Once a dominant tree throughout the Northeast, prized for the timber from its long, unbranched trunks, the American variety (Castanea dentata) stopped providing those famed chestnuts roasting on an open fire long before Bing Crosby's time. The tree bore sweet nuts, but they were small, and the quest for a meatier nut led to the species's downfall. Such notables as Thomas Jefferson in the 18th century and Luther Burbank in the 19th century imported European and Asian chestnut varieties. By 1904, the fungus Cryphonectria parasitica was identified in New York City, and the blight had begun.
Thought to have come from Japan, the fungus causes cankers that disrupt nutrient movement and eventually girdle the stem. By midcentury, mature natives were virtually wiped out, although multiple stems often still could sprout from the roots and grow until the fungus struck again. This cycle of sprouting, early death, and resprouting relegated the once-lofty chestnut to a shrub in the forest's understory.1
European varieties had similar problems, but a naturally occurring recovery in Italy was attributed in 1965 to "hypovirulence," in which fungal strains infected with a virus had less-than-normal ability to kill chestnut trees. Viruses in these strains, called hypoviruses, could spread into lethal fungi, transforming them into nonlethal strains.2 Hypovirulent strains subsequently were introduced artificially to French chestnuts, ridding plantations of the blight within a decade. Some stands of American chestnut, particularly in Michigan, were later discovered to have survived the blight due to hypoviruses.3
Upper Left: William L. MacDonald (right) and Mark Double examine a strain of the chestnut fungus. Lower Left: Sandra L. Angnostakis collects American chestnut twig samples. Right: Nonlethal cankers on American chestnut trees in Hamden, Conn.
At the Connecticut Agricultural Experiment Station in New Haven, efforts that had been proceeding since 1930 to cross-breed American and foreign chestnut varieties to obtain resistance to the fungus took on a new focus. Agricultural scientist Sandra L. Anagnostakis imported hypovirus-containing fungal strains from Italy and began field-testing them in 1973. She continues such work today, despite numerous ecological and phenotypical variables that have complicated it.
A major problem has involved the process of vegetative anastomosis, during which fungi exchange cytoplasmic material by fusing their threadlike vegetative filaments called hyphae. Hypovirus genetic information can be transmitted through such hyphal fusion, but only if the fungal strains are of the same vegetative compatibility (vc) group. Seven genes are thought to determine vc groups, and these genes can appear in many combinations through sexual reproduction. With 128 or more vc groups, widespread cytoplasmic transmission of the hypovirus was stymied.
Another way to transmit virus is through asexual spores or conidia, which can be disseminated overland. But, like mycoviruses in general, hypoviruses are not infectious in the classical sense of being transmissible between fungal strains by any extracellular route. It seemed the only way to get around the vc blockade was if the hypovirus could be made sexually transmissible. This is because the fungus has just two mating types, which are determined by genetic loci and cellular structures different from those responsible for the vc groupings.
In 1984, it was discovered that hypoviruses have a genetic makeup composed of RNA that accumulates in infected fungal cells in a double-stranded form.4 In 1992, construction of the first infectious complementary DNA clone of the viral RNA that confers hypovirulence was reported.5 With hypovirus genetic information incorporated into the fungus's chromosomes, it could now be sexually transmitted. After mating and meiosis, the hypovirus DNA would assort to sexual spores independently of vc fate. Once that happened, it could be transmitted by anastomosis.
Photo 1: An American chestnut sprout against a split-rail fence. Photos 2 and 3: By 1904, the fungus Cryphonectria parasitica was identified and the blight had begun.
"The important point is that viruses transmitted through mating end up in the background of the different vc groups," notes the coauthor of that paper, Donald L. Nuss, now director of the Center for Agricultural Biotechnology at the University of Maryland Biotechnology Institute, and a key figure throughout this decade in constructing transgenic hypovirulent fungal strains.
In 1994, the first such transgenic strains were introduced to stems in two forest plots, one in Connecticut by Anagnostakis's group and the other in West Virginia by researchers led by William L. MacDonald, a forestry pathology professor at West Virginia University. "For a couple of decades, we have been working with these viruses as agents that occur in the cytoplasms of the fungus, not the nuclei," MacDonald observes. Although the first field trials with a transgenic strain were only sustained over two years, he says, "We now know engineered strains will survive over winter, will mate, and will carry the virus into the DNA of sexual spores."
To improve conditions for biological control, the prototypic transgenic hypovirulent strain was introduced again in 1998 by Anagnostakis and colleagues to a plot clear-cut of all but chestnut stems. "We wanted to do it in a rapidly growing environment, and put as much hypovirus out as we could, for exposure to at least as much hypovirulent inoculum as virulent inoculum," she says. "I can see the difference already."
But the eventual goal is to succeed in the mixed environment of a forest. To help achieve that end, Nuss and collaborators have engineered another hypovirus that is more aggressive in colonizing chestnut stem tissue and has higher levels of asexual sporulation than its prototype.6 He estimates it will take a year to make more of the new transgenics, test their stability, and get permission to field-test them. Risk also will be assessed, but there currently is no indication that the hypovirus will spread to any pathogen other than the fungus or to any nonwoody species. Ultimately, the new transgenic variety might be able to replace the population of wild-type, lethal chestnut blight strains. "Our next question would be to see if they will radiate out to other forested areas," he says.
The American Chestnut Foundation
Web site: chestnut.acf.org
"Progress can be judged in a number of ways," Nuss muses. "One way is that this research has given us a very powerful experimental system to examine virus-host interactions, and what regulates fungal pathogenesis."
Another way is that, after a long struggle, the American chestnut may one day flourish in forests again.
Steve Bunk (firstname.lastname@example.org) is a contributing editor for The Scientist.
- S.L. Anagnostakis, "The pathogens and pests of chestnuts," in: Advances in Botanical Research, vol. 21, J.H. Andrews and I. Tommerup, eds., New York, Academic Press, 1995, pages 125-45.
- J. Grente, "Les formes Hypovirulentes d' Endothia parasitica et les espoirs de lutte contre le chancre du châtaignier," Académie d'Agriculture de France, Extrait du Proces-verbal de la Séance, 51:1033-7, 1965.
- W.L. MacDonald, D.W. Fulbright, "Biological control of chestnut blight: use and limitations of transmissible hypovirulence," Plant Disease, 75:656-61, 1991.
- D.W. Fulbright, "Effect of eliminating dsRNA in hypovirulent Endothia parasitica," Phytopathology, 74:722-4, 1984.
- G.H. Choi, D.L. Nuss, "Hypovirulence of chestnut blight fungus conferred by an infectious viral cDNA," Science, 257:800-3, 1992.
6. B. Chen, D.L. Nuss, "Infectious cDNA clone of hypovirus CHV1-Euro7: a comparative virology approach to investigate virus-mediated hypovirulence of the chestnut blight fungus Cryphonectria parasitica," Journal of Virology, 73:985-92, February 1999.