Group Title: Agricultural research (Washington, D.C.)
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Title: Agricultural research
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Full Text
U.S. Department of Agriculture Agricultural Research Service October 1996

Agricultural Research
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Agricultural Pests:
No Shortage in the
The cover story in this issue shows
a few snapshots from the 33-year
research album of Interregional
Research Project No. 4, or IR-4. The
name tells nothing about IR-4's
unique role in protecting our food
and fiber supply from a seemingly
limitless supply of pests.
Through IR-4, ARS and other
federal and state scientists conduct
field trials and collect data to support
registration or reregistration by the
U.S. Environmental Protection
Agency of certain pesticides and
Mostly, these are pest controls for
use on a minor crop-that is, a crop
grown on less than 300,000 acres
nationwide. But IR-4 projects also
address minor-use pest controls
applied to multimillion-acre crops
like wheat and corn.
Every day, you very likely eat or
drink some of the rich array of foods
available through IR-4's labors-
even if you consider only the so-
called minor crops. These crops
occupy a fraction of the nation's 975
million farmland acres. But they
include lettuce, carrots, fresh-market
tomatoes, broccoli, cucumbers,
melons, apples, pears, peppers,
onions, and dozens of other food,
fiber, and ornamental crops.
IR-4 scientists also seek EPA
permission for wide-scale field trials
of new anti-pest technologies. That's
why, for example, ARS will soon
conduct new tests of a natural
weapon against aflatoxin, a grain
contaminant that can threaten food
and feed safety.
Without IR-4's "minor" activities,
Americans might have to import
more of the variety of foods needed
for a diverse, healthful diet. And our

farm economy would be malnour-
ished if it lost a significant part of the
42 percent of total sales that minor
crops bring in.
"About the only minor things
about minor crops are their relative
acreage and their contribution to total
pesticide use," sums up ARS nema-
tologist Bill Johnson. He heads a
field and lab program for IR-4 at Tif-
ton, Georgia. Studies by Johnson and
colleagues in Tifton's Nematodes,
Weeds, and Crops Research Unit fo-
cus on the environmental fate of pes-
ticides and other farm chemicals.
Farmers decide to grow any crop-
minor or major-only if the risks are
tolerable. Given the smorgasbord of
pest problems, it seems a wonder
anything arrives safely to our dinner
plates. Worldwide, over 600 insects
cause enough agricultural damage to
make controlling them worthwhile.
Additional threats include thousands
of destructive weeds, nematodes,
fungi, bacteria, and viruses.
Last summer's epidemic of Karnal
bunt fungus in American wheat is the
latest example of how fast and furi-
ously pests can strike. Within weeks,
efforts to contain the fungus caused
world wheat stocks to dip to the low-
est level in two decades.
Fully 30 percent or more of the
world's potential crop production
may be stolen in advance by pests,
according to some estimates.
Meanwhile, population growth
lays an ever-increasing burden on
farmland resources. With world pop-
ulation now "only" 5.8 billion, hun-
dreds of millions of people go
hungry. Over the next 40 years, pop-
ulation may rise another 2.8 billion-
the same increase that doubled our
numbers over the past 40 years.
Pest control can scarcely become
less important. But today's important
public debates over how best to
achieve it often ring chords of uncer-
tainty about the future of pesticide

management research. To chart this
future, ARS' National Program Staff
(NPS) will sponsor a workshop for
agency researchers in 1997.
NPS and more than 100 ARS re-
searchers began planning the work-
shop last spring through an Internet
discussion group set up by Tifton
chemist Don Wauchope. The scientists
soon received new food for thought.
The National Research Council's
Board on Agriculture announced plans
for a 20-month study of the future of
pesticides in U.S. agriculture.
At the workshop, ARS researchers
will grapple with many of the issues to
be addressed by the NRC study. For
example, the study is expected to make
recommendations on which chemical
controls likely will continue to be
needed, what opportunities exist for
reducing health risks, and what federal
role is appropriate to support develop-
ment and use of chemical controls.
"Perhaps the NRC will find our
workshop's report useful," Wauchope
says. "ARS has a large research pro-
gram on nonconventional pesticides
and on reducing pesticide amounts and
unwanted impacts. Our overall strat-
egy is to minimize pesticide use and to
define where such use is appropriate,
while controlling pests effectively,
safely, and economically.
"It's appropriate for us at ARS to
examine all those aspects of pest con-
trol that are essential for the public
good but will not be addressed by the
private sector," says Wauchope. "We
feel we can bring unique expertise and
experience to this research."
This rule of thumb has guided ARS
researchers in finding solutions to a
host of problems for four decades. It's
still the best guide for the future.

Jim De Quattro
ARS Information Staff

Agricultural Research/October 1996

October 1996
Vol. 44, No. 10
ISSN 0002-161X

Agricultural Research is published monthly by
the Agricultural Research Service, U.S.
Department of Agriculture, Washington, DC
The Secretary of Agriculture has determined
that this periodical is necessary in the transac-
tion of public business required by law.
Dan Glickman, Secretary
U.S. Department of Agriculture
Catherine Woteki, Acting Under Secretary
Research, Education, and Economics
Floyd P. Horn, Administrator
Agricultural Research Service
Robert W. Norton, Director
Information Staff
Editor: Lloyd McLaughlin (301) 344-2514
Assoc. Editor: Linda McElreath (301) 344-2536
Art Director: William Johnson (301) 344-2561
Photo Editor: John Kucharski (301) 344-2900
Assoc. Photo Ed.: Anita Daniels (301) 344-2956
Information in this magazine is public property
and may be reprinted without permission. Non-
copyrighted photos are available to mass media
in color transparencies. Order by photo number
and date of magazine issue.
Subscription requests should be placed with
New Orders, Superintendent of Documents,
P.O. Box 371954, Pittsburgh, PA 15250-7954.
See back cover for order form.
Complimentary 1-year subscriptions are
available to public libraries, schools, employees
of the U.S. Department of Agriculture, and the
news media. Send requests or comments to:
Editor, Agricultural Research Magazine, Room
408, 6303 Ivy Lane, Greenbelt, MD 20770. E-
To visit Agricultural Research magazine on the
Internet, go to and select
News and Information.
This magazine may report research involving
pesticides. It does not contain recommendations
for their use nor does it imply that uses
discussed herein have been registered. All uses
of pesticides must be registered by appropriate
state and/or federal agencies before they can be
Reference to any commercial product or service
is made with the understanding that no
discrimination is intended and no endorsement
by the U.S. Department of Agriculture is
USDA prohibits discrimination in its programs
on the basis of race, color, national origin, sex,
religion, age, disability, political beliefs, and
marital or familial status. (Not all prohibited
bases apply to all programs.) Persons with
disabilities who require alternative means for
communication of program information
(Braille, large print, audiotape, etc.) should
contact the USDA Office of Communications
at (202) 720-2791. To file a complaint, write
the Secretary of Agriculture, U.S. Department
of Agriculture, Washington, DC 20250, or call
(202) 720-7327 (voice) or (202) 720-1127
(TDD). USDA is an equal employment
opportunity employer.

Agricultural Research/October 1996

Cover: The interagency IR-4 program
ensures the safety of so-called minor-use
chemicals before they are approved for
commercial agricultural production. At
Salinas, California, ARS agronomist
Sharon Benzen displays test-plot-grown
broccoli that will be used to determine
pesticide residue levels. Photo by Scott
Bauer. (K7429-11)

Ozone and other air pollutants take their
toll on sensitive crops. (Story on page 20.)

IR-4 Projects Protect "Minor" Crops
For more than three decades, this collaborative effort has helped relatively small specialty crops
deliver big economic and nutritional payoffs to farmers and consumers.

agricultural chemical pro-
ducers readily test and seek
U.S. Environmental Protec-
tion Agency (EPA) approval for new
pesticides for blockbuster crops like
corn and wheat.
That's because there's a potential
to market a product that can be used
on from 70 to 80 million acres. The
chemicals industry recoups its invest-
ment and makes a profit.
Other, smaller crops like mint and
cucumbers are generally not worth
the industry's attention. But these
minor crops-defined as those grown
on 300,000 acres or less-are helped
by a federal-state project known as
Interregional Research Project No. 4,
or IR-4. Its charge is to conduct field
trials and collect data needed for EPA
approval of so-called minor-use
"But 'minor' can be misleading,"
says Richard T. Guest, national direc-
tor of the IR-4 program. He's sta-
tioned at the New Jersey Agricultural
Experiment Station at Rutgers Uni-
versity in New Brunswick.
"According to the most recent cen-
sus of agriculture, 11 million acres of
minor crops are grown annually in
the United States," says Guest. "They
have a combined value of $32 billion
and represent 42 percent of all crop
sales. In 27 states, these minor crops
exceed the value of all the other ma-
jor crops including corn, cotton, soy-
beans, and wheat."
"Minor use" can also mean the in-
frequent use of a chemical product on
high-acreage crops like corn and
wheat. Last year, the IR-4 project
was responsible for 104 minor-use
pesticide clearances; in 1994, there
were 141.

At the Yakima Agricultural Research
Laboratory in Wapato, Washington,
technician Tom Treat applies a test
pesticide to a rapeseed variety being grown
for canola oil production. (K7433-13)

Agricultural Research/October 1996

Since its inception in 1963, IR-4
has assisted with more than 4,400
clearances on some 208 crops, from
acerola (Barbados cherry) and alfalfa
to yam and youngberry. While some
of these crops have odd-sounding
names like canistel (a tree fruit grown
in Florida) and kenaf (a plant that is
being used for newsprint), most can
be found in the fresh produce section
of any supermarket.
The IR-4 Ornamentals Research
Program, which was added in 1977,
has resulted in more than 3,600 addi-
tional pesticide clearances for 263
commercially grown floral and nurs-
ery crops-from abelia and acacia to
zebra plant and zinnia.
The program took on even more
importance when its role was ex-
panded in 1982 to include registra-
tion of biopesticides. This is a com-
mitment to develop alternatives to
chemical pest controls. IR-4 interacts
with the U.S. Department of


cid Impc Asssmn Program is to -rmt inore rgl-

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wha hapn if a petcd is no loge avial fo use ona
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esiae the cot to famr -a-d cosmr of loigpetcd
uss whl tain int accun an isusrltdt uan
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of adqut pes conro tool. Alhog th tw prgrm are
seaae thi como iners ha le to coopeatio. Whl
IR.. is chre wit sekn reitaino r- ere*g~eist CBSn

ne use of exitin chmial on inrcop, NAIPgahr
an anlye inomto on petcie aled approvedforSboth

"Ou douet are deige to pr -vie ubaed infrmaio

Field research director Sharon Benzen
collects romaine lettuce from an IR-4 test
plot in Salinas, California. The samples
will be frozen and shipped to a laboratory
for pesticide residue analyses. (K7431-17)

Agricultural Research/October 1996

At Wapato, technicians Kathryn Morford (left foreground) and Ronah Grigg analyze
extracts of crops grown on IR-4 test plots to determine pesticide residue levels. Eric
Fendell (left background) and Kim Foster verify findings with a mass spectrometer.

Agriculture (USDA), the U.S. Food
and Drug Administration (FDA), and
EPA to determine what data will
need to be collected.
As a result of this cooperation, 68
biopesticides have been approved.
One of these is granulosis virus to
control codling moths that attack
apples, pears, walnuts, and plums.
Another is cinnamaldehyde for the
control of Verticillium spot and dry
bubble disease of mushrooms.
"The program is important to pro-
tect growers and consumers. It en-
ables farmers and ranchers to use
pesticides judiciously against weeds,
diseases, and insects. Otherwise,
some might be tempted to spray any-
thing that works and at incorrect
rates. Because of IR-4, consumers get
foods that are wholesome, safe, and
relatively inexpensive," says Paul H.
Schwartz, Jr.
Schwartz coordinates the IR-4
program for USDA's Agricultural
Research Service at Beltsville,
Maryland. USDA's Cooperative
State Research, Education, and
Extension Service is the lead agency.
Regional laboratories servicing
satellite locations are Davis, Califor-
nia; East Lansing, Michigan; Geneva,
New York; and Gainesville, Florida.

"As a matter of fact," says
Schwartz, "we believe the program
may actually reduce overall pesticide
use. Once we have all the data, we
learn the proper doses farmers should
be using. If that information weren't
on the product label, they might ap-
ply more than they actually need.
That would raise their costs and put
more chemicals into the environ-

Plenty of Grower Input
"One of the reasons IR-4 is so suc-
cessful is that it's a real grass roots
program," says Guest. "We have
workshops open to growers, grower
groups, researchers, and any interest-
ed parties. They tell us what pest
problems they have, and that's what
we work on."
And, he adds, having a close
working relationship with EPA
streamlines communication and pro-
duces results more quickly.
Each trial looks at how effective
the chemical is, how it might affect
the crops, how much to apply, and
how much, if any, of the chemical
remains on harvested crops. There
are 10 ARS test sites across the coun-
try representing diverse climates.

These are at Wapato and Prosser,
Washington; Corvallis, Oregon; Sali-
nas, California; Urbana, Illinois;
Wooster, Ohio; Weslaco, Texas;
Beltsville, Maryland; Charleston,
South Carolina; and Tifton, Georgia.
Scientists at three ARS analytical
chemical residue labs-at Wapato,
Tifton, and Beltsville-determine the
amount of residue remaining on com-
modities after treatment, so a toler-
ance level can be established. The
minute amount of chemical residue
allowed to remain on commodities
falls within a safety margin set at
least 100-fold below a no-effect level.
Data from laboratory research and
replicated field trials is incorporated
by IR-4 headquarters into petitions
and submitted to EPA. The petitions
are a request that a tolerance or ex-
emption be established for a specific
product on a specific crop. The pro-
cess generally takes from 3 to 5
years, depending on the difficulty of
the study and EPA review time.
During the fiscal year that ended
September 1996, $8.3 million in fed-
eral funds supported the IR-4 pro-
gram. State agricultural experiment
stations contribute additional fund-
ing. Private industry also contributes.

Testimonials From IR-4 Fans
"Survival," says Ann George, "is
what the IR-4 program means to the
hops industry. We're still in busi-
ness." And that's important, for to-
day's U.S. hops industry is a healthy
one with an annual crop valued at
$137 million-about 28 percent of
the world's production.
But back in the late 1980's, things
looked pretty bleak when hops grow-
ers lost registration on the primary
miticide, herbicide, and insecticide
they needed to stay competitive in a
worldwide market.
"After using emergency exemp-
tions from EPA on a year-by-year

Agricultural Research/October 1996

basis, we have now obtained full
registrations for a key insecticide and
a miticide," says George. She is
administrator of the Washington Hop
Commission, as well as administrator
of the U.S. Hop Industry Plant
Protection Committee in Yakima,
George says the pesticide industry
was unwilling to gather data and peti-
tion EPA for labels that would permit
use of the products on 42,000 acres
of U.S. hops-a drop in the bucket
compared to the market potential of
nearly 80 million acres of corn.
Charles Matthews has a similar
story. He's a representative with the
Florida Fruit and Vegetable Associa-
tion in Orlando.
"The IR-4 Program assisted in pro-
viding data for certain uses of an im-
proved insecticide-imidacloprid,"
says Matthews.
"Growers are now allowed to use a
single application of this chemical to
control silverleaf whitefly on their to-
matoes and melon thrips on peppers.
Some other previously registered in-
secticides required up to 20 sprays to
knock down insect populations.
"And," he adds, "imidacloprid is
not harmful to beneficial insects and
is compatible with integrated pest
management programs."
But when EPA required that its
use entail a crop rotation, many
growers were reluctant to switch
from growing high-value crops to
less-income-producing ones. Back
came IR-4 with data supporting the
safety of planting high-value crops
like cucumbers, squash, and melons
after the tomatoes and peppers were
"This is an excellent example of
what growers, IR-4, university scien-
tists, private industry, and EPA can
accomplish when we work toward a
common goal," says Matthews.
In dry onions, pendimethalin was
approved for weed control. It is more

The IR-4 program also checks out chemicals applied to ornamentals like these dahlias
being examined by technician Tom Treat for evidence of damage. (K7433-12)

effective and controls a broader spec-
trum of weeds than the major alterna-
tive herbicide, and growers need ap-
ply only about one-tenth as much.
While it costs more per unit, the net
result is that farmers pay only one-
fifth of what they used to pay for the
Gene Batali, who grows 250 acres
of spearmint near Wapato, says, "We
need to control weeds in our mint
fields. If any are harvested with the

crop, we end up extracting their oils
along with that from the mint.
"Now we have control over our
weeds because IR-4 was the vehicle
we used to get the required herbicide
registration."-By Dennis Senft, ARS.
Paul H. Schwartz, Jr., is on the
USDA-ARS Pesticide Research Staf
Bldg. 1072, 10300 Baltimore Ave.,
Beltsville, MD 20705-2350; phone
(301) 504-8256, fax (301) 504-8142. *

A Brief IR-4 Timeline

1963-Interregional Research
Project No. 4 organized by directors
of state agricultural experiment
stations in cooperation with U.S.
Department of Agriculture.
1976-ARS program formally
established to assist IR-4 with
backlog of clearance requests.
1977-IR-4 expanded to include
commercially grown ornamentals,
such as floral and foliage plants,
woody nursery stock, Christmas
trees, and turf grass.
1982-Scope expanded to include
research supporting registration of bi-
opesticides, such as microbial and
biochemicals. More recently, geneti-

cally engineered material was
added to the mission.
1989-IR-4 strategic plan de-
veloped to gather data for reregis-
tering by 1997 nearly 1,000 minor
use labels mandated by amend-
ments to Federal Insecticide,
Fungicide, and Rodenticide Act.
1995-Goals identified for the
next 7 years, providing for a shift
in program emphasis to increase
the number of registrations for
both biological pest control agents
and less potent products needed in
integrated pest management

Agricultural Research/October 1996

Improving Ethanol Yield From Corn

The goal of microbiologist Robert Hespell and a team of Feoria, Illinois, scientists is to
devise a way to ferment corn fiber into ethyl alcohol, potentially increasing yield by 0.3
gallon per bushel. (K7413-14)

E thanol to fuel cars early in
the 21st century may come
from fiber-laden crop
residues-instead of feed grains-if
a vision of agricultural researchers at
Peoria, Illinois, pans out.
But before that vision is fully
realized, science and technology will
most likely enable ethanol producers
to squeeze a bit more ethanol from
corn-the main source at present.
Through fermentation of starches
and sugars found inside the grain,
modern ethanol plants now produce
2.5 gallons of ethyl alcohol-
ethanol-from each bushel of corn,
says Rodney J. Bothast. He's an ARS
microbiologist at the National Center
for Agricultural Utilization Research
(NCAUR) in Peoria, Illinois.
Bothast and his colleagues have
their sights set on making fiber in the
grain's outer layer yield nearly 0.3
additional gallon per bushel.

In 1994, about 1.3 billion gallons
of fuel ethanol were produced in the
United States from corn, with more
than 60 percent obtained through wet
milling. The wet-milling process
involves soaking, or steeping, the
corn in water, grinding it, and
separating high-protein germ, oil, and
fiber from the starchy endosperm that
is fermented to produce ethanol.
The current practice is to mix the
fiber fraction with fermentation
solubles before drying it and forming
animal feeds.
Agricultural engineer Michael R.
Ladisch and colleagues at Purdue
University in West Lafayette, Indi-
ana, teamed up with the NCAUR
scientists to assess ways to increase
ethanol production from corn.
"We estimate that if the fiber were
also processed into ethanol, a corn
wet-milling facility that produces 100
million gallons of ethanol per year

could generate an additional $4 to $8
million of annual income," says
microbiologist Robert B. Hespell. He
is project leader for ethanol research
in Bothast's Fermentation Biochem-
istry Research Unit.

Stilling a Criticism
The increased efficiency of corn
and ethanol production that has
evolved over the last 10 years may
also help to subdue criticisms that
petroleum used to produce corn and
process it into ethanol requires more
energy than is released when the
ethanol is burned.
According to "Estimating the Net
Energy Balance of Corn Ethanol," a
report published last year by USDA's
Economic Research Service (which
now includes the Office of Energy
and New Uses), the ethanol energy
now produced from each bushel of
corn is 25 percent greater than the
amount of energy used to grow and
harvest the corn and distill it into
ethanol. This is thanks to today's
higher corn yields, more energy-
efficient fertilizer production, and
improved distillation technology.

Unlocking Fiber's Energy Potential
Hespell says his team's research
strategy for economically converting
fiber to ethanol is three-pronged.
They hope to:
find better ways to physically
and chemically treat the fiber to
expedite its conversion to sugars,
find enzymes that better convert
the fiber to sugars, and
custom engineer suitable
microbes to ferment the sugars D-
glucose, D-xylose, and L-arabinose.
To achieve these goals, the re-
searchers are combining their efforts
with those of other ARS scientists
and university cooperators.
At College Station, Texas A&M
University researcher Mohammed

Agricultural Research/October 1996

Fungal physiologist Shelby Freer examines yeasts under high magnification, projected on
monitor, for their ability to make enzymes needed for ethanol production. (K7416-8)

Moniruzzaman and chemical engi-
neer Bruce E. Dale, who is now with
Michigan State University, applied a
pretreatment called ammonia fiber
explosion (AFEX) to corn fiber to

unlock its potential for fermentation.
With it, NCAUR scientists found
they could convert some 50 to 60
percent of the pretreated fiber's
components-cellulose, hemicellu-


A small-scale bioreactor enables chemical engineer Bruce Dien (left) and microbial
geneticist Herbert Wyckoff to see how well new genetically engineered microorganisms
will produce ethanol in fermentation. (K7415-1)

lose, and starch-into fermentable
sugars called monosaccharides,
while converting an additional 20 to
30 percent of the fiber into short
sugar polymers.
In the AFEX process, a slurry of
water and corn fiber is mixed with
highly pressurized liquid ammonia.
Quickly releasing the pressure splits
the fiber's bundles of carbohydrate
components that are normally rather
inaccessible to chemicals or microbes
because they are so tightly glued
together by lignin.
Researchers treated the lignin-
freed polymers with mixtures of
commercial enzymes. Some enzymes
called amylases and cellulases
thoroughly hydrolyzed, or split apart,
chains of starch and cellulose into
links of simple fermentable sugars
such as glucose, each with a back-
bone of six carbon atoms. Today's
ethanol plants typically use bakers'
yeast-Saccharomyces cerevisiae-
to produce ethanol only from these 6-
carbon sugars called hexoses.
From the corn hemicellulose, or
arabinoxylan, xylanase enzymes
clipped off at least 25 percent of the
component pentoses or 5-carbon
sugars-monosaccharides such as
arabinose and xylose. That success
was enough to spur a search for
xylanases that might be recruited to
enhance ethanol production.
"If we can find bacteria that
produce more active xylanases, this
ethanol research might also lead to
improving the efficiency with which
ruminant livestock such as cattle and
sheep digest hemicellulosic crop
residues," says Hespell. He is also
involved in research on ruminant

Drawing On Archival Research
Another impetus for the current
focus on freeing up pentoses for
ethanol production is recent success

Agricultural Research/October 1996

by biotechnologists in transforming
single microbial species to subsist on
both hexoses and pentoses.
Seeking an alternative pretreat-
ment of the fiber to free up more
simple sugars, the researchers took
note of work done by John W.
Dunning and Elbert C. Lathrop at
NCAUR in one of the earlier ethanol
research programs dating back more
than 50 years.
Recognizing the large amount of
sugars in corn cobs and other agricul-
tural residues, Dunning and Lathrop
hydrolyzed hemicellulose with mild
sulfuric acid treatments, forming a
solution of mostly pentoses.
But further costly processing, such
as deacidifying and removing the
toxic byproduct furfural, was re-
quired before microorganisms could
use the sugars.
ARS chemist Karel Grohman of
Winter Haven, Florida, and Bothast
reasoned that formation of furfural
and other chemicals that inhibit
fermentation could be reduced by a
two-stage process. First, they quickly
hydrolyzed the fiber with hot, mild
acid; then they quickly cooled it and
added a mixture of cellulase and
amyloglucosidase enzymes before
further hydrolysis.
In the laboratory, Grohman found
that the sequential treatment on
batches of low-starch corn fiber
resulted in about 85 percent of all
polysaccharide becoming sugars in
monomer forms.
Within 2 days, genetically engi-
neered Escherichia coli bacteria
fermented these sugars into solutions
of more than 2 percent alcohol.
The hydrolysis is now being scaled
up as a continuous process at
NCAUR. A goal is to complete the
acid hydrolysis phase within 2

After successfully finding ways to
break down corn fiber into hexoses
and pentoses, the researchers' next
challenge is to identify or develop
strains of microorganisms that will
convert both of these sugar types to
ethanol as efficiently as bakers' yeast
makes it commercially from hexoses.
The genetically engineered E. coli
strain K011 that Grohman and
Bothast used to produce ethanol from

multiple sugars of acid-hydrolyzed
fiber was developed by microbiolo-
gist Lonnie O. Ingram at the Univer-
sity of Florida in Gainesville. The
evaluation of K011 was conducted by
ARS under a cooperative agreement.
Starting with KO11, microbiologist
Herbert Wyckoff and chemical
engineer Bruce S. Dien at NCAUR
worked with Hespell and Bothast to
further transform the microbe. Unlike

Microbiologist Rodney Bothast (left) and technician Loren Iten add starter
microorganisms to pilot-plant-size bioreactors used for brewing ethanol from sugar
mixtures derived from corn fiber. (K7408-13)

Agricultural Research/October 1996

the older version, the new one does
not need antibiotics to survive in
anaerobic fermentation environments
like those of commercial ethanol
plants. The scientists are scaling up
their laboratory research to the pilot
Another recombinant microbe that
can ferment both glucose and xylose
is a strain of S. cerevisiae yeast
developed by geneticist Nancy Ho at

Purdue. It was also evaluated under
an ARS cooperative agreement.
Because this microbial species has
long been used to make ethanol, a
modified version too might someday
work well for the industry, says
Bothast. Further research, however,
is needed to develop Ho's strain into
one that can survive better under
industrial conditions, produce ethanol
from other sugars such as arahinse.
and quickly produce larger volumes
of ethanol.

Fungi, Too, Might Join the Effort
In addition to bacteria and yeasts,
genetically transformed filamentous
fungi could become ethanol plant
At NCAUR, microbiologists
Christopher D. Skory and Shelby N.
Freer envision harnessing industrial
and food processing fungi for a "one-
pot" method of producing ethanol.
The microbes prodigiously spew out
enzymes that efficiently break down
the corn fiber's cellulose and hemi-
cellulose while producing tiny
amounts of ethanol from the resulting
sugars. "Through both mutagenesis
and genetic engineering, we hope to
increase their ethanol production,"
says Skory.
Similar genetic research could lead
to one-pot production of lactic acid
that is valuable in food processing
and for industrial applications, such
as making biodegradable plastics.
Developing such a wet-milling
coproduct would help offset ethanol
production costs, since ethanol is
fairly low in economic value.
Considering alternative fermenta-
tions of glucose, Freer is screening
part of the ARS Culture Collection
located at the NCAUR for Brettano-
myces yeasts that efficiently produce
acetic acid from glucose. In earlier

screening of the collection, Freer
identified a microbe with a gene
responsible for producing a beta-
glucosidase enzyme that breaks down
small cellulose polymers into fer-
mentable sugars. Skory has cloned
the gene and inserted it into several
other microbes, including ethanol-
producing ones.
In still another screening, microbi-
ologist Badal C. Saha has identified a
yeast that produces a heat-stable
beta-glucosidase that works in
environments high in glucose. He is
mutating the yeast to try to increase
its production of the enzyme so that it
can be used to efficiently convert
cellulose to sugars.
In addition to trying to get more
ethanol from a bushel of corn, the
NCAUR researchers hope to increase
the usefulness of other ethanol
fermentation coproducts.
One abundant low-value product
of fermentation is carbon dioxide.
NCAUR plant physiologist Brent
Tisserat is evaluating the ability of
different CO2 concentrations to speed
the growth of plant tissue cultures.
He envisions using such cultures one
day to produce food flavorings and
high-value pharmaceuticals.
At NCAUR, other scientists are
researching potential value-added
products that can be made from wet-
milling coproducts.-By Ben
Hardin, ARS.
Rodney J. Bothast heads the
USDA-ARS Fermentation Biochemis-
try Research Unit, National Center
for Agricultural Utilization Research,
1815 N. University St., Peoria, IL
61604; phone (309) 681-6566, fax
(309) 681-6686, e-mail *

Agricultural Research/October 1996

Two Strategies for Protecting Poultry

From Coccidia

f ever organisms lived up to the
; label "parasite," it is those
'W belonging to the order Coccidia.
Not only do the single-cell proto-
zoans of the Eimeria genus infest the
nation's poultry flocks, costing
American producers an estimated
$600 million-plus annually in medi-
cation costs and lost production; they
also invade and take shelter in the
very cells marshaled by the chicken's
immune system to defeat them.
Though a vaccine is available in
this country against coccidiosis, its
key ingredient is a low dose of the
live parasite, which stimulates
protective immunity. But Hyun SCOTn
S. Lillehoj, who is an immu-
nologist with the ARS Immu-
nology and Disease Resistance
Laboratory at Beltsville,
Maryland, says the presence of
the live parasite may pose
"The live parasite can cause
disease in the bird," Lillehoj
contends. "If the bird's im-
mune system isn't functioning
properly for some reason, the
live parasite in the vaccine can
overcome the immune system. A fi
Also, there can be a negative anal
interaction within the bird with imm
feed contaminants such as
mycotoxins or with other
infections that might be
present, such as salmonella or
Complicating the vaccine situation
is the existence of seven different
species of Eimeria.
"An effective vaccine needs to
incorporate elements from all seven
species," says Lillehoj. "The vaccine
that has the live parasite uses many
of the seven strains. But the inci-
dence of variant species of Eimeria in
the field is increasing, and the live
coccidia vaccine cannot protect
effectively against all of them. It's

also very labor-intensive to produce
the live parasites."
Lillehoj favors a different ap-
proach. She and her research team-
including support scientist Marjorie
B. Nichols and technician Melody B.
Lowe-have devised a two-pronged
strategy to thwart coccidia.
"The chicken's immune system
produces cytotoxic T-cells whose
function it is to target and destroy
infected cells," explains Lillehoj.
"That's part of nature's protective
immune mechanism against this

ow cytometer used by immunologist Hyun Lillehoj
yzes intestinal lymphocytes that indicate chickens'
lune response to coccidia exposure from vaccination
iral infection. (K5130-3)

"But there is a phase of the
coccidia life cycle when the parasites
are called sporozoites. These actually
get inside the cytotoxic cells, which
then cannot kill them, but instead
deliver the parasites to the part of the
intestine called the crypt epithelium,
where they exit to develop."
Once nestled in crypt epithelial
cells, the thriving coccidia wreak
havoc in the intestinal lining and
interfere with the chicken's ability to
absorb nutrients from the feed it has
eaten. Result: The bird doesn't gain
weight and may die.

In 1993, Lillehoj and ARS immu-
nologist James M. Trout observed
coccidia's commandeering of cyto-
toxic cells firsthand when they used
two fluorescent, color-stained mono-
clonal antibodies to cling to and track
movement of both parasites and cyto-
toxic cells inside chicken intestines.
Green-stained monoclonal anti-
bodies allowed them to see where the
coccidia went; red-stained antibodies
pinpointed the presence of the
cytotoxic cells. Overlapping red and
green colors proved the coccidia
invaded the very cells that were
supposed to protect the chick-
en against them.
Part of Lillehoj's plan is to
block the initial invasion of
those crucial infection-fighting
cytotoxic cells by the coccidia.
"The sporozoite has to bind
to the cytotoxic cell to get
inside it," she explains. "Once
it binds, it makes a little dent
on the cell. The parasite has a
retractable structure called a
conoid that makes this dent.
Then enzymes from the
parasite act on the cell to make
an opening for the parasite to
get in.
or "We've developed and have
applied for a patent on a
chicken monoclonal antibody
that identifies a protein that the
sporozoite uses to cling to the
cytotoxic cell. In laboratory tests, this
antibody actually blocks the sporozo-
ite invasion of the cytotoxic cell."
Lillehoj is working with ARS
molecular biologists Mark C. Jenkins
and Kang D. Choi on ways to use the
protein recognized by the antibody as
a potential vaccine. Also promising
as potential weapons are cytokines,
substances produced naturally by the
bird's white blood cells.
We have shown in laboratory tests
that some cytokines inhibit develop-
ment of the parasite," says Lillehoj.

Agricultural Research/October 1996

Parasite: "An organism that grows, feeds, and is sheltered on or in a different organism while contributing
nothing to the survival of its host," says Webster's New World Dictionary. The word comes from the Greek
"parasitos," meaning "one who eats at another's table."

"They also enhance cytotoxic
activity by turning precursor cytotox-
ic cells into active cytotoxic cells.
Once activated, these cytotoxic cells
kill parasite-infected host cells.
Cytokines also activate white blood
cells called macrophages to devour
the parasites."
Starting in 1995, Lillehoj collabo-
rated with ARS molecular biologist
Dante S. Zarlenga and scientists at
Korea's Seoul National University to
clone the chicken gene that controls
manufacture of a cytokine called
gamma-interferon. The research team
has produced genetically engineered
chicken gamma-interferon and is
testing its protective powers in live
chickens. Early results look promis-
ing, Lillehoj says.
"If this works, a bird that's treated
with the gamma-interferon might still
get infected with coccidia, but it
might not lose as much weight or get
as bad a case of coccidiosis," she
explains. "You wouldn't want to
completely block the infection,
anyway, because then you wouldn't
stimulate the bird's immune system
to provide natural immunity against
future coccidia infections or other
opportunistic pathogens."
Gamma-interferon may prove
useful in the battle against coccidia in
other ways as well, Lillehoj adds.
"Antigens are proteins from the
parasite that stimulate an immune
response from an animal's immune
system," she points out. "It's been
shown in mammalian cells that when
you add gamma-interferon to a weak
antigen, you get a greater immune
response than if you just vaccinate
with the antigen alone. Plans are
under way to use gamma-interferon
as an adjuvant to enhance the action
of the vaccine."
Mass production of gamma-
interferon may be tricky, Lillehoj
says. In lab tests, attempts to repro-

duce the substance by inserting the
gene for its production into fast-
multiplying E. coli bacteria fell short
of the scientists' expectations.
"The protein was not very effec-
tive when expressed in E. coli,"
Lillehoj admits. "But once you have
a gene that expresses the protein, you
can raise it in a mammalian cell line."
One possible solution to the
protection dilemma might be to
identify an antigen common to all
strains of coccidia and use that as the
basis for a new vaccine.
"We know of one such segment,
but we're not ready to test it yet as a
vaccine," says Lillehoj.
The more common game plan-
waiting to clean up coccidiosis in
flocks after it occurs-is rapidly
becoming a risky proposition,
according to Lillehoj.
"The major problem is that the
parasite develops drug resistance
very quickly," she notes. "The main

emphasis for control has been on
drugs, but the coccidia have devel-
oped resistance to all the drugs ever
tested against them."
The ARS research team's multi-
faceted efforts come down to one
simple goal: to mimic nature.
"In the field, once birds have been
exposed to coccidia, they develop
immunity," says Lillehoj. "We've
been trying to figure out how chick-
ens get that immunity. Over the
years, we've learned a lot about how
the parasite invades cells and stimu-
lates natural immunity."-By Sandy
Miller Hays, ARS.
Hyun S. Lillehoj is at the USDA-
ARS Immunology and Disease
Resistance Laboratory, Bldg. 1043,
10300 Baltimore Ave., Beltsville, MD
20705-2350; phone (301) 504-8771,
fax (301) 504-5306, e-mail
hlilleho@ggpl.arsusda .gov *

Support scientist Marjorie Nichols (right) and technician Melody Lowe examine leghorn
chickens of the TK strain for signs of coccidiosis. The one on the right is infected with
Eimeria tenella, which results in a smaller comb, diarrhea, and weight loss. (K7396-8)

Agricultural Research/October 1996

Fire Ants Find Grains Tasty

N o one would ever confuse
John Morrison with mystery
story detectives Sam Spade
or Hercule Poirot.
An agricultural engineer by train-
ing, Morrison's world is the sun-
baked fields of east-central Texas,
where he devises workable conserva-
tion tillage techniques for clay soils
at the ARS Grassland, Soil, and Wa-
ter Research Laboratory at Temple.
But in the early 1980's, in the
midst of a planting study, Morrison
unexpectedly joined the ranks of
those whose job it is to unravel
mysteries-and he uncovered a
whole new threat to farmers' eco-
nomic well-being.
"We had field plots where only 20
to 25 percent of the plants were
coming up, so we thought we'd better
dig up the seeds and take a look,"
says Morrison. "Since we were using
experimental no-till planters for this
area's sticky clay soil, we thought the
planters might somehow be damaging
the seeds in planting."
Morrison did find damaged seeds
in abundance. But the menace wasn't
"Fire ants were just invading this
area," he notes. "When we dug up the
seeds, we found their hearts had been
eaten out by fire ants. In some cases,
we'd actually find the seed with a fire
ant burrowed into it, eating away,
with just its tail sticking out."
Accidentally imported from South
America half a century ago, the fire
ant species S. invicta can be found
today from Texas to Florida and as
far north as Tennessee and Virginia.
The ants pose a threat to animals and
humans alike. So researchers at the
ARS Medical and Veterinary Ento-
mology Research Laboratory at
Gainesville, Florida, are pursuing a
range of weapons against the biting
pests. [See "Fighting the Fire Ant,"
Agricultural Research, January 1994,
p. 4.]

Although Morrison first discov-
ered the fire ants ravaging seeds in
his no-till cotton field plots, later
studies showed that cotton suffered
the least from the foraging pests.
Tests at the Temple lab have
shown the ants will damage dry
wheat seed at a rate of about 11 per-

cent per day-capable of wiping out
an entire planting in 10 days' time.
Damage on dry corn seed runs about
6 percent, grain sorghum about 7 per-
cent, soybeans about 1 percent, and
cotton a mere 0.5 percent per day.
Fortunately for farmers, a possible
deterrent is at hand.
"At about the time we discovered
the ants, we were just starting to use
liquid starter fertilizer in our no-till
furrows at about 100 pounds per
acre," Morrison recalls. "When we
used that liquid fertilizer, our plants
emerged without fire ant damage.
That's a 'green' solution, because
we'd be putting fertilizer on the soil

The seed needs protection mostly
until germination, according to Mor-
rison. Although fire ants will crunch
on tender stems once plants have
broken through the soil, their allure
is lessened and so is ant damage.
"There's a race between the rate
at which the fire ants eat the seed
and the rate at which the seeds can
take in water, germinate, and emerge
from the soil," Morrison says. "In
our studies, as the seed gains water
and softens, damage from the fire
ants is more likely."
Insecticides are also an option for
averting the ants. But treating seeds
with insecticides can lower their
germination rates by 5 to 8 percent,
warns Morrison.
Oddly, the liquid fertilizer's
effectiveness was significantly less
in greenhouse studies in 1994-95. In
the greenhouse, it controlled only
about 50 percent of fire ants-
compared to 80 to 90 percent in the
field-so the Temple team plans
more field tests. But Morrison says
the work has already provided an
important warning for farmers in
areas with fire ants.
"You have to use either insecti-
cides or other effective repellents to
keep these ants from eating your
seeds," he notes. "Fertilizer may be
one solution, but others may be
found in the future."-By Sandy
Miller Hays, ARS.
John E. Morrison is in the USDA-
ARS Natural Resources Systems
Research Unit, Grassland, Soil, and
Water Research Laboratory, 808 E.
Blackland Rd., Temple, TX 76502;
phone (817) 770-6507, fax (817)
770-6561, e-mail morrison @brcO. *

Agricultural Research/October 1996

Cool Nightlife

Bad for


Plant physiologist Don Ort will insert the
leaf of a tomato plant exposed to cool night
temperatures into the airtight sample
chamber of a device designed and
constructed in his laboratory. Light guides
and hoses lead to instruments that
simultaneously measure photosynthetic
activities in living plants. (K7405-7)

imagine a footrace in which the
runner's feet moved in opposing
directions. Mission impossible?
Scientists in the ARS Photosyn-
thesis Research Unit at Urbana,
Illinois, have discovered a drop in the
overnight temperature below 50F
can create a biochemical version of
mission impossible for some crops
like tomatoes, soybeans, and corn.
The result is less efficient photosyn-
thesis, reduced yields, and an expla-
nation for the geographic limits
imposed on these plants because of
their temperature sensitivity.
Don Ort, a plant physiologist at
Urbana, says the warm-weather
evolutionary origins of plants like
tomatoes and corn make them more
sensitive to changes in temperature
during the growing season.
"Plants have an inborn time-
keeping mechanism-a circadian
rhythm played out over 24 hours-
during which specific chemical
reactions take place," Ort says.
The circadian rhythms are impor-
tant because they regulate the timing
of processes within the plant, he
adds. "There are specific reactions
that are timed to occur at a given
period of day or night." If allowed to
occur simultaneously, they would
compete and stall photosynthesis-

just as competing foot movements
would paralyze a runner.
In plants such as tomatoes, low
temperature disrupts the circadian
clock. "The mistiming of the expres-
sion of certain genes upsets photo-
synthetic metabolism, giving rise to
the characteristic chilling sensitivity
of these crops," says Ort.
Low night temperatures inhibit
daytime photosynthesis in these types
of plants by effectively delaying until
after dawn those reactions and
processes that would normally take
place at night.
For example, in tomatoes, if the
nighttime temperature were to drop
below 50F at 10 p.m. and not warm
up until 8 a.m. the next day, the plant
would behave as if it were still night
and continue nighttime activities
during daylight hours. At the same
time, the plant would initiate daytime
processes that compete with such
ongoing nighttime processes as the
breakdown of starch into sugars.
The regulation of phosphoprotein
phosphatase gene transcription gives
rise to the circadian pattern in
activity of sucrose phosphate syn-
thase and nitrate reductase. It is the
effect of low temperature on the
transcription of this gene that causes
delay in the circadian activity pattern
of these two key enzymes. Ort says it

is very likely that what differentiates
a chilling-sensitive plant from a
chilling-tolerant one has to do with
expression of phosphatase genes.
"What makes it doubly intriguing
is, if you look at the same things in a
native plant, you don't see this
effect," he says.
Using this information, scientists
hope to narrow the focus of their
research to a specific realm of the
photosynthetic process and to use
molecular engineering to override
low-temperature sensitivity.
"If we're successful, it could have
a significant impact on several
economically important crops," says
Ort. "For instance, an improvement
of even one or two degrees Fahren-
heit in temperature tolerance would
significantly expand the geographic
range of these crops to new regions,
as well as dramatically improve the
year-to-year consistency of yields
where the crops are currently grown."
-By Dawn Lyons-Johnson, ARS.
Donald Ort is in the USDA-ARS
Photosynthesis Research Unit, 190
ERML, 1201 W. Gregory Drive,
Urbana, IL 61801; phone 217-333-
2093, fax 217-244-0656, e-mail d- *

Agricultural Research/October 1996

People who have had bacte-
rial food poisoning may
have potential for illness
other than just the temporary incon-
venience of diarrhea and vomiting.
"Certain individuals may suffer
chronic joint diseases, such as
reactive arthritis, after being infected
with bacteria ingested with food,"
says James L. Smith.
Smith, who is retired from ARS'
Eastern Regional Research Center at
Wyndmoor, Pennsylvania, has been
studying the relationship between
foodborne bacteria and arthritis. Now
a collaborator and microbiologist
emeritus associated with the center's
Microbial Food Safety Research
Unit, he has come up with some
fascinating findings.
Infections from four rather com-
mon foodborne pathogens-Campy-
lobacter, Salmonella, Shigella, or
Yersinia-may lead to reactive
arthritis, he says.
"When we think of bacterially
induced arthritis, it's usually the
septic type in which the infecting
organism is present in the joint," says
Smith. "As it grows there, it can
cause inflammation and destruction
of joint tissue.
"In sterile, or reactive, arthritis,
organisms are not present in the joint.
Antigenic components of the
infecting bacteria are there, but
viable, living organisms are not,"
Smith explains.
It is interesting, he says, that an
infection in the gastrointestinal tract
by Campylobacter, Salmonella,
Shigella, or Yersinia bacteria can
somehow lead to inflammation of an
organ or joint that is far removed
from the site of infection.
"Genetic makeup may predispose
individuals to reactive arthritis,"
Smith says. "No one knows why, but
individuals carrying the gene respon-
sible for producing the human

leucocyte antigen HLA-B27 are more
susceptible to arthritis.
"We do know that the molecular
structure of antigens from these
particular foodborne bacteria mimic
the HLA antigen. It has been suggest-
ed that this antigen mimicry could be
a mechanism for causing arthritis."
An antigen is a substance that,
when introduced into the body,
causes the body to produce antibod-
ies to fight the foreign substance and
create immunity to it. Antigens can
be toxins, bacteria, viruses, or foreign
blood cells. T-cells are immune cells
that activate other cells that directly
destroy pathogens.





Agricultural Research/October 1996


The HLA-B27 gene is found in
about 10 percent of healthy Cauca-
sians, 1 percent of Japanese, and up
to 4 percent of North American
blacks but is absent from African
and Australian blacks. While only
about 2 percent of the people who
get food poisoning develop arthritis,
about 20 percent of those exposed
who have the HLA-B27 gene get it.
Smith cites a recent outbreak of
milkborne salmonellosis in the
United States that made about
198,000 people ill. After that infec-
tion, arthritis was found in about 2.3
percent-more than 4,500-of those
actually infected with the bacteria.
Foods that can carry Campylo-
bacter, Salmonella, Shigella, or
Yersinia bacteria include raw and
undercooked meat, poultry, eggs,
shellfish and other seafood, unpas-
teurized milk, fruit, and vegetables.
Untreated drinking water and
household pets are also sources.
Although they are the most
commonly reported, bacterially-
caused foodborne illnesses are the
easiest to prevent. To kill bacteria,
consumers should always cook foods
(especially meat, poultry, and eggs)
thoroughly, keep raw and cooked
foods separate, and promptly refrig-
erate cooked foods in shallow
containers. Raw fruits and vegetables
should be washed with water.
"For most people, an infection
from foodborne bacteria just means
feeling rotten for a day or so. But for
an unfortunate few, it can mean the
severe hardship of arthritis," Smith
says.-By Doris Stanley, ARS.
James L. Smith can be reached at
the USDA-ARS Microbial Food
Safety Research Unit, Eastern
Regional Research Center, 600 East
Mermaid Lane, Wyndmoor, PA
19038-8551; fax (215) 233-6568; e- *

This Salmonella typhimurium bacterium is
about half a micrometer (millionth of a
meter) wide. (K7482-1)

At Long Last-

A Producer-Friendly Brucellosis Vaccine

major hurdle has been
cleared in the long battle
'.* against brucellosis, a
contagious bacterial disease that
costs U.S. cattle producers some $30
million annually.
The latest weapon is a new vac-
cine called RB51. Its name is taken
from a strain of the Brucella abortus
bacterium that causes brucellosis. But
unlike longstanding vaccines that use
B. abortus strain 19, the RB51
vaccine doesn't stimulate the ani-
mal's immune system to produce
antibodies that interfere with the
diagnosis of B. abortus. K
The presence of such antibod-
ies in subsequent blood tests has
sometimes been viewed as a sign
of infection, rather than of
vaccination-making some cattle
producers reluctant to vaccinate
their herds.
USDA's Animal and Plant
Health Inspection Service (APH-
IS) granted a provisional license
in February 1996 to Colorado
Serum Company in Denver to A
make and sell a vaccine contain- b
ing RB51. ARS researchers at the
National Animal Disease Center
(NADC) in Ames, Iowa, have a
vested interest in the success of this
new vaccine, which is gradually
being adopted as the official vaccine
to replace B. abortus strain 19.
Brucellosis, or Bang's disease as
it's often called, reduces cattle
fertility, causes abortions, and
reduces milk production in beef and
dairy cattle. People can become
infected if they handle, slaughter, or
consume unpasteurized milk products
from infected cattle. In humans, the
disease is called undulant fever and
causes flulike symptoms, weakness,
and loss of appetite and weight.
No treatment or prophylactic drug
has ever been developed for cattle

For over 50 years, veterinarians
have tested cattle. Those animals
found to be infected were separated
from the herd or slaughtered.
Since the early 1940's, vaccines
based on B. abortus strain 19 have
been the chief defense against this
devastating disease.
"But the problem with using strain
19 in a vaccine has always been that
it induced an antibody response,
making identification of truly infect-
ed animals more difficult," says ARS
veterinarian Steven C. Olsen.

animal caretaker Terry Krausman raised this bison
rom a 1-day-old calf for use in research to develop a
rucellosis vaccine for bison. (K7078-7)

Olsen is a member of the research
team responsible for developing the
new vaccine. The RB51 strain was
first identified and isolated in the
early 1980's by Gerhardt Schurig, a
microbiologist at Virginia Polytech-
nic Institute and State University in
Blacksburg, Virginia.
Lack of antibody response isn't the
only advantage offered by the RB51
vaccine. In studies conducted by
veterinarians and researchers in
Alabama, Kansas, Georgia, Texas,
and Florida, the vaccine appeared
clinically safe for use in pregnant
cows. Only one pregnant animal in
1,000 aborted after vaccination with
the live RB51 vaccine.

To help APHIS speed up brucello-
sis eradication, the ARS research
team of Olsen, Mark G. Stevens,
Mitchell V. Palmer, Shirley M.
Halling, Betsy J. Bricker, and Nor-
man F. Cheville, who was formerly
with ARS, were responsible for
developing an improved vaccine.
"These researchers performed
years of work behind the scenes-
vaccinating calves, raising them to
breeding age, waiting until they were
pregnant, and exposing them to the
bacteria to see if the vaccination
worked," says veterinary medical
officer Carole A. Bolin, who is in
charge of NADC's Zoonotic
Diseases Research Unit.
The problem of brucellosis
extends well beyond the bound-
aries of this country's cattle
ranches. About 50 percent of the
bison leaving Yellowstone
National Park during the winter to
forage in cattle-populated areas of
Montana, Wyoming, and Idaho
test positive for brucellosis,
according to Olsen.
He and fellow ARS researchers
are participating in a multiagency
program to study the disease in
free-ranging bison in Yellow-
stone. Olsen is cooperating in the
program with APHIS; the Montana
Department of Fish, Wildlife, and
Parks; and the U.S. Department of
the Interior's National Park Service
and the Biological Resources Divi-
sion (formerly the National Biologi-
cal Service), U.S. Geological Survey.
The Park Service hopes to eliminate
the disease from Yellowstone bison
by 2010.-By Linda Cooke, ARS.
Steven C. Olsen is in the USDA-
ARS Zoonotic Diseases Research
Unit at the National Animal Disease
Center, P.O. Box 70, Ames, Iowa
50010; phone (515) 239-8200, fax
(515) 239-8458, e-mail solsen@nadc *

Agricultural Research/October 1996

New Alfalfa Powers Cows and Houses

Center, top: Leaves from a single alfalfa
plant (right) bred for both livestock feed
and electric power generation. Top, left:
Leaves from four plants (bottom, left)
grown for livestock feed. Center, bottom:
Pelletized alfalfa leaves for feed. (K7479-2)


Plant physiologist Deborah Samac
genetically engineers alfalfa for increased
leaf retention, disease resistance, higher
chlorophyll, and delayed flowering. Some
of these features may be bred into the
variety of high-biomass alfalfa held by
dairy scientist Hans Jung. (K7479-3)

y the turn of the century, a
dairy cow munching on a
mouthful of alfalfa leaves
could be helping to take the bite out
of energy costs.
A team of Agricultural Research
Service scientists and University of
Minnesota researchers are working to
develop a new alfalfa variety to be
used as both a high-protein feed
source for dairy cattle and an environ-
mentally friendly energy source to
generate electricity for Minnesota
Carroll Vance, a plant physiologist,
says the role of ARS in the project is
to provide a plant material that will
work as a combustible energy source
and be economically practical for
farmers to produce.
"Our mission is twofold: Develop
a variety that meets energy needs for
electric power and provide farmers
with an economic incentive to pro-
duce it," says Vance.
The first generation of the new al-
falfa variety is taller, stronger, and
thicker stemmed than alfalfa pro-
duced for use as animal feed. ARS
scientists crossed European varieties
bred for lodging resistance-the abili-
ty to stand up until harvest-with
modern alfalfa varieties developed as
feed for dairy cattle.
The original parental line was
identified as the best of the lodging-
resistant alfalfa varieties after a sum-
mer storm flattened test plots planted
for the study, says plant geneticist
JoAnn Lamb.
"We had high winds the night be-
fore, and everything was down except
for six rows of tall, thick-stemmed
plants," she says. "These rows became
a parent line for the new variety."
Hans Jung, a dairy scientist and co-
ordinator for the ARS portion of the
energy project, explains that the new
variety serves several purposes.
"First and foremost, the new vari-
ety provides a large, new market for

alfalfa," he says. "It will be grown
and marketed by a newly formed
southwestern Minnesota farmers co-
operative. Marketing the alfalfa this
way will be more simple and similar
to marketing grain.
"But it won't compete with
existing alfalfa markets," says Jung,
"because it isn't being produced
strictly as animal feed. And it intro-
duces a legume into the crop rotation,
which will lessen farmers' need for
nitrogen fertilizer, reduce tillage, and
enhance the environment.
A feasibility study shows it would
be profitable to market the stems for
energy production and the leaves for
meal. Whole-plant alfalfa brings
growers about $100 per ton in Minne-
sota; selling the alfalfa leaves as a
value-added product could bring them
additional profits.
A special farmer cooperative has
already been formed to grow and
market the new alfalfa, once it be-
comes available for commercial pro-
duction. The Minnesota Valley Alfal-
fa producers cooperative has over 400
members and has raised over $2 mil-
lion by selling shares in the coopera-
tive to produce and market the alfalfa.
The co-op has a 51 percent share in a
planned biomass system with Polsky
Energy Corporation, a Chicago-based
power systems developer.
The farmer cooperative and its
partner plan to build a power plant to
burn the alfalfa stems and then sell
the electricity it generates. The leaves
will be separated from the plant and
processed as animal feed. "We be-
lieve there is a good market for the
leaf meal in many areas of the live-
stock industry, especially dairy cat-
tle," says Jung.
"As currently planned, the project
will generate 75 megawatts of elec-
tricity, which will require the stem
material from about 600,000 tons of
alfalfa hay annually," he says. "It will
take about 180,000 acres to produce

Agricultural Research/October 1996

this amount of al-
falfa hay in
Minnesota. The
power plant is
creating the pro- N
duction demand. ,
"Alfalfa leaf
meal is a valuable
byproduct of the
biomass energy
system," says
Jung. "Neither the power plant nor
the leaf meal can survive without the
other; the leaf meal is needed to make
the economics work for generating
power from alfalfa, and the power
plant is needed as a disposal mecha-
nism for the stems generated in the
production of the leaf meal."
Researchers at the University of
Minnesota will begin feeding trials
with raw alfalfa leaf meal in the next
6 months. They will try to determine
which processing methods will yield
the best quality bypass protein source.
This protein bypasses part of the
cow's internal fermentation process-
es, making more protein available to
the animal's system.
Bypass proteins are more readily
utilized by dairy cows. However,
Jung says the leaf meal-whether raw
or processed-will need to be pel-
letized for ease of handling.
"Before processing, we will try
physical treatments like heat and
pressure, chemical treatments like
acid and alcohol, and combinations of
leaf meal with other crop byproducts
such as distillers grains, beet pulp,
and whey. All of the research is tar-
geted toward developing a more valu-
able feed ingredient, both in terms of
nutritional quality and price."
Developing alfalfa plants with
good leaf retention is the job of ARS
plant pathologist Deborah Samac.
"In some locations, alfalfa varieties
can lose up to 70 percent of their
leaves before harvest because of envi-

- Leaves -



Plant geneticist JoAnn Lamb, soil scientist
Michael Russelle (kneeling), and plant
physiologist Carroll Vance compare the
biomass of a single alfalfa plant selected for
use in electric energy production (left) with
several smaller alfalfa plants bred for use
as livestock feed. (K7479-1)

ronmental factors and disease," she
says. "We want to build excellent leaf
retention into the new variety so farm-
ers can raise a more profitable crop."
Using biotechnology techniques
like gene cloning and plant transfor-
mation, a process which allows scien-
tists to transfer specific new genes into
plants, Samac hopes to boost produc-

tion of cytokinin,
a plant growth
regulator that can
delay leaf aging.
"If we can in-
4 crease the produc-
tion of cytokinin
in older leaves far-
ther down in the
plant canopy, we
think we can trick
the leaves into
staying young longer," she says.
"Younger leaves are less prone to
many diseases and make a higher
quality animal feed. Along with tall,
rigid stems, it is critical to incorporate
good disease resistance in the variety
at the same time."
To achieve improved leaf reten-
tion, Samac is also working to isolate
the specific gene controlling alfalfa
leaf senescence, to modify production
of the plant growth regulator.
The usefulness of the new alfalfa
variety extends beyond feed and fiber.
ARS soil scientist Michael P.
Russelle will evaluate the fertilizer
value of ash left over from burning
the alfalfa stems. "We will examine
particle size, conduct chemical analy-
ses, and determine both the toxicity of
the ash and the biological availability
of the nutrients in it," he says.
Samac points out that the new al-
falfa variety being developed for the
biomass project in Minnesota can also
be used all over the Midwest for both
renewable energy and animal feed.
The dual-purpose alfalfa project
has received U.S. Department of En-
ergy funding.-By Dawn Lyons-
Johnson, ARS.
Carroll Vance, Hans Jung, and
other scientists mentioned are in the
USDA-ARS Plant Science Research
Unit, 411 Borlaug Hall, University of
Minnesota, St. Paul, MN 55108;
phone (612) 624-0763, fax (612) 649-
5058, e-mail *

Agricultural Research/October 1996

Electricity From Alfalfa


,6 TV/w 'm

The Year Sugar Baby Died

Air pollution, ozone take their toll on crops.

T he year was 1983, the place
was Indiana, and something
terrible was happening to
Sugar Baby.
A mysterious blight attacked this
popular, super-sweet, table-sized
watermelon, causing its leaves to
yellow and the plant to fail just as the
plump green fruits were developing.
This melon malady ravaged
between 6,000 and 7,000 acres in
southwestern Indiana, damaging
several melon varieties and hitting
about 100 farmers hard in the pocket-
book. Panicked growers were looking
for anything to stop the disease they
called yellow crown blight.
"Southwestern Indiana is one of
the larger melon producing areas east
of the Mississippi," says Purdue
professor James E. Simon, who is
with the university's research and
extension unit. "And since we're the
largest melon-producing area in the
Midwest, we had a real incentive to
solve this problem fast."
To find the cause of the melon
blight, Simon enlisted a team of

scientists including Agricultural
Research Service plant pathologist
Richard A. Reinert. The team's
diagnosis: acid soil conditions
leading to nutritional imbalance
(magnesium deficiency and manga-
nese toxicity), along with ozone-a
major air pollutant.
When Reinert and his colleagues
talk about ozone, they don't mean the
kind in the stratosphere that protects
living things from harmful ultraviolet
radiation. The ozone at ground level
comes from auto emissions reacting
with sunlight. This kind damages
plants by robbing them of the power
to synthesize food. Estimated cost of
the damage for all crops: $4 to $5
billion annually in the United States.
By 1990, Indiana growers were on
the mend, thanks to different water-
melon varieties, such as Crimson
Sweet, and improved soil nutrition.
The very ozone-sensitive varieties
like Sugar Baby are no longer grown.
Simon says the state's growers
earned $7.8 million from water-
melons in 1994, putting Indiana

upen-lop cnamoers at naleign, nornm aronlna, allow plain pPrlysaui gaIs Jusepn v1llner
(left) and North Carolina State University (NCSU) technician Walter Pursley to measure
cotton plants' photosynthesis and gas uptake through leaf pores with a portable infrared
gas analyzer. (K7440-16)

NCSU research associate Chantal Reid
performs laboratory photosynthesis
measurements on soybean plants grown
under different ozone and carbon dioxide
concentrations in open-top field chambers.
This helps identify specific photosynthetic
processes affected by the combined gases
and explain how they may alter plant
growth. (K7444-12)

among the nation's top 10 producers.
Texas, Georgia, and California head
the list, producing $60, $58, and $55
million worth, respectively.
"Not only did dedicated research
by Richard Reinert help solve this
complex environmental problem, but
also by correctly identifying its cause,
he helped save growers thousands of
dollars in unnecessary sprays they
had been using," Simon says.

Dealing With an Unseen Menace
Indiana was not the only place
where ozone damaged watermelons.
"My colleagues from as far away as
Spain and as close by as here in the
Carolinas saw similar problems,"
Reinert says.
But the scientists faced another
challenge after learning the cause of
yellow crown blight: They had to
convince many growers that some-
thing they could not see or smell was
injuring their crops. Some farmers,
believing they were dealing with a
new biological threat, poured on
agricultural chemicals.
An important lesson learned from
the melon case was the need to

Agricultural Research/October 1996

correctly diagnose a problem before
trying to solve it, Simon emphasizes.
"It was a real education and
outreach effort-in addition to the
science," adds Reinert.
Yellow crown blight was a dra-
matic example of how a combination
of factors, including air pollution, can
damage crops. But ozone is usually a
more subtle thief of farm profits than
in the Sugar Baby case.
"With most crops grown in the
field, you're not likely to see evi-
dence of damage," says plant pathol-
ogist Allen S. Heagle. He is Reinert's
colleague in the ARS Air Quality-
Plant Growth and Development
Research Unit at Raleigh, North
Carolina. "Visible injury looks like
normal aging, but there is a consis-
tent decrease in yield each year."
David A. Westenbarger, an
economist with USDA's Economic
Research Service, has been tracking
the cost of air pollution damage using
data provided by ARS. In a paper
published in the October 1995
Agricultural and Resource Econom-
ics Review, he credits Walter W.
Heck, also with the Raleigh lab, and
other ARS scientists with laying a
scientific foundation for his eco-
nomic research.
Westenbarger's study suggests
tighter air pollution controls in the
eastern United States could bring
between $17 and $82 million in farm
profits-mainly in Virginia, Mary-
land, and North Carolina. North
Carolina has seen new businesses and
a growing population, which benefit
the state's economy but may have
contributed to ozone problems.
"Ozone is not the greatest threat to
U.S. agriculture," Westenbarger
cautions. "But when it's your farm
and your yield not reaching top
potential, it suddenly becomes
He says ozone damage is not
limited to farms near major urban

areas. Weather patterns can move
ozone well beyond city lines-even
to remote farms and forests. And
stagnant air can cause pollutants to
linger, adding to the effect.
"In the case of the melons in
southwestern Indiana, the ozone
damage came from being downwind
of St. Louis and other urban areas,"
says Simon. "We had very high
temperatures, lots of humidity, and
stagnant air-all of which played a

Pinning Blame-for Sure
Proving ozone was the culprit
required special equipment.
The Raleigh unit where Reinert
works has open-top chambers where
researchers can study a pollutant's
effects in detail.
These chambers don't have a roof.
A mechanical system in the chamber
draws air from the outside, pushing it
from the bottom to the top. Because
the system is moving and filtering the
air, outside pollutants don't get

"Open-top chambers offer scien-
tists a special advantage in that
outside pollutants can be filtered
out," says plant physiologist Joseph
E. Miller, who oversees the research
unit. "There are other systems for
predicting pollution damage, some of
which are used by ARS scientists.
But this method allowed us to mirror
Indiana air conditions."
When Sugar Baby watermelons
were grown in the open-top chambers
and exposed to the same ozone levels
found in Indiana, they also developed
yellow crown blight. This helped
confirm ozone damage.
Reinert, Simon, and others then
grew other watermelon varieties in
the same environment that killed
Sugar Baby melons to find a variety
that was not damaged.
Open-top chambers allow scien-
tists to combine pollutants and can be
easily adapted to researchers' study
focus. "As global air pollution
changes, this kind of flexibility
becomes important," says Miller.

Agricultural engineer Kouen rnlDeCK aujusEs me system mat aenvers, controls, ana
monitors ozone and carbon dioxide concentrations in open-top field growth chambers.

Agricultural Research/October 1996

"In recent experiments, scientists in our unit
studied the combined effects of ozone and carbon
dioxide-an important aspect of global change."
While seed industry executives say ozone resis-
tance doesn't top their list of desired plant traits,
they acknowledge that it could become more im-
portant in the future if air pollution levels continue
to rise. They also agree that ozone-resistant vegeta-
bles could fill a certain market niche.
"I'll tell you where ozone resistance could be of
some use-truck farms," says John M. Reich, who
is with Cal-West seed company in Woodland, Cali-
fornia. "They grow fresh produce just outside ma-
jor urban areas. New Jersey, for example, is a big
truck farm area for New York City. Those produc-
ers are going to need ozone resistance sooner than
the rest of us."
"In my experience, cantaloupes have been more
environmentally sensitive than watermelons," says
Paul Chung, senior plant breeder for Peto Seeds,
also in Woodland. "In Europe-particularly in the
greenhouses of France and Spain-we see canta-
loupes that fail to thrive because of many factors,
including air quality."
Miller adds there are already areas in the United
States where ozone-sensitive vegetable crops can-
not be grown.
There may be more demand for ozone resistance
in the future as farmers come to appreciate its sig-
nificance. But developing resistant plants doesn't
happen overnight. Heagle has been studying crops
and air pollution for most of his 27-year career.
"For example, in studies with clover, I identified
20 clones that proved better at withstanding
elevated ozone levels, but that took a lot of time,"
Heagle says.
And he and his colleagues will keep testing vari-
ous crops for varieties that can thrive in a more
polluted world. Business and growth, both positive
things, caught up with Sugar Baby, so it moved on.
But in its place are melons made of tougher stuff.
And if a similar problem happens to any crop-
anywhere-Miller and his team of researchers
hope to be ready.-By Jill Lee, ARS.
Joseph E. Miller, Richard A. Reinert, and Allen
S. Heagle are in the USDA-ARS Air Quality-Plant
Growth and Development Research Unit, 1509
Varsity Dr., North Carolina State University, Box
7632, Raleigh, NC 27695-7632; phone Miller and
Reinert at (919) 515-3311, Heagle at (919) 515-
3728, fax (919) 515-3593. *

New Test Quantifies Aflatoxin in Grain

There's no place to hide for a crop-damaging fungus that
attacks corn, thanks to a new laboratory test that unmasks the
extent of the fungus' forays in the corn seed.
The target is Aspergillusflavus, the culprit behind a highly
toxic grain contaminant called aflatoxin. Federal law prohibits
the sale of grain for human consumption if it contains more than
20 parts per billion of aflatoxin, or 200 parts per billion in feed
for nonlactating animals.
The new test developed by ARS microbiologist Thomas E.
Cleveland and plant pathologist Robert L. Brown can demon-
strate the fungus' ability to grow in various corn kernels-
valuable information for commercial plant breeders. The test has
generated interest at Mississippi State, Mississippi, where ARS
geneticist Paul Williams and plant pathologist Gary L. Windham
are concentrating on breeding corn lines that fend off A. flavus.
"Scientists have suspected for a long time that some corn
varieties carry natural resistance, but until now we haven't been
able to select for them carefully," says Cleveland, who is based at
ARS' Southern Regional Research Center in New Orleans.
Previous tests verified A. flavus' presence in corn, but they
took days to complete and didn't necessarily indicate the amounts
of the fungus present. With the new test, A. flavus activity in corn
kernels inoculated with the fungus can be quantitatively detected
in a single day.
A. flavus is naturally present in all soils, but problems occur
when its metabolic byproduct, aflatoxin, accumulates in the
tissues of crops such as corn or peanuts. It can also contaminate
cottonseed, an important ingredient in feed for beef and dairy
cattle. Fungicides offer some protection but must be used in such
large quantities that they're not economical.
To track the fungus, researchers attach foreign genetic material
called a reporter, or marker, gene to a portion of an A. flavus gene
involved in cell division and growth. ARS scientists are working
closely with university cooperators-among them, Gary Payne at
North Carolina State University-in the construction of reporter
gene-containing strains of the fungus. A. flavus strains are then
used to inoculate corn breeding lines.
To check the growth of the fungus in a particular corn kernel,
the kernel is sliced open and soaked in a special chemical solu-
tion that reacts with a measurable enzyme produced by the
inserted reporter gene. Activity is indicated by a blue stain.
"If plant breeders are going to develop new corn hybrids with
resistance to the fungus, it's very important to be able to measure
the amount of the fungus' growth in the seed," Brown con-
cludes.-By Jill Lee, ARS.
The scientists are in the USDA-ARS Food and Feed Safety
Research Unit, Southern Regional Research Center, 1100 Robert
E. Lee Blvd., New Orleans, LA 70179; phone (504) 286-4531, fax
(504) 286-4419, e-mail *

Agricultural Research/October 1996

Science Update

Global Address for New
About 13,000 summaries of ARS
research findings can now be
searched in the agency's TEKTRAN
database. It's been available on the
World Wide Web since April.
TEKTRAN is a new online window
to ARS research labs-and to farm,
food, environmental, and industrial
technologies and products of the
future. ARS adds new summaries to
TEKTRAN after scientists submit
manuscripts to scientific journals.
Summaries are removed after 3 years.
Browsers can conduct a full-text
search of the summaries, including
titles, keywords, and author informa-
tion. They can also search by catego-
ries such as nutrition, germplasm,
pests, and soil management. Some
summaries are not posted-to
safeguard intellectual property rights
of ARS inventors and cooperators.
The Internet version of TEKTRAN
was developed by the Technology
Transfer Information Center (TTIC)
of ARS' National Agricultural
Library in cooperation with ARS'
Office of Technology Transfer (OTT)
and National Program Staff. ARS is
steadily improving TEKTRAN's
scope and convenience. The TTIC
home page (http://www.nal.usda.giv/
ttic) offers links for investigating new
ARS technologies available for
licensing. C. Andrew Watkins,
USDA-ARS, Office of Technology
Transfer, Beltsville, Maryland, MD,
phone (301) 504-5734; and Kate
Hayes, USDA-ARS, Technology
Transfer Information Center, Nation-
al Agricultural Library, Beltsville,
Maryland, phone (301) 504-6875.

Cantaloupes, sometimes called
muskmelons. (K7355-11)

Cantaloupe: More Nutritional
Clout for a Sweet Favorite
Growers and home gardeners have
three options for making sweet,
succulent cantaloupe a brighter beta
carotene star. The fruit's content of
this important nutrient is independ-
ently influenced by soil texture, fruit
size, and melon variety. ARS scien-
tists made this discovery through a 2-
year field test. Nutritionists recom-
mend eating plenty of foods rich in
beta carotene that the body converts
to vitamin A. Beta carotene and
lesser known, naturally present
substances in foods can boost defens-
es against cancers and other diseases.
Melons-cantaloupe, honeydew, and
watermelon-rank second of the 10
most popular fresh fruits. But for
supplying beta carotene, cantaloupe
is in a league by itself. On average, a
cupful provides 160 percent of the
Recommended Daily Intake of 5,000
International Units (vitamin A value).
That's quadruple the average in the
closest competitor, fresh peaches. In
the test, researchers measured
cantaloupe beta carotene levels as
high as 13,295 I.U. Levels were
generally higher in cantaloupe grown
on silty clay loam than on coarser,
sandy loam. Among six size classes,
bigger cantaloupe meant higher beta

carotene, with one exception. The
largest fruits (over 5 pounds) had 25
percent less beta carotene than the
second-largest class of 4- to 5-
pounders. Differences among com-
mercial varieties were less pro-
nounced, but Mission and Cristobal
had the highest beta carotene levels
of six varieties tested. Gene Lester,
USDA-ARS Crop Quality and Fruit
Insects Research Unit, Weslaco,
Texas, phone (210) 565-2647.

Sensing New Agricultural Tools
Instruments for espionage and war
might be refashioned into hi-tech
agricultural eyes, ears, and noses.
This is the intent of ARS and Ad-
vanced Information Management and
Movement, Inc., of Starkville,
Mississippi. Scientists will examine,
for example, whether computer
systems that detect dangerous chemi-
cals on the battlefield can be adapted
to sense insect pheromones. The
pheromones may signal an impend-
ing attack on crops by six-legged
invaders. There are several other
potential applications of the scien-
tists' effort, conducted under a
cooperative research and develop-
ment agreement. A few examples:
radar used to measure cotton growth,
devices to alert growers to snoop on
pesky insects' conversations (rustling
crop leaves as they feed), and modi-
fied weather-charting systems to
detect plant stress and soil moisture.
Jim McKinion, USDA-ARS Crop
Science Research Laboratory,
Mississippi State, Mississippi, phone
(601) 324-4376.

Agricultural Research/October 1996

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- Nutrition researchers are
discovering that the rainbow of
color pigments in fruits and
vegetables may do more than
attract attention or please the

,- Uncommon plants in USDA's
special legume collection may
be an untapped medicine chest
for the future.

,- A new treatment for grapefruit
promises fumigant-free destruc-
tion of fruit fly pests while the
fruit is being shipped to market.

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