Newhouse, Joseph R. "Chestnut Blight" Scientific American July 1990, pp. 106-111.

Rankin, W. Howard. Manual of Tree Diseases. The Macmillan Company. 1929, pp. 140-148.

Bramble, William C. "Reaction of Chestnut Bark to Invasion by Endothia Parasitica." American Journal of Botany. Vol. 23, Issue 2, Feb. 1936,
pp. 89-94.

Cook, Melville Thurston ad Wilson, Guy West. "The Influence of the Tannin Concentration of the Host Plant on Endothia parasitica and related species." Botanica Gazette. Vol. 60, No 5 Nov 1915, pp. 346-361.

Griffin, G. J., Elkins, J. R., Hebard, F. V. "Host-Parasite Interactions of Endothia Parasitica on Chestnut Species: State of the Art." "Proceedings of the USDA Forest Service American Chestnut Cooperators' Meeting." West Virginia University Books, Morgantown 1982, pp.184-192.