Mycologist Amy Rossman and Steve Rehner select strains of
Trichoderma for DNA "fingerprinting" and sequence analysis.
When it comes to savoring mushrooms, ants have a 23-million-year jump on
ARS scientists recently discovered that about 200 species of a
fungus-farming ant group called attines have long cultivated a fungus related
to the parasol mushroom prized by connoisseurs. Attines include the destructive
leaf-cutter ants of Central America.
The fungus can do what the insects cannot: break down indigestible leaf
pieces harvested by the leaf-cutting ants and turn them into absorbable
nutrients. In turn, the ants provide the fungi with vegetable matter on which
to grow, protect them from competing organisms, and propagate them by cloning,
which allows the fungi to reproduce without sex.
"We found parallel patterns of genetic development between many ant
species and their favorite fungievidence that the two have been
coevolving for millions of years," says ARS mycologist Steve Rehner, a
He made the discovery with ARS mycologist Ignacio H. Chapela and scientists
at Cornell University in Ithaca, New York.
Their fungus research answered a century-old mystery for mycologists. In
relying on the ants for reproduction, the fungus had stopped making spores and,
instead, had come to depend on the ants to propagate it. So it was impossible
to determine the species or relationships of the ant-farmed fungus through
traditional taxonomic meansanalysis of its fruiting bodythe
mushroom cap where the spores are produced.
Instead, Rehner and Chapela took genetic samples from fungi isolated from
the nests of 19 species of attine ants. Then they compared those genes with the
DNA of free-living-fungi not tended by the ants.
Leaf-cutting ants, such as this foraging worker of Atta
cephalotes, are the primary herbivores of tropical areas such as Central
America. they can be serious agricultural pests.
To identify the fungal species, they relied on specimens that Chapela had
collected. These are now part of the 1 million specimens in the U.S. National
Fungus Collections at ARS' Beltsville (Maryland) Agricultural Research Center.
200-Year-Old System Still Useful
Morphological characteristics, which rely on observable form and structure,
have served systematists as the primary basis for classifying agriculturally
important organisms for more than 200 years. "These will continue to
provide the majority of systematic information, since they are easily and
inexpensively obtained," says ARS mycologist Amy Rossman, who is in charge
of the Systematic Botany and Mycology Laboratory.
But according to Rossman, "two new computer-based toolsDNA
fingerprinting and expert systemsare now revolutionizing how systematists
classify organisms, including our ability to accurately identify, catalog and
DNA sequencing uses molecular characteristics to develop
"fingerprints'" for genetic material found in all living organisms.
This new technology can differentiate between organisms based on their genes,
rather than their morphology. (See "DNA Sequencing Is New Tool for Insect
Detectives," Agricultural Research, Feb. 1995, pp.12-18.)
"For the first time, this powerful tool makes it possible to
distinguish between so-called cryptic species that appear identical," says
Douglass R. Miller, who works in the ARS Systematic Entomology Laboratory in
Beltsville, "DNA fingerprinting can prove if two visually similar species
are related, different, or new."
Rehner adds, "Until now, precisely identifying fungal species and
defining relationships have been difficult because many fungi look alike.
Neither DNA fingerprinting nor modern phylogenetic analysis, as a means for
determining the pattern of evolutionary history and common ancestry among
species, existed until about 10 years ago."
Tracking Trichinosis Carriers
Entomologist Douglass Miller gathers and analyzes systematic
information on a mealybug species.
The practical benefits of precise classification can be seen in the case of
Trichinella spiralis, the round-worm that causes trichinosis. The
disease was discovered in 1835 and for almost 150 years was believed to be
caused by one species.
Says Ralph Lichtenfels, "Because we couldn't distinguish between
parasites of this genus, field studies on the transmission patterns of wild
strains and their impact on food safety were hindered." Lichtenfels heads
the ARS Biosystematics and National Parasite Collection Unit at Beltsville.
Trichinella spiralis is a parasitic nematode with a complex life
cycle. Because it can infect nearly all meat-eating animals, it poses a public
health risk worldwide.
Despite a very low estimated trichinellosis infection rate in pigsand
even though thorough cooking or freezing makes pork safe to eatconsumer
confidence in pork products suffers and swine producers lose sales. In 1985,
the National Pork Producers Council estimated that being able to assure
consumers of trichina-free pork would boost domestic demand by 2 percent and
exports by 33 percentgains worth about $450 million yearly to pork
"Although most control strategies can effectively prevent transmission
within hog farms, concern persists over the risk posed by infections from wild
animalsespecially by roundworm species not killed by freezing,"
He and ARS scientists Darwin Murrell and Dante S. Zarlenga, working with two
Italian parasitologists, used DNA fingerprinting and other biochemical and
statistical technologies to compare 300 Trichinella samples taken from
various animals from around the world. They discovered that instead of one,
there are at least five species in the genus Trichinella that appear
This basic systematic research confirmed biological and epidemiological
evidence developed by Murrell and his cooperators that Trichinellain
domestic pigs is a species that infects wildlife coming in contact with pig
farms, causing the wildlife to become reservoirs for reinfection of swine.
However, several other species and genetic types of the parasite that are
commonly present in wildlife have a very low infectivity for pigs; thus,
natural infections are extremely rare.
The other breakthrough technology for systematists is expert systems.
Rossman says electronic technology now provides a means of synthesizing and
making available to users vast quantities of detailed information that exists
on agriculturally important organisms.
To be developed as an expert system, information must not only be converted
to an electronic format and made available to all users, but must also be
reevaluated for accuracy.
According to Miller, "At present, our knowledge of the systematics of
less conspicuous organismssuch as fungi, insects, nematodes, and
bacteriais grossly limited. It's so limited, we often don't have even the
most elemental systematic understanding of their existenceno inventory,
checklist, or way of identifying them."
For example, because of a lack of systematic information, there is confusion
between the pathogenic organism that causes dogwood anthracnose in the United
States and a similar fungus isolated from dogwood trees. The real pathogen was
unknown to science until about 10 years after it started to devastate U.S.
Mycologist David Farr examines some of the larger fungi that
are a part of the U.S. National Fungus Collections.
After a careful study of both organisms, the pathogenic dogwood fungus was
described and classified as a new species in 1991 by ARS mycologist Scott
Rossman thinks that DNA fingerprinting may yield clues about the fungus'
distribution and the means of its introduction into the United
Statesinformation that cannot be obtained through conventional
"However, before we can apply modern technology to answer these
questions, we need fundamental systematic knowledge about the organism,"
"And to facilitate breeding new dogwood trees resistant to this fungus,
rapid identification tools are needed."
The elusiveness of this fungus is not unique.
"Mycologists estimate there are at least 1.5 million species of
fungi," says Rossman. "Of these, only 150,000 speciesabout 10
percenthave been discovered and named.
And the situation is similar for insects and parasites. Many of these
organisms are important to agriculture but remain undescribed or relatively
In 1993, Plant Protection and Quarantine officers with USDA's Animal and
Plant Health Inspection Service (APHIS) intercepted about 37,000 non-native or
quarantine-significant insects on commodities entering the United States.
"And," reports the agency's national identification branch chief,
Rebecca Bech, "over 500,000 quarantine-significant diseases and 8
quarantine-significant nematodes were intercepted on incoming materials."
Curator of the U.S. National Parasite Collection, Ralph
Lichtenfels retrieves a specimen, one of several hundred that are lent to
researchers worldwide each year.
Accurately identifying fungi and insects is essential but difficult. Between
25 and 50 percent of the specimens submitted to the ARS systematic laboratories
cannot be accurately identified at the species level. Many belong to large
genera that have never been systematically described and classified. So
identifying them at the species level is nearly impossible.
One reason for accurately identifying these organisms is to prevent the
introduction of exotic diseases and pests. Some disastrous accidents have
occurred in the past, such as the introduction of Dutch elm disease into North
America and the release of Japanese beetles, as well as the importation of
dogwood anthracnose, oak wilt, chestnut blight, and scleroderris canker that
attacks pine trees.
"If we had had available more knowledge of the causal organisms of
these diseasestheir identity, range of hosts, and geographic
distributionperhaps those inadvertent introductions could have been
avoided," says Rossman.
A Separate Kingdom
Since the third century B.C., when Aristotle attempted to order life forms
from simplest to most complex into a "great chain of being," fungi
have been lumped in with plants.
Mycologist Gary Samuels records microscopically visible details
of a wasp species to use in its classification.
Occurring everywhere and on all living matter, fungi have coevolved with and
flourished inside diverse hosts.
Though they've been recognized for more than 20 years as a separate kingdom
thought to be closely related to plants, DNA sequencing now places them closer
"The implication of the revelation that mushrooms are more closely
related to humans than to orchids is that biologists will start looking more
closely at characteristics that fungi and animals sharelike biochemical
pathways," says Rossman.
The fact that fungi are related to animals makes it difficult to treat
fungal infections in humans, livestock, and domestic animals. Fungi are
becoming more and more common as human pathogens. But antibiotics capable of
destroying them are also likely to harm human patients.
Recently, Rehner and colleague ARS mycologist Gary Samuels have been
studying the relationships of an important beneficial fungus. Previously
misclassified as Gliocladium virens, this species is used as a
biocontrol agent against several root pathogens, including those that cause
damping-off diseases. These diseases rot seeds, seedlings, and cuttings in
cotton, beans, carrots, and other crops at a cost of more than $1 billion
"DNA sequencing has shown that the genus Gliocladium is an
unnatural group," says Samuels. "What was called Gliocladium
virens is actually a species of Trichoderma. All members of the
genus Gliocladium do not share a recent common ancestor. So putting the
organisms together using morphology, as has been done in the past, has proven
This discovery gives scientists looking for more effective biocontrol agents
a clue about where to look.
"Because Trichoderma virens is not known to reproduce sexually,
traditional breeding techniques can't be used to develop stable strains that
might be even more effective in controlling damping off diseases," says
Samuels. "But we now see that T. virens is related to species with
a sexual cycle that can he manipulated for genetic improvement."
He thinks DNA sequencing will reveal those links.
"What's significant for mycologists is that after nearly 200 years of
having a separate classification for fungi known to reproduce sexually versus
those that reproduce asexually, we can now use molecular characters to unity
the classification of both types of organisms," says Rehner.
"In so doing," he adds, "we can learn more about the
evolutionary relationships in our classifications and better understand the
roles of sexual and asexual reproduction in the evolution of
fungiespecially those of economic interest that are either beneficial or
pathogenic to plants and insects." By Hank Becker, ARS.
Samuels are at the USDA-ARS Systematic Botany and Mycology Laboratory,
10300 Baltimore Ave., Beltsville, MD 20705; phone (301) (301) 504-5364.
Miller is at the USDA-ARS
Entomology Laboratory, 10300 Baltimore Ave., Beltsville, MD 20705; phone
(301) 504-5895, fax (301) 504-6482.
J. Ralph Lichtenfels is in the USDA-ARS
Biosystematic and National
Parasite Collection Unit, 10300 Baltimore Ave., Beltsville, MD 20705; phone
On Chalcid Corner
Entomologists Eric Grissell (right) and Mike Schauff work over
the final details of illustrations that will become part of comprehensive
identification keys to North American parasitic wasps.
Parasitic wasps are the single most important group of organisms currently
used for biological control. Nearly half of all biocontrol successes are
attributed to them, says ARS entomologist Eric E. Grissell, who is quartered at
the National Museum of Natural History in Washington, D.C.
Practically every insect pest has a wasp enemy that can be used to control
it. At just about every stage in an insect's life cycle, one or more species of
wasps will parasitize it.
"Beneficial wasps," entomologist Michael E. Schauff says,
"are tremendously important as natural enemies of insect pests like the
Colorado potato beetle and the Mexican bean beetletwo of the most
devastating pests of potatoes, tomatoes, beans and eggplants."
For the last 4 years, Schauff and Grissell have been working with an
international group of taxonomists on a pictorial key of a superfamily of
beneficial parasitic wasps, the Chalcidoidea. With several hundred pages, the
key will have over 1,500 illustrations of more than 700 genera.
"This is the first time so much information about a wasp superfamily
has been compiled. It includes over 2,500 species of Nearctic Chalcidoidea
wasps that belong to 20 different families. Each genus has illustrations
showing peculiar characteristics, including male and female, winged and
wingless, and exceptional forms," Schauff says.
The illustrated key is also the basis for a new interactive expert system
called Chalcid Corner.
The system incorporates graphics, sound, visual effects, and other features
to help users identify members of this important group of beneficial insects.
"What is needed to increase the use of wasps as an alternative to
chemical controls are some basic, easy-to-use guides to help identify adult
wasps and summarize pest species that wasps will attack," he says.
Chalcid Comer should fill that need because it is geared to nonexpert
audienceslike farmers, home gardeners, and others not knowledgeable about
The program allows users to choose from a list of alternative questions
about chalcids, then proceed through a series of screens to help identify a
wasp. Users can confirm tentative identifications, look at biological and
taxonomic information on a particular family, or review the superfamily as a
Schauff says one version of the system is now available, free, by sending a
high-density 3-½-inch disk to the address below. He is working on an
updated, interactive version of the key that will make identification even
Available electronically on the Internet by the end of 1995, the updated
version of Chalcid Corner will have a voice component that warns users to
"be careful," if they are making an error in their search. By
Hank Becker, ARS.
Where Identifications Are Made
Five reference collections are curated by more than 30 Agricultural Research
Service systematists at Beltsville, Maryland. These specialists discover, name,
describe, and classify biota consisting of plants, insects and mites,
nematodes, other parasites, and fungi.
During 1994, experts in the Beltsville systematic collections processed
about 85,000 specimens, categorized roughly as follows:
- Systematic Botany and Mycology Laboratory (two collections) identified
about 350 seed samples and 2,500 specimens of fungi;
- Systematic Entomology Laboratory identified about 80,000 insect and mite
- Biosystematics and National Parasite Collection Unit added about 1,000 new
samples of parasites;
- USDA Nematology Laboratory examined more than 300 samples of nematodes.
Each nematode sample included several different genera and species of both
plant-parasitic and soil nematodes.
" Reinventing Systematics" was published in
the May 1995 issue
of Agricultural Research magazine.