U.S. Department of Agriculture Agricultural Research Service April 2012

Tuning In to Technology Benefits of Biotech, Computational Tech, and Nanotech

Agricultural Research Service‡6ROYLQJ3UREOHPVIRUWKH*URZLQJ:RUOG

B$SULOLQGG 30 ³ZH QHHG WR SXW D SUHPLXP RQ 2QH '1$ YLUXV JHQRPH VHTXHQFH LQ ³'LJLWDO'HWHFWLYHV'HFLSKHU,QJUHGLHQWV´ FUHDWLQJLQQRYDWLYHVROXWLRQVWRDGGUHVV SDUWLFXODUKDVWKHPH[FLWHG,WLVDSUHYL SDJH  RXUFXUUHQWDQGIXWXUHSUREOHPV´²86 RXVO\XQGLVFRYHUHGEDFWHULRSKDJHQDPHG )LQDOO\LQIRRGVDIHW\$56VFLHQWLVWV 'HSDUWPHQWRI$JULFXOWXUH6HFUHWDU\7RP ³SKL&$´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²PHWDJHQRPLFVJHQRPLF FDQWDUJHWDQGGHVWUR\SDWKRJHQLFEDFWHULD VHOHFWLRQPHWDERORPLFVDQGPRUH In plant breeding efforts, ARS scientist LQ QDWXUH 7ZR VXFK EDFWHULD DUH PDMRU Jean-Luc Jannink at the Robert W. Holley SDWKRJHQVRIGDLU\FDWWOH²6WDSK\ORFRFFXV (DFK SURMHFW UHSUHVHQWV D IXVLRQ RI Center for Agriculture and Health, in VSS DQG 6WUHSWRFRFFXV VSS LQFOXGLQJ OHDGLQJHGJH VFLHQFH DQG WHFKQRORJLFDO Ithaca, New York, is leading the charge PHWKLFLOOLQUHVLVWDQW 6WDSK\ORFRFFXV LQQRYDWLRQWKDWLVKHOSLQJWRVKDSH$56¶V for enhancing future crop designs with the DXUHXV RU 056$ 7KLV UHVHDUFK LV UHVSRQVHWRWKHJURZLQJIRRG¿EHUDQG use of “genomic selection”—thestatistical H[SHFWHGWREHUHSRUWHGLQDQXSFRPLQJ IXHOQHHGVRID86DQGZRUOGSRSXODWLRQ analysis of genomewide marker data to LVVXHRI$JULFXOWXUDO5HVHDUFK IRUHFDVWHG WR H[FHHG  PLOOLRQ DQG  predict the breeding value for selected ELOOLRQLQGLYLGXDOVUHVSHFWLYHO\E\ /LNHWKHQHZJHQHUDWLRQRIK\EULGYH progeny, including small- and large- KLFOHVK\EULGDQWLPLFURELDOVFRPELQHWKH 7DNHIRUH[DPSOHWKH¿HOGRIPHWDJH effect trait genes (see “A New Approach EHVWIHDWXUHVRIWKHLUSDUHQWFRPSRXQGV² QRPLFV DQ H[FLWLQJ QHZ GLVFLSOLQH WKDW to Molecular Plant Breeding,” page 13). O\WLFDFWLYLW\DQGVSHFLHVVSHFL¿FLW\²WKXV LQYROYHV VDPSOLQJ WKH '1$ FRGH RU -DQQLQN DQG XQLYHUVLW\ FROODERUDWRUV UHGXFLQJWKHSUREDELOLW\WKDWEDFWHULDZLOO JHQRPH VHTXHQFHV IURP D FRPPXQLW\ VKRZHGWKDWUHFHQWDGYDQFHVLQJHQRPLF GHYHORSUHVLVWDQFH RIPLFURRUJDQLVPVHQPDVVH7KDWFRP VHOHFWLRQ FDQ GUDPDWLFDOO\ LQFUHDVH JH PXQLW\PD\KDYHGLYHUVHRULJLQV²IURP $OO SURMHFWV KLJKOLJKWHG LQ WKLV LVVXH QHWLFJDLQVSHUXQLWRIWLPHDQGFRVWIRU DGURSRIRFHDQZDWHUWRDSLQFKRIVRLORU RI $JULFXOWXUDO 5HVHDUFK ZRXOG QRW EH ZKHDWDQGEDUOH\:KHQFRPELQHGZLWK HYHQDVZDEIURPWKHLQWHVWLQDOWUDFWRID SRVVLEOH ZLWKRXW WKH FRQWULEXWLRQV IURP \HDUURXQG QXUVHULHV DQG LQH[SHQVLYH GLVHDVHGIDUPDQLPDO WKH LQQRYDWLYH VFLHQWLVWV DQG VWDII RI KLJKWKURXJKSXW JHQRW\SLQJ SODWIRUPV $56 ZKR DUH H[SORLWLQJ QHZ VFLHQWL¿F $56VFLHQWLVWV/DV]OR=VDNDQG0LFKDHO JHQRPLFVHOHFWLRQZLOOHQDEOHEUHHGHUVLQ GLVFRYHULHVDQGWHFKQRORJLHVWRGHYHORS 'D\ERWKDWWKH6RXWKHDVW3RXOWU\5HVHDUFK WKHIXWXUHWRGHYHORSQHZYDULHWLHVPRUH VROXWLRQVIRUDJULFXOWXUH¶VJUHDWFKDOOHQJHV /DERUDWRU\LQ$WKHQV*HRUJLDDQG%ULDQ TXLFNO\DQGHI¿FLHQWO\ DQGSULRULWLHV 2DNOH\DQG%UXFH6HDORIWKH5LFKDUG% ,QKXPDQQXWULWLRQ$56VFLHQWLVW-DPHV 5XVVHOO5HVHDUFK&HQWHUWKHUHXVHGWKH +DUQO\DWWKH%HOWVYLOOH>0DU\ODQG@+XPDQ PHWDJHQRPLFDSSURDFKWRDQDO\]H51$ 1XWULWLRQ5HVHDUFK&HQWHUUHVSRQGHGWR DQG'1$YLUXVHVDVVRFLDWHGZLWKWZRPDMRU -DFN2NDPXUR DFULWLFDOQHHGLQWKHGLHWDU\VXSSOHPHQWV HQWHULFGLVHDVHVLQSRXOWU\SRXOWHQWHULWLV 1DWLRQDO3URJUDP/HDGHU LQGXVWU\IRUYDOLGDWHGDQDO\WLFDOPHWKRGVWR FRPSOH[DQGSRXOWHQWHULWLVPRUWDOLW\V\Q &URS3URGXFWLRQDQG3URWHFWLRQ LGHQWLI\GLYHUVHPHWDEROLWHVLQVXSSOHPHQWV GURPH VHH³0HWDJHQRPLFV2IIHUV,QVLJKW FRQWDLQLQJERWDQLFDOPDWHULDOV %HOWVYLOOH0DU\ODQG ,QWR3RXOWU\'LVHDVHV´SDJH  7KHDGDSWDWLRQRIPHWDERORPLFV²KLJK .D\6LPPRQV 7KH\IRXQGDQHZXQH[SORUHGXQLYHUVH UHVROXWLRQ DQDO\VLV RI PHWDEROLWHV XVLQJ 'HSXW\$GPLQLVWUDWRU RI YLUXVHV IURP FRPPRQ 51$ YLUXVHV PDVV VSHFWURPHWU\²WRDQDO\]H GLHWDU\ &URS3URGXFWLRQDQG3URWHFWLRQ DVWURYLUXVUHRYLUXVDQGURWDYLUXVHV WR VXSSOHPHQWVKDVSURYLGHGLPSRUWDQWQHZ DQDEXQGDQFHRIXQH[SHFWHGYLUXVHV SL %HOWVYLOOH0DU\ODQG LQVLJKW LQWR WKH FKHPLFDO FRPSRVLWLRQ FRELUQDYLUXVHVDQGFDOLFLYLUXVHV  DQGTXDOLW\RIWKHVHSRSXODUSURGXFWV VHH

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B$SULOLQGG 30 April 2012 Vol. 60, No. 4 ISSN 0002-161X Solving Problems for the Growing World

PHOTO BY SUNGHYUN NAM, COLORIZED BY STEPHEN AUSMUS (D2468-1) Agricultural Research is published 10 times a year by the Agricultural Research Service, U.S. Department of Agriculture (USDA). The Secretary of Agriculture has determined that this periodical is necessary in the transaction of public business required by law. Tom Vilsack, Secretary U.S. Department of Agriculture Catherine E. Woteki, Under Secretary Research, Education, and Economics Edward B. Knipling, Administrator Agricultural Research Service Sandy Miller Hays, Director Information Staff Editor: Robert Sowers (301) 504-1651 Associate Editor: Sue Kendall (301) 504-1623 Art Director: BA Allen (301) 504-1669 Photo Editor: Tara Weaver-Missick (301) 504-1663 Scanning electron microscope image of silver nanoparticles on Staff Photographers: the surface of cotton. The silver helps cotton fabric resist microbial Peggy Greb (301) 504-1620 growth. Magnification about 12,000x. Story begins on page 14. Stephen Ausmus (301) 504-1607 Most information in this magazine is public property and may be reprinted without permission (except where copyright is noted). Non -copyrighted articles and high- 4 A Bit of Gold and Nanotechnology Bring Viruses to Light resolution digital photos are available at ars.usda.gov/ar. Paid subscriptions are available from the U.S. 6 Digital Detectives Decipher Ingredients Government Printing Office (Superintendent of Documents). See back cover for ordering infor mation. 8 SERS: High-Tech Tactic May Newly Expose Stealthy Complimentary 1-year subscriptions are available directly from ARS to public libraries, schools, USDA employees, Salmonella and the news media. Call (301) 504-1638 or e-mail [email protected]. 10 What’s in Your Blood? The Ongoing Hunt for This magazine may report research involving pesticides. Metabolites It does not contain recommendations for their use, nor does it imply that uses discussed herein have been 12 Protein Biomarkers Identify Disease-Carrying Aphids registered. All uses of pesticides must be registered by appropriate state and/or federal agencies before they can be recommended. 13 A New Approach to Molecular Plant Breeding Reference to any commercial product or service is made with the understanding that no discri mination is intended 14 Cotton Gets Nanotech and Biotech Treatment in and no endorsement by USDA is implied. New Orleans The USDA prohibits discrimination in all its programs and activities on the basis of race, color, national origin, 16 Barcoding Insects To Control Them age, disability, and where applicable, sex, marital status, familial status, parental status, religion, sexual orientation, genetic information, political beliefs, 18 Metagenomics Offers Insight Into Poultry Diseases reprisal, or because all or part of an individual’s income is derived from any public assistance program. (Not 20 Longstanding Erosion Calculator Enters 21st Century allprohibited bases apply to all programs.) Persons with disabilities who require alternative means for 23 Locations Featured in This Magazine Issue communication of program information (Braille, large print, audiotape, etc.) should contact USDA’s TARGET Center at (202) 720-2600 (voice and TDD). To file a complaint of discrimination, write to USDA, Director, Office of Civil Rights, 1400 Independence Avenue, S.W., Cover: At ARS’s Food Composition and Methods Development Laboratory, in Beltsville, Washington, D.C. 20250-9410, or call (800) 795-3272 Maryland, postdoctoral associate Jianghao Sun prepares for HPLC/MS analysis of (voice) or (202) 720-6382 (TDD). USDA is an equal extracts from teas and supplements made from teas. The purpose is to evaluate the opportunity provider and employer. differences in the ingredients in the teas and supplements. Story begins on page 6. Photo by Peggy Greb. (D2473-2)

Agricultural Research O April 2012 3

B$SULOLQGG 30 NOAH PORITZ (K4705-12) Mosquitoes like this female $HGHV 2FKOHURWDWXV sp., sucking blood to get nourishment for her eggs, can transmit the viruses that cause West A Bit of Gold Nile fever and Rift Valley fever. ease Research Unit. “The assay is easier and quicker in detecting West Nile virus.” and In an initial study, researchersused gold nanoparticles and a Raman label of methylene blue dye to detect West Nile Nanotechnology virus and develop a nucleic acid assay. “We were having difficulty in getting a really clean signal,” Wilson says. “We could demonstrate signals, but they weren’t of the intensity that we would like.” Even- tually, however, the scientists were able to clean up the signals. Confirming a Detection Tool Gold nanoparticles have the ability to enhance light scatter, which can be taken Bring Viruses to Light advantage of in detecting virus-infected cells. This is the principle behind SERS that the researchers have used to develop an immunoassay and test its ability in detecting antibodies to West Nile virus. ll that glitters is not gold, but can be detected with a spectroscope. When Gold nanoparticles were coated with a it isA in the case of nanotechnology that’s Raman spectroscopy is used, molecules West Nile virus protein, and a malachite being used to develop detection tools for give off Raman spectra that have sharp green-conjugated protein A/G was used viruses that affect animals and people. peaks, making them more distinguishable as a Raman reporter. Protein A/G binds to Scientists at the Agricultural Research than with fluorescence, where molecules immunoglobulin molecules (antibodies). Service’s Center for Grain and Animal emit broad peaks. If a molecule is moved When antibodies specific to viral proteins Health Research (CGAHR) in Manhattan, in close proximity to a metal like gold or are present, the proteinA/G-antibody com- Kansas, and the University of Wyoming silver, as with SERS technology, the signal plex then binds to the viral protein-coated are using surface-enhanced Raman scat- is enhanced maybe up to a millionfold. gold nanoparticles. tering (SERS) with gold nanoparticles to Using SERS, scientists designed a “Because the protein A/G was labeled design tests that rapidly identify the virus nucleic acid diagnostic assay that cap- with a Raman dye, that would bring the that causes West Nile fever. tures Raman dyes—fluorescent tags—and Raman dye in close proximity with the The West Nilevirusis spreadby infected brings them close to the gold nanoparticles gold nanoparticles and we would get the mosquitoesand can cause headaches, fever, in a compound solution. They also devel- enhancement from the Raman signal,” and body aches. In some cases, it causes oped an immunological assay that rapidly Wilson says. a more serious and sometimes fatal neu- detects antibody responses. roinvasive disease—aseptic meningitis, “The advantage of SERS is that it In the future, a pen-side diagnostic test for Rift Valley fever and other diseases of cattle may arise encephalitis, or acute flaccid paralysis. amplifies the signal without requiring an from SERS research. Symptoms include disorientation, muscle enzymatic reaction or long incubation weakness, and paralysis. and can be done in a complex solution SERS technology is based on theconcept without multiple washing steps,”says that molecules have their own unique Ra- microbiologist William Wilson in the man scattering signal—wavelengths that CGAHR Arthropod-Borne Animal Dis-

SCOTT BAUER (K7612-17)

4 Agricultural Research O April 2012

B$SULOLQGG 30 KEITH WELLER (K4166-5) The results proved that gold nanopar- ticles in Raman scattering could beused as a detection tool in an antibody-based system. Still, researchers wanted to increase the signal’s intensity and improve the quality. “Instead of just using a gold nanopar- ticle, we used a magnetic particle, which has a capture probe,” Wilson says. “We capture the West Nile target RNA or DNA and come in with a second probe that detects the second region of the West Nile target.” The second probe is a gold nanoparticle that already has the reporter molecule at- tached to it, he explains. “We pull that into an area and focus the laser on it to see the signal,” Wilson says. “So it’s a way of enhancing our signal’s in- tensity for the end solution hybridization.” Gold and magnetic nanoparticles are coated with capture antibodies or DNA in a dry reagent form to avoid having to process blood samples, says Patrick John- son, assistant professor of chemical and petroleum engineering at the University of Wyoming. “At most, you may have to remove ARS scientists are using surface-enhanced Raman scattering with gold nanoparticles to design tests for just the cells from the blood, put the identifying viruses that cause West Nile fever and Rift Valley fever (RVF). RVF is spread by mosquitoes remaining fluid with the dry reagent of to humans and to sheep, cattle, and some other livestock in Africa and the Middle East. nanoparticles, mix it for a short period, and expose the sample to a strong mag- “It would allow physicians to do quick to other pathogens, Johnson says. Future net,”he says. “You can then read the tests at the bedside of patients, and veteri- studies will involve examining how this magnetic pellet with a laser.” narians couldpotentially use it for pen-side technology can be used to identify multiple If virus proteins are present, they will tests at farms,” Wilson says. disease agents in one test. Available pen- be detected through SERS spectroscopy, Researchers are using SERS with West side diagnostic tests are generally agent Johnson adds. Nile virus as well as Rift Valley fever specific. virus, but the process could be adapted “The sensitivity of this test is much Taking It to the Field greater than the current pen-side system,” Scientists are using a bench-top com- Wilson says. “It’s potentially as good as puter system in laboratories to analyze amplification technology, such as PCR and report the signal. [polymerase chain reaction]. It’s faster “The goal is to bring laboratory-level ´7KHDVVD\ and also gives us the potential to easily analytical sensitivity to the field for por- do multiple-pathogen detection.”—By table care devices,”Johnson says. “You LVHDVLHUDQG Sandra Avant, ARS. will be able to take a blood sample, put it in a small vial, and read it with a hand- TXLFNHULQ This research is part of Animal Health, held device.” an ARS national program (#103) described Systems exist that could easily be GHWHFWLQJ :HVW at www.nps.ars.usda.gov. adapted to field-type instruments, Wilson William Wilson is in the USDA-ARS says. The device would be more like an 1LOHYLUXVµ Arthropod-Borne Animal Diseases Re- advanced dipstick that could be used dur- search Unit, Center for Grain and Animal inga disease outbreak to rapidly determine Health Research, 1515 College Ave., areas where the outbreak is occurring. ³:LOOLDP:LOVRQ Manhattan, KS 66502; (785) 537-5570, [email protected].*

Agricultural Research O April 2012 5

B$SULOLQGG 30 ingredients in supplements to be identified. To answer the increased demands for valid analytical methods, Harnly chaired an expert review panel that established Digital “Guidelines for Validation of Botanical Identification Methods”for AOAC International (Association of Analytical Communities)—a nonprofit association Detectives dedicated to excellence in analytical methods. Pei Chen, a chemist with the ARS food- composition lab, headed a study of the dif- ferences between phytochemicals in green tea supplements andgreen tea leaves used for brewing beverages. Green tea-based dietary supplements have gained popular- ity in the U.S. market in recent years. But when it comes to sipping green tea versus taking the dietary supplement form, the Decipher Ingredients jury is out as to the better choice relative to health. HRMS can be very useful in identifying the differences. Chen and BHNRC colleagues Jianghao Sun and Long-Ze Lin analyzed “extractions” of 20 commercially available green tea elevision shows featuring crime lites, of cell processes. These individual dietary supplement products (12 tablets sceTne investigators have been keeping molecules are produced by active, living or capsules and 8 liquid samples) and viewers intrigued for years. But the cells during the metabolic process. Lipids, 8 dry green tea leaf samples, and they Agricultural Research Service’s intriguing fats, sugars, amino acids, and flavonoids, compared the chemical constituents of “food composition investigators”are just as for example, are all metabolites. The plant the samples using an analytical technique innovative at deciphering truth from fiction metabolome furnishes nutrients necessary called “HPLC/MS.” relating to ingredients of plant-basedfoods for human health. and dietary supplements. The researchers Metabolomics requires bioinformatics are at the ARS Food Composition and toolsfor data analysis, visualization, and ARS scientists are using mass spectrometry of Methods Development Laboratory, which integration. The researchers are using newly red grapefruit juice to determine differences in fruit grown conventionally versus organically. is headed by research leader James Harnly. procured equipment—high-resolution The laboratory is part of the Beltsville mass spectrometers (HRMS)—to conduct [Maryland] Human Nutrition Research metabolomic studies. “High resolution” Center (BHNRC). means the technology gives a high level They’re using new equipment and a of information. For example, with HRMS, metabolomics approach to discovercom- glucose has a measured mass of 179.0556 pounds and to accurately identify ingredi- atomic mass units (amu), compared with ents in foods and supplements. They are also 179 amu as detected by a regular mass looking atchemicalcomposition patterns to spectrometer. HRMS has the potential for find differences between cultivars, growing identifying every metabolite in a sample. years,andlocations—knowledgeimportant Green Tea: Sip it or Supp it? for consumers, farmers, and marketers. The U.S. Food and Drug Administration Metabolomics is the analysis of all the issued “current good manufacturing intermediate and end products, or metabo- practices” that require all botanical

PEGGY GREB (D2492-1)

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B$SULOLQGG 30 Brand names were not disclosed in the questionable from a chemical-composition were analyzed by mass spectrometry with published study, but the researchers noted point of view, and the results “demonstrate no separation of the molecules. The over- that most major dietary supplement manu- the urgency of quality control for green tea lapping mass spectra of all the molecules, facturers were represented. dietary supplement products,”wrote the or the “spectrometric fingerprint,” is very The study demonstrated that flavonol authors. The 2011 study was published in complex and, like human fingerprints, is glycosides were degraded and the com- the Journal of AOAC International. analyzed by looking at the overall pattern. pound catechin had oxidized during manu- Organic and Traditional Differences ANOVA-PCA was used to determine facturing and storage for many of thegreen Switching to fresh fruit, the BHNRC whether a distinction could be made tea supplement samples studied. They also “Rio Red Grapefruit” study showed between the two cultivation methods and found some additives in the supplements that MS fingerprinting, when combined the three harvest dates (growing phases) by that were not listed on the labels. with a pattern-recognition method called analyzing the MS spectral fingerprints of The BHNRC team found a signifi- “ANOVA-PCA,” could clearly establish the grapefruit juices. The analysis showed cant chemical variance across the range that there are chemical differences between that the chemical patterns of the fingerprints of green tea dietary supplements they grapefruit samples in terms of growing were statistically different among the sampled. They also noted that the daily year, harvest time, and farming method farming modes, growing years, and times of intake amount recommended by the labels (conventional or organic). harvest, regardless of the MS method used. varied significantly. They concluded that For the study, Harnly and Chen studied The results are important in demonstrat- although there are some good green tea samples of Rio Red grapefruit furnished by ing that conventional and organic products dietary supplement products, there is no Gene Lester of the ARS Food Quality Labo- have different chemical compositions, way for the consumer to know the quality ratory, also in Beltsville. The grapefruits although it is unknown at this time if these from reading the labels. More importantly, were grown using conventional and organic differences have significance to consumers. the consumer may ingest other botanical cultivation methods. They were harvested The 2010 study was published in the Jour- extracts unintentionally, and the quality of at three growing phases nal of Agricultural and Food Chemistry. those green tea products varies significantly. (early, mid, and late Let’s Not Forget Herbals The claim that a green tea dietary supple- season) during 2005 American ginseng is one of the most ment is a good alternative to tea leaves is and 2006. The juices commonly used herbal medicines in the world. But discriminating between gin- sengs grown in different countries is dif- ficult using traditional methods. Chen headed a study involving MS fin- gerprints and pattern-recognition analysis methods to discriminate between American ginseng grown in theUnited States and that grown in China. They studied 15 Ameri- can ginseng samples grown in Wisconsin and 25 samples grown in China. The MS fingerprints, representing the chemical compositions of the samples, made it possible to distinguish between samples grown in the two different locations. The 2011 study was published in Analytical and Bioanalytical Chemistry.—By Rosalie Marion Bliss, ARS. This researchis part of Human Nutrition, an ARS national program (#107) described at www.nps.ars.usda.gov. James Harnly and Pei Chen are with the USDA-ARS Food Composition and Meth- ods Development Laboratory, 10300 Bal-

Chemist Pei Chen prepares timore Ave., Beltsville, MD 20705-2350; extracts from teas and tea-based (301) 504-8569, ext. 261 [Harnly], (301) dietary supplements to study 504-8144, ext. 238 [Chen], james.harnly@ differences in phytochemicals ars.usda.gov, [email protected]. (health-protecting compounds) in * the two forms of tea.

PEGGY GREB (D2474-1)

Agricultural Research O April 2012 7

B$SULOLQGG 30 small quantities of bacteria in a complex, real-world background, such as a food or beverage sample. That’s especially important, since it’s apparently possible for as few as 10 Salmonella cells to cause SERS illness, depending on the strain involved and other factors. Nobel Laureate’s Work is Basis of Today’s SERS High-Tech Tactic Developed over the past two decades or so, SERS has expanded the power and potential of Raman spectroscopy, its parent technology. Raman spectroscopy is based May Newly Expose on discoveries made in the early 1900s by Sir Chandrasekhara V. Raman, who received the 1930 Nobel Prize in physics for his research. Stealthy Salmonella Ina SERS analysis, a specimen is placed on a surface, such as a stainless steel plate, silicon wafer, or glass slide, that has been “enhanced”—or changed from smooth to rough. For example, a glass slide or silicon wafer could be etched, to roughen it, or a small stainless steel plate could be coated with an extremely thin “nanolayer” t laboratories of the future, their in-house quality-control labs to help of minuscule particles of a metal, such as evenA the smallest quantity of Salmonella ensure that their products are free of unsafe silver, gold, or copper. bacteria may be easily detected with a levels of this or other harmful bacteria. In some of its research, Park’s team technology known as “SERS,” short for For Salmonella testing, labs have many enhanced the surface of stainless steel “surface-enhanced Raman scattering.” choices. They can opt for well-established plates by coating them with tiny spheres, Agricultural engineer Bosoon Park, technologies, such as monoclonal antibody made up of a biopolymer encapsulated in the Agricultural Research Service’s assays, ELISA (enzyme-linked immuno- with silver nanoparticles. Quality and Safety Assessment Research sorbent assay), or PCR (polymerase chain Unit in Athens, Georgia, is leading reaction) tests. Rough surfaces and colloidal metals, such as silver, can enhance the scattering of exploratory studies of this analytical Park’s team is evaluating the strengths light that occurs when a specimen, placed technique’s potential for quick, easy, and and shortcomings of SERS in relation on this “nanosubstrate,” is scanned with reliable detection of Salmonella and other to these popular analytical methods and the Raman spectrometer’s laser beam. The foodborne pathogens. several newer ones as well, including scattered light that comes back to the spec- According to the U.S. Centers for Dis- atomic force microscopy and surface troscope forms a distinct spectral pattern ease Control and Prevention, Salmonella plasmon resonance. causes more than 1 million cases of illness According to Park, a SERS analysis is in this country every year. relatively simple to perform, an advantage Transmission electron microscope image of If SERS proves successful for cornering when labs are swamped with samples biopolymer spheres coated (encapsulated) with silver nanoparticles, which might make an ideal Salmonella, thetechniquemightbe used at from an outbreak of foodborne illness or surface (substrate) for SERS detection of food- publichealth laboratories around thenation a product recall. borne pathogens in food and beverage samples. to rapidly identify pathogens responsible With further research, SERS may for outbreaks of foodborne illness. Tomor- offer superior detection capabilities, row’s foodmakers might opt to use SERS at making it faster and easier to find very

JAYA SUNDARAM (D2477-1)

8 Agricultural Research O April 2012

9359_April2012-r3-14.indd 8 3/14/12 12:44 PM JERRY HEITSCHMIDT (D2475-1) Postdoctoral research associate Jaya “SERS is one of several candidate tech- Sundaram (left) and agricultural engineer nologies we are currently testing,” says Bosoon Park are determining whether SERS signatures can be used to reliably identify Park. “If we find that it continues to offer pathogenic foodborne bacteria. major advantages over other options, our goal would be to develop a SERS-based would be posted on the Internet for public system that wouldn’t require extensive access by researchers and analytical labs training and would use affordable SERS worldwide. instruments.” In follow-up studies, Park intends to use Park has presented some of these find- less-concentrated samples of Salmonella ings at an SPIE (Society of Photo-Optical serotypes to morerigorously challengethe Instrumentation Engineers) conference in known as a “Raman spectral signature,” technology’s ability to detect and quantify Orlando, Florida. His collaborators include or a “Raman scattered signal.” the pathogen. These tests may ultimately Yao-Wen Huang and Yiping Zhao of the “The idea of using a substrate of silver reveal SERS’s sensitivity, or detection University of Georgia-Athens; Arthur nanoparticles for Raman spectroscopy is limit—the smallest number of Salmonella Hinton, Jr., Kurt C. Lawrence, Jaya Sun- not new,”says Park. “But in SERS studies cells, in a given concentration of solution, daram, William R. Windham, and Seung to detect foodborne pathogens, our use that can be accurately and reliably detected Chul Yoon, all with ARS at Athens; and of a substrate of silver nanoparticles that with a given combination of variables. Yongkuk Kwon of South Korea’s Animal, encapsulate a biopolymer is novel, to the Those variables might include the types Plant, and Fisheries Quarantineand Inspec- best of our knowledge.” of SERS instruments, sample preparation tion Agency. Though SERS research with biological method, sample nanosubstrate, power The team’s studies may do much to specimens is still in comparatively early and duration of the laser beam scanning, keep us safe from some of the world’s stages, researchers expect to prove the and more. worst foodborne pathogens.—By Marcia concept that all molecules, such as those In an earlier experiment, using adifferent Wood, ARS. that make up Salmonella, have their silver-based substrate, the team showed This research supports the USDAprior- own unique Raman spectral signature. that SERS can differentiate live Salmonella ity of food safety and is part of Food Safety, In the future, the Raman signature of an cells from dead ones. That’s significant. It an ARS national program (#108) described unidentified biological specimen could be could help quality-control labs evaluate the at www.nps.ars.usda.gov. compared to known Raman signatures to performance of today’s food-sterilization find a match and thus identify the unknown methods, such as high-pressure process- Bosoon Park is in the USDA-ARS specimen. ing, irradiation, and thermal processing. In Quality and Safety Assessment Research Unit, Richard B. Russell Research Center, Park’s team has already developed addition, reliable differentiation can speed 950 College Station Rd., Athens, GA preliminary Raman spectral signatures and simplify the work of researchers who 30605; (706) 546- 3396, bosoon.park@ for two common pathogenic kinds, or are developing and testing new techniques ars.usda.gov. serotypes, of S. enterica—Enteritidis to keep food safe to eat. * and Typhimurium—collected from raw chicken. Bosoon Park (right) and Jaya Sundaram examine a hyperspectral microscope image of an experimental substrate developed for SERS food-safety analyses. Silver-Encapsulated Spheres Tested as a Nanosubstrate For this research, the group used a biopolymer encapsulated with silver as the nanosubstrate, and they worked with comparatively large concentrations of the serotypes (about 108 colony-forming units per milliliter of solution). The high concentrations simplified detection and showed, apparently for the first time, that SERS can differentiate thetwo Salmonella serotypes by their Raman signatures. Plans call for developing a database, or library, of Raman spectral signatures of these Salmonella serotypes and other major foodborne pathogens at various concentrations. Ideally, the signatures

JERRY HEITSCHMIDT (D2476-1)

Agricultural Research O April 2012 9

B$SULOLQGG 30 associate adjunct professor of nutrition at the University of California-Davis, where the center is located. The ARS team and scientists at five other What’s in centers each used different analytical tech- niques, or “platforms,”to examine blood samples from healthy adult volunteers and from adults with cardiovascular disease. Your Blood? Several Significant Firsts Most of the metabolites documented in the database are not new to science. None- theless, the database represents several important firsts. The Ongoing To begin with, it documents an estimated 84 percent of human serum metabolites. As such, it “represents the most compre- hensive coverage of serum metabolites ever offered, in one convenient, reliable Hunt for Metabolites resource,”says Newman. What’s more, the database pulls together information on serum metabolites not only from the participating labs, but also from an impressive array of sources in the scientific literature—more than 2,000 in all. Careful t any given time, blood metabolomics might use metabolic finger- review of these sources led to the addition circAulating through your body carries printing to assess whether experimental of nearly 700 metabolites to the database, thousands of small molecules known diet- and physical activity-based strategies augmenting those detected and quantified as “metabolites.” Medical and nutrition can prevent the onset of disorders such as in the laboratory analyses. researchers are eager to discover more type 2 diabetes or cardiovascular disease. The sources were gleaned from the about these compounds—amino acids, Medical and nutrition researchers who project’s computer-assisted data mining sugars, fats, and more—that are formed are investigating serum metabolites for of some 19 million scientific abstracts. in and by our bodies. these and other promising applications An additional first: The database is the Metabolites are of interest because the have a remarkable new resource for their most extensive electronically searchable presence and concentrations of some of studies. compendium available today of the human them can provide meaningful profiles, And it’s only a mouse click away. sometimes referred to as “metabolic sig- It is the Serum Metabolome database natures” or “fingerprints.” (www.serummetabolome.ca), a compre- In the future, for instance, yourmetabolic hensive, first-ever public catalog of 4,229 fingerprint—detected in a small sample metabolites in human serum. of blood taken for your annual physical A team of 24 researchers in Canada and examination—might prove to be a reliable the United States—including Agricultural indicator of your health and a predictor of Research Se rvice investigators John W. certain diseases. This information could Newman and Theresa L. Pedersen—par- then be used to create personalized recom- ticipated in this international project. mendations about what you should eat for Newman, a chemist, and Pedersen, a optimal health. support scientist, are with the ARS West- In another futuristic scenario, scientists ern Human Nutrition Research Center specializing in the field of nutritional in Davis, California. Newman is also an ARS nutrition researchers are analyzing the metabolites in the liquid component of blood.

PEGGY GREB (D2478-3)

10 Agricultural Research O April 2012 serummetabolome. This search capability A peer-reviewed article, published in greatly speeds and simplifies its use by 5HJXODWRU\OLSLGPHWDEROLWHV 2011 in PloS One, documents the serum metabolome project. The research was researchers worldwide. ´DUHQXPHURXVDQGWKH\SOD\ led by David S. Wishart, who is with both The database lists the detected metabo- LPSRUWDQWUROHVLQWLVVXHJURZWK lites and, if known, their association with the University of Alberta and Canada’s DQGUHSDLUEORRGFORWWLQJ diseases. The quantities,or concentrations, National Institute for Nanotechnology. of metabolites are presented as ranges that LQӿDPPDWLRQSDLQSHUFHSWLRQ Funding was provided primarily encompass the values determined during DQGDSSHWLWHUHJXODWLRQ by the Canadian Institutes for Health the study and values from the curated 7KH\DUHFULWLFDOWRRXUKHDOWKµ Research, Alberta Advanced Education literature. ³-RKQ:1HZPDQ and Technology, Genome Alberta, Genome Analytical Methods: How Do They BC, the Alberta Ingenuity Fund, and the University of Alberta, but also by ARS, the Compare? help researchers select analytical tools in U.S. Department of Veterans Affairs, and The project “presented an invaluableop- the future.” the National Institutes of Health. portunity to learn more about the strengths Newman and his team are international The endeavor is part of the ambitious and shortcomings of various analytical authorities on nutritional metabolomics Human Metabolome Project, also led by platforms by comparing the metabolite and the use and improvement of advanced Wishart, which aims to document all of the coverage and values obtained from each,” technologies to detect and accurately metabolites in our fluids and tissue.—By says Newman. quantify lipids in blood, tissue, food, and Marcia Wood, ARS. Five different analytical approaches other materials. Hisgroup is interested were used in the project. Newman’s team in determining how the kinds and This researchis part of Human Nutrition, worked with two: UPLC-ESI-MS/MS concentrations of lipids in our bodies an ARS national program(#107) described (ultra-performance liquid chromatogra- are influenced by our eating habits, at www.nps.ars.usda.gov. phy-electrospray ionization-tandem mass physical activity patterns, and genetic John W. Newman is with the USDA-ARS spectroscopy) and GC-MS (gas chroma- and environmental factors and the relation Western HumanNutrition ResearchCenter, tography-mass spectroscopy). of these lipids to obesity and its adverse 430 W. Health Sciences Dr., University His lab’s analyses focused on lipid effects on health. The serum metabolome of California, Davis, CA 95616; (530) metabolites involved in regulating is, not surprisingly, a perfect tool for these 752-1009, [email protected]. biological processes. These metabolites are investigations. * formed by the body from the fats and oils in Researchers John Newman and Theresa Pedersen review a summary of lipid metabolites in blood. foods such as nuts, dairy products, meats, Our bodies form these metabolites from fats and oils in foods such as nuts, dairy products, meats, and fish. According to Newman, regulatory and fish. lipid metabolites “are numerous, and they play important roles in tissue growth and repair, blood clotting, inflammation, pain perception, and appetite regulation. They are critical to our health. “Our targeted analyses of these metabo- lites provided a swath of coverage that the other platforms used in this study didn’t capture,”hesays. “In particular, morethan 80 percent of the metabolites found on our UPLC-ESI-MS/MS platform were not detected by the other techniques.” Hesays thatthestudy “provides astrong starting point for determining the best future combinations of various analytical platforms to achieve maximum coverage of the serum metabolome. We know that some metabolites can be measured on mul- tiple platforms and that different platforms have different advantages. Some are less expensive, whileothersaremoresensitive, for example. We think our comparisons of results from different approaches will

©UC REGENTS/THOMAS USHING (D2471-1)

Agricultural Research O April 2012 11 KENT LOEFFLER (D2459-1) Close-up of greenbug aphid, 6FKL]DSKLVJUDPLQXP showing the piercing-sucking mouthparts it uses to Protein feed and inject virus into plants. and Other Field Crops Research Unit in Stillwater, Oklahoma. The Ithaca researchers found the field- Biomarkers collected aphids consistently transmitted yellow dwarf virus only when they carried most, if not all, of the nine key biomarker proteins. “The aphid does not need all nine to spread the yellow dwarf virus, but some are essential,”Cilia says. Identify The discovery in the lab was published in the March 2011 issue of the Journal of Virology, and the study confirming the Disease-Carrying biomarkers in the field was reported in Proteomics in June 2011. The findings mark the first time that Aphids protein biomarkers have been linked to an insect’s ability to transmit viruses, and the discovery is expected to lead to development of a test to identify potential disease vectors. Cilia and Gray are also collaborating phids can transmit viruses They knew from previous work that for on an effort to test whether the biomarker thaAt cause crop diseases and reduce aphids to pick up and transmit viruses, the proteins can predict disease-vectoring the quality and quantity of fresh foods. virus must be able to interact with specific ability in other insects. Collaborators Spraying insecticides can control aphids aphid proteins that direct movement of the include Michael MacCoss and Michael and reduce the spread of viruses, but virus through the insect and back into a Bereman at the University of Washington; spraying is expensive and can harm the plant during feeding. In laboratory stud- Alvin Simmons at ARS’s U.S. Vegetable environment. Additionally, not all aphids ies of greenbug aphids, they discovered Laboratory in Charleston, South Carolina; transmit viruses. So a key question for that the laboratory-raised insects’ ability and Lava Kumar and Rachid H anna at the growers is knowing when and what to to transmit yellow dwarf viruses could be InternationalInstituteof TropicalAgricul- spray to control viral diseases. predicted by the presence or absence of ture in Africa. The project is being funded Agricultural Research Service scientists nine different biomarker proteins in the by theNational Science Foundation Basic Michelle Cilia and Stewart Gray, in the insect cells. Research toEnableAgricultural Develop- Biological Integrated Pest Management To see if their lab findings would prove mentprogramthroughCornellUniversity, Unit at the Robert W. Holley Center for true in the field, they analyzed greenbug with support from the Bill & Melinda Gates Agriculture and Health in Ithaca, New aphids collected from cereal crops and Foundation.—By Dennis O’Brien,ARS. York, have found a way to distinguish noncultivated grasses around the United aphids that spread viruses from those that States by ARS colleagues John Burd and This research is part of Plant Diseases, don’t—by studying the aphid’s proteins. Melissa Burrows, of the Wheat, Peanut, an ARS national program (#303) described at www.nps.ars.usda.gov.

Left: Plant pathologist Stewart Gray and molecular biologist Michelle Cilia examine greenhouse plants To reach scientists featured in this for virus symptoms. Right: Greenbug aphid feeding on an oat leaf infected with yellow dwarf disease. article, contact Dennis O’Brien, USDA- ARS Information Staff, 5601 Sunnyside Ave., Beltsville, MD 20705-5129; (301) 504-1624, [email protected].*

KENT LOEFFLER (D2460-1) KENT LOEFFLER (D2458-1)

12 Agricultural Research O April 2012 JACK DYKINGA (K7878-1)

n ARS scientist in Ithaca, New YorkA, is using a new statistical approach to A New help speed the development of improved varieties of crops. Plant breeders constantly strive to breed Approach new varieties that yield more, resist emerg- ing pests and pathogens, tolerate heat and drought, and grow in marginal soils and environments. Increasingly, molecular tools are used to speed those efforts. By to Molecular identifying genesassociated with desirable traits, scientistsdon’t have to wait for time- consuming field observations. “To grow wheat and evaluate it for Plant traits in the field takes 5 to 9 years. Us- ing genomic data, we can do it in about 6 months,”says Jean-Luc Jannink, who is Breeding in the ARS Plant, Soil, and Nutrition Re- search Unit at the Robert W. Holley Center for Agriculture and Health on the Cornell To grow wheat and evaluate it for traits in the field takes 5 to 9 years. Using genomic data, ARS University campus, in Ithaca. scientists can do it in about 6 months. Though molecular tools speed up the Jannink’s approach exploits more technology, Jannink says.—By Dennis process, they also require analyzing mas- genetic data by including all of the small- O’Brien, ARS. sive amounts of data. Jannink has dem- effect QTLs and estimating the effects of This research is part of Plant Genetic onstrated how new statistical approaches all of the known genetic markers in a plant Resources, Genomics, and Genetic Im- should help plant breeders handle all that population. In a recent study, Jannink and provement, an ARS national program data. Analysis of the data enables “genomic his colleagues constructed statistical mod- (#301) described at www.nps.ars.usda.gov. selection” (GS) of untested breeding lines, els, using both GS and MAS approaches, Jean-Luc Jannink is with the USDA-ARS using predictions about which will perform and compared how well they could pre- Robert W. Holley Center for Agriculture best. Dairy cattle breeders have begun to dict the values of 13 agronomic traits in and Health, 538 Tower Road, Cornell use GS, and with Jannink’s help, it will crosses made from a “training population” University, Ithaca, NY 14853; (607) 255- catch on among plant breeders. assembled for the study. The accuracy of 5266, [email protected]. Even with modern molecular tools, the models was measured by comparing * breeders face challenging traits like their predictions with field observations of drought tolerance and high yield, which 374 lines of wheat, characterized by Mark result from the combined action of mul- Sorrells, a collaborator and wheat breeder tiple genes, each having a small effect. at Cornell University. In their study, Jan- These genes, called “quantitative trait nink and colleagues were able to use all loci” (QTLs), are stretches of DNA that the available data, including all of the trait affect an observable trait. The conventional data, along with the genomewide marker marker-assisted selection (MAS) approach data that captured the genetic values of has limited power when a trait is affected the wheat lines. by small-effect genes. The sheer volume The results, published in the March 2011 of data involved forces MAS to exclude issueof The Plant Genome,showed that the some small-effect QTLs when individual GS approach was more accurate at predict- plants are selected for further breeding. ing trait values than the MAS method was. Jannink reported similar success in another study, this time of oats, published in June 2011 in The Plant Genome. The work is expected to speed up molecular breeding efforts and might prove extremely useful, given the rapid pace of advances in DNA Wheat seed heads.

DOUG WILSON (K3945-7)

Agricultural Research O April 2012 13

B$SULOLQGG 30 (D2467-1) Made of water-soluble polymers, na- noscale clay particles, and other “green” ingredients, the ARS-TAMU flame retar- Cotton Gets dant is applied as a nanocoating that reacts to open flame by rapidly forming a swollen, charred surface layer. This process, known as “intumescence,” stops the flame from Nanotech and reaching underlying or adjacent fibers. Ateam led by Jaime Grunlan at TAMU’s Department of Mechanical Engineer- Biotech ing, in College Station, Texas, originally developed the intumescent nanocoating using a layer-by-layer assembly. In this procedure, alternating layers of positively and negatively charged ingredients, in- Treatment in cluding clay particles 50-100 nanometers wide, are deposited onto the surface of a desired material. The result is a striated nanocoating that, when viewed under a New Orleans scanning electron or other high-powered microscope, resembles the stacked layers of a brick wall. Condon’s interest was piqued after listening to Grunlan discuss his team’s research at a recent American Chemical cientists at the Agricultural Foreseeable applications range from the Society meeting, and he approached the ReSsearch Service’s Cotton Chemistry and purely functional—like better shrink re- TAMU professor about potential benefits Utilization Research Unit (CCUR) in New sistance—to the truly futuristic, such as to cotton. That conversation, in turn, led Orleans, Louisiana, have a long history of fabrics made of cotton-optical fiber blends to a cooperative research project enabling research successes leading to advances that can change color. Condon and Chang to evaluate the nano- in the use, manufacturing, and quality of Flame-Retardant Coating coating at CCUR. cotton fiber. In one ongoing project, Condon and Treating cotton for flame resistance isn’t For example, groundbreaking studies CCUR chemist SeChin Chang are col- arecentconcept, adds Condon, whose lab is led by chemist Ruth Benerito at the Cotton laborating with Texas A&M University part of the ARS Southern Regional Research Chemical Reactions Laboratory (CCUR’s (TAMU) scientists to evaluate a first-of- Center in New Orleans. In fact, some of predecessor), starting in the 1950s, gave its-kind, environmentally friendly flame the most successful early treatments were rise to easy-care, permanent-press clothing retardant for cotton apparel and durable born of research conducted by Benerito and other consumer-friendly improvements goods. and colleagues there several decades that helped cotton better compete with Halogenated flame retardants have been ago. (See “Cross-Linking Cotton,” synthetic fibers, like polyester and nylon. among the most widely used chemical Agricultural Research, New challenges and consumer demands treatments for cotton. But there’s been a February 2009, have since emerged, but the ARS lab’s push to find alternatives that are not only pp. 10-11.) tradition of excellence and innovation in more benign, but that also avoid imparting Condon research continues. the same stiffness to fabric characteristic of coauthored Under the leadership of Brian Condon, some chemical treatments. For these and a 2011 CCUR researchers today are leveraging other reasons, “the textiles industry would Top of page: the latest developments in nanotechnol- like to move away from using halogenated Cross-section of a ogy to bring cotton into the 21st century. flame retardants,” says Condon. cotton fiber with clay nanoparticles attached. Magnification about 11,000x, with transmission electron microscope. Right: Closer view of a clay nanoparticle coating.

Photos on this page courtesy of Jaime Grunlan, Texas A&M University. Colorization by Stephen Ausmus.

(D2466-1)

14 Agricultural Research O April 2012

B$SULOLQGG 30 BRIAN CONDON (D2462-1) ACS Nano paper on the potential of before application. “This is a leg intumescent coatings together with up for cotton over the synthetics,” Chang, Grunlan and his TAMU team, and which have not been amenable Alexander Morgan of the University of to silver nanoparticle treatment, Dayton Research Institute in Ohio. says Condon. Early trials of the nanocoating using From Nanotech to Biotech standard flame-resistance tests are promis- The CCUR scientists have also ing. In one case, 95 percent of treatedcotton been busy on the biotech front. fabric remained intact after exposure to In one project, CCUR chemist flame, whereas the untreated fabric used Vince Edwards, together with for comparison was completely destroyed Condon, devised atreatment for “What we’re investigating now is how impregnating nonwoven cot- well it will perform after repeated launder- ton fabrics with lysozyme, an ings oftreated fabric,”says Condon. “After enzyme that slices open the cell all, the coating contains clay, and that’s walls of microorganisms, killing something detergents aremade to remove.” them—including microbes that Even if the coating does eventually cause infection or odor. That same wash out and the treated fabric loses its cell-slicing capacity may also be Chemist Sunghyun Nam examines suspensions of flame resistance, the nanotech approach used for biodefense applications silver nanoparticles. Fabrics can be treated with these could still be used to protect textiles that deactivate nerve agents—es- nanoparticles to add antimicrobial qualities. and durable goods that aren’t frequently sentially by chewing them up, or washed, such as upholstery, mattress pads, “hydrolyzing” them, he adds. By adding also work to improve the conversion of box spring covers, automotive interiors, the lysozyme to cottons, the resulting cotton sugars into biofuels—a potential and firefighter coats. nonwovens could have these bactericidal value-added market for 2 million tons of Tapping Silver’s Antimicrobial Properties and detoxifying properties. U.S. cotton gin waste generated annually. On another nanotech front, Condon and In another biotech project, Condon and Besides publishing their findings in sci- CCUR engineer Sunghyun Nam are inves- CCUR chemist Michael Easson are ex- entific journals, Condon’s team is actively tigating a way to inhibit microbial growth perimenting with equipment that generates seeking commercial partners to help usher in cotton using silver particles ranging in an ultrasonic field of mechanical energy these advances into the marketplace—all size from about 2 to 6 nanometers. Silver (similar to that used to clean jewelry) to with an eye towards assuring the viability nanoparticles have been used as an anti- improve the biobased processing of raw, of America’s $25 billion cotton industry microbial agent in many products, includ- or “greige,” cotton using enzymes, like at a time of increasing production costs, ing clothes, plastic food containers, and cellulase. Chemical processing agents are dwindling resources, and global competi- medical textiles. The methods of producing now used to strip away waxes, pectins, fats, tion.—By Jan Suszkiw, ARS. them, however, have mostly relied on the and other fiber components that can hinder This research is part of Quality and use of toxic agents and organic solvents. subsequent dying procedures and diminish Utilization of Agricultural Products, an Condon’s team has explored an alterna- the quality of finished cotton products. But ARS national program (#306) described tive approach using an environmentally the waste these “wet chemistry” methods at www.nps.ars.usda.gov. generate is a concern. friendly agent, polyethylene glycol, and Brian Condon is in the USDA-ARS Cot- water as a solvent to generate silver “We’re interested in enzymes as an ton Chemistry Research Unit, Southern nanoparticles of the desired size. Condon environmentally friendly alternative, and Regional Research Center, 1100 Robert E. and Nam reported on this method in the we found that subjecting a solution of the Lee Blvd., New Orleans, LA 70124; (504) Journal of Nanoparticle Researchtogether enzymes to a field of ultrasonic energy can 286-4540, [email protected]. with engineer Dharnidhar Parikh, formerly speed up their reaction rates,”says Condon. * with CCUR. Also under investigation is a In experiments with lint fibers, for ex- Following flame exposure, the untreated fabric (top) is completely destroyed, but the fabric new method by which the nanoparticles ample, use of ultrasonic energy increased treated with clay nanoparticles is intact. form directly on cotton fiber, eliminat- the bioprocessing efficiency of cellulase ing handling and storage of the particles by 22 percent. The same concept can

SeCHIN CHANG (D2465-1)

Agricultural Research O April 2012 15

B$SULOLQGG 30 PEGGY GREB (D1518-1) Adults of the native carabid beetle /HELDJUDQGLV are voracious predators of Colorado potato beetle eggs and larvae.

Barcoding four insects that previous studies showed were the most common potato beetle predators. They fed them laboratory-raised Insects potato beetles and looked at the digestion rates of each of the four insects to determine the Colorado potato beetle’s DNA “half life”—defined as the point at which at least some DNA of the potato beetle could still be found in half of the fed individuals of each predator species. They used the potato beetle’s barcoded DNA to detect it in the predators’ guts. The results, published in the journal EntomologiaExperimentalis et Applicata, show the importance of taking digestion rates into accountwhen considering differ- To Control Them ent insect predators as biocontrol agents. They may also provide guidance to growers on the most effective control strategies for combating a voracious pest. “Different pesticides have different ention barcodes and it often damaging insect pest of potatoes in the effects on different predators, and not all briMngs to mind the sales tags and scanners eastern United States. It also damages predators are equally susceptible to all found in supermarketsand otherstores. But tomatoes and peppers and is known for insecticides. Based on what you learn, Agricultural Research Service scientists developing resistance to any pesticides you might delay spraying insecticides, are using “DNA barcodes” in their search used to control it. rule out the use of insecticides that harm for waysto control and monitor insects that Greenstone is trying to find the insects your most important biocontrol agents, or pose the greatest threats to crops as diverse that are the beetle’s worst nightmare. limit spraying to certain times, depending as wheat, barley, and potatoes. Numerous studies have analyzed the on the predator’shabits,”Greenstone says. In DNA barcoding, scientists sequence gut contents of predator insects to ARS researchers are also using barcod- a designated part of an organism’s genome evaluate their ability to control pests. ing to understand and track the threat of and produce a barcode from it for a sys- But predators eat and digest prey at various biotypes of Russian wheat aphid, an tematic comparison with the sequenced different rates, so simple gut analysis is insect about the size of a sesame seed, that DNA of other closely related species. insufficient for accurately comparing is a major worldwide pest of wheat, barley, DNA barcodes are being developed on a predator effectiveness, Greenstone says. and other cereals. Since it appeared in 1986 wide range of plants and animals as part He has fine-tuned that approach and used in Texas, it has cost U.S. wheat growers of a global effort to catalog the diversity barcoding to come up with a way to factor alone about $200 million each year. of life on Earth. in how quickly different predatory insects Gary Puterka, an entomologist in the At the Invasive Insect Biocontrol and Be- actually digest the Colorado potato beetle. ARS Wheat, Peanut, and Other Field Crops havior Laboratory in Beltsville, Maryland, “Scientists often use barcoding to dis- entomologist Matthew Greenstone is using tinguish one closely related species from Russian wheat aphid adult next to its young. This DNAbarcodes in an unconventional way: another. We’re using it to identify prey in aphid gives live birth to young that are identical clones of itself during the asexual phase of its to identify insect predators best equipped the gut of an insect predator, and in a sense, lifecycle. to control the Colorado potato beetle. The that’s an atypical use,” says Greenstone. Colorado potato beetle is the single most Greenstone and his colleagues collected

GARY PUTERKA (D2491-1)

16 Agricultural Research O April 2012

B$SULOLQGG 30 PEGGY GREB (D2486-1) Research Unit in Stillwater, Oklahoma, periodically surveys Russian wheat aphid populations across eight western states to provide guidance to wheat growers on infestation levels and on the range of biotypes so they can decide whether to implement control measures. He works closely with ARS entomologist Kevin Shufran, who is also in Stillwater, on efforts to control the aphid. Until recently, the Russian wheat aphid, in North America, was believed to reproduce strictly by asexual cloning, which made it particularly susceptible to resistance mechanisms bred into wheat and barley crops. But Puterka’s surveys have turned up evidence showing that it is reproducing sexually. If the Russian wheat aphid is sexually reproducing, the result- ing genetic recombination would produce new “bioytpes” that will be better equipped to counter resistant crops by giving each new generation a varied genetic tool kit, increasing the likelihood that offspring will be able to overcome the plant’s resistance mechanisms, feed on it, and survive to reproduce resistant off- Entomologist Matt Greenstone examines DNA analysis results of Colorado potato beetle removed from the gut of an insect predator spring. Sexual reproduction could also broaden the pest’s range, collected from the field. He is looking for evidence that the predator enabling it to lay eggs as cooler weather approaches, like other mainly consumes Colorado potato beetle. alpine aphids do, and survive harsh winters. that it would work, they extracted DNA from the eggs of 10 Researchers found a new biotype of Russian wheat aphid in previously identified species of aphids, including several of the Colorado in 2003 that spread so quickly and caused so much Russian wheat aphid’s closest relatives. They sequenced the damage—overcoming a new resistance gene that breeders had first 640 base pairs of a gene known as “CO1.” In a blind test, recently developed for wheat growers—that its success was Shufran compared DNA from eggs provided by Puterka, who seen as a new introduction or evidence that the pest had “gone masked the identities of the different species. With help from sexual.” Subsequent surveys have turned up additional evidence various aphid genetic databases, Shufran was able to correctly of new biotypes, an evolving threat. distinguish the different aphid species by comparing their CO1 Puterka’s survey showed thatthe Colorado biotype now makes sequences. Results were published in Annals of the Entomologi- up 90 percent of the population in eight major wheat-producing cal Society of America. states. Another study by Puterka found a small, localized popula- With the new tool, Puterka and Shufran will be able to identify tion of Russian wheat aphids in a Colorado field that were the Russian wheat aphid eggs for the first time and can better track offspring of male and female parents. This population of nymphs the biotypic diversity of an aphid that poses a major threat to had a signature trait—missing antenna segments—that was wheat and other crops.—By Dennis O’Brien, ARS. evidence they hatched from eggs and were the result of sexual reproduction. Thirty-nine new biotypes were detected when This research is part of Crop Protection and Quarantine, an these nymphs were increased and screened. ARS national program (#304) describedat www.nps.ars.usda.gov. “These populations are continually shifting in terms of dif- To reach scientists mentioned in this article, contact Dennis ferent biotypes, and as natural selection dictates, the biotypes O’Brien, USDA-ARS Information Staff, 5601 Sunnyside Ave., that are the fittest are the ones that will survive and dominate,” Beltsville, MD 20705-5129; (301) 504-1624, dennis.obrien@ says Puterka. ars.usda.gov.* But the extent of the threat posed by the Russian wheat aphid largely remains a mystery, and the field surveys are time consum- ing. For accurate results, Puterka must make clone colonies from each adult collected and screen them by exposing them to nine different types of resistant germplasm to confirm their biotype. The most efficient way to determine whether Russian wheat aphids are sexually reproducing would be to find their eggs. When the females reproduce asexually, they give birth to live females, but they lay eggs when they reproduce sexually. The problem is that the eggs of all aphid species look alike, so the scientists cannot distinguish Russian wheat aphid eggs from other aphids’ eggs. Shufran and Puterka have developed a process that uses DNA Adult Colorado potato beetle on a potato plant. Adults and larvae barcoding to tell the different aphid eggs apart. To establish eagerly consume potato foliage.

PEGGY GREB (D1515-1)

Agricultural Research O April 2012 17

B$SULOLQGG 30 SCOTT BAUER (K7038-1) (polymerase chain reaction) test to detect these novel parvoviruses in commercial poultry flocks. Unlocking a Treasure Trove With help from industry producers and veterinarians, microbiologist Michael Day and research leader Laszlo Zsak, in SEPRL’s Endemic Poultry Viral Diseases Research Unit, collected intestinalsamples from five different turkey flocks affected by enteric disease. To identify and char- Metagenomics acterize viruses using metagenomics, they prepared intestinal homogenates from the samples. The homogenates were filtered to remove larger constituents, like bacteria, Offers Insight and leave the smaller particles, like viruses. Metagenomics techniques were then used to sequence nucleic acid of all the RNA Into Poultry Diseases viruses present in the samples. “I was expecting to find RNA sequences from viruses that had not been described before in the poultry gut,”Day says. “It turned out that there were quite a number of viruses in that particular sample.” ess than 10 years ago, the world against enteric diseases for scientists at the Acomparison to similar viruses in com- maLrveled at the completion of the human Agricultural Research Service Southeast puter databases showed that the intestinal genome project, which involved traditional Poultry Research Laboratory (SEPRL) in virus metagenome contained thousands of technology to identify all the genes in a Athens, Georgia. Disorders like poult en- pieces of nucleic acid representing many single organism—the human. Today, a teritis mortality syndrome, poult enteritis groups of known and unknown turkey more powerful technology is being used complex, and runting-stunting syndrome viruses. Common avian viruses such as to detect thousands of organisms in an cause diarrhea in birds, resulting in de- astrovirus, reovirus, and rotavirus were entire community. creased weight, mortality, and increased confirmed. Many RNA viruses, like mem- Unlike traditional gene sequencing, production costs. bers of the Picornaviridae family, were the new molecular technique—metage- Research has revealed that several also detected. nomics—eliminates the need to cultivate viruses may be responsible for enteric dis- An unexpected discovery was an and isolate individual microbial species. eases, yet a single causative agent has not abundance of previously unknown turkey Scientists can apply genomic analysis to been identified. Metagenomics research viruses, such as picobirnavirus, a small, mixed communities of microbes instead may help solve that mystery. of to just one organism. Scientists at SEPRL are using meta- For example, researchers examining genomics to uncover vast amounts of viral enteric (intestinal) diseasesin poultry known and previously unknown viruses can take intestinal samples from different in poultry. They have discovered and poultry flocks. The material can be pro- sequenced the complete genome of a cessed to sequence all the viral nucleic new bacteriophage (phage) that might acid—RNAand DNA—in the sample and have future antimicrobial applications, then analyzed as a single genome. described for the first time the complete Learning more about how genes inter- genome of new chicken and turkey In studies of act is extremely important in the battle parvoviruses, and developed a PCR intestinal samples from turkeys with enteric diseases, ARS scientists have discovered a new virus that may have future antimicrobial applications.

SCOTT BAUER (K7043-16)

18 Agricultural Research O April 2012

B$SULOLQGG 30 double-stranded RNA virus implicated those enzymes in an organism generally treatments, and improve management in enteric disease in other agricultural recognized as safe, like yeast for example, practices to help control costly animal and animals, Day says. A calicivirus—the kind we can put them in feed to help reduce plant diseases, Day says. associated with human enteric diseases— certain types of bacteria that cause disease.” The beauty of metagenomics is that was also identified in poultry. Chipping Away at Chicken Viruses viruses do not have to be isolated or Prospects of a Novel Phage In earlier studies, Zsak and Day used identified. Small pieces of nucleic acid “Because metagenomics is so powerful, metagenomics to identify and analyze the can be sequenced from samples taken we generated and continued to analyze genome of a novel chicken parvovirus, from mixed communities—a process that additional data from these samples and ChPV ABU-P1. allows scientists to discover new enzymes discovered a new bacteriophage,” Zsak “This was the first in-depth characteriza- and proteins and look for genetic markers says. “Until now, no one had described this tion and analysis of the full-length genome for disease-resistant traits or genes with kind of phage in turkey enteric samples.” sequence of the chicken parvovirus,”Day possible antimicrobial applications. The virus, called “phiCA82,”belongs says. “Comparisons were made to other “We need some way to understand a to a group known as “microphages”and members of the Parvovirinae subfamily community and interrogate the nucleic is the type of virus that naturally kills that infect mammals and birds.” acids in that community to see who’s there bacteria, Zsak says. Phages are important Scientists also developed a PCR assay and what they’re doing,”Oakley says. because they can potentially be used as to detect the virus in turkeys and chickens “Are there pathogenic bugs in there? Are alternatives to antibiotics and as weapons and used thetestto examineentericsamples there genes associated with pathogenesis? against multi-drug-resistant pathogens. collected fromU.S. commercialturkey and Metagenomics does that.”—By Sandra Zsak and Day found a short sequence of chicken flocks across different regions. Avant, ARS. the phage DNA and designed a technique “PCR proved to be highly sensitive and This research is part of Animal Health to sequence its entire genome. Colleagues specific in detecting parvoviruses in both (#103) and Food Safety (#108), two ARS Brian Oakley and Bruce Seal, both micro- clinical samples from infected birds and national programs described at www.nps. biologists in the Poultry Microbiological field samples from turkeys and chickens ars.usda.gov. Safety Research Unit of the ARS Richard with enteric diseases,”Zsak says. To reach scientists mentioned in this B. Russell Agricultural Research Center, Advantages of a Community Approach article, contact Sandra Avant, USDA- also in Athens, helped analyze the data. The overall goal is to use metagenomics ARS Information Staff, 5601 Sunnyside One task was to find out whether the new technology to develop and update Ave., Beltsville, MD 20705-5129; (301) phage was related to similar viruses. diagnostic tools, identify effective new 504-1627, [email protected]. “That’s a question you would have with * the discovery of any new kind of organ- Microbiologist Michael Day examines the validation results of a new molecular diagnostic assay for a ism,”Oakley says. turkey picobirnavirus. Day used a metagenomic approach to detect the novel picobirnavirus RNA in Oakley downloaded all publicly turkeys experiencing enteric (intestinal) disease. available viral genome sequences and used bioinformatics—the application of computer science and information technology to the field of biology—to compare the newly discovered genome to previously discovered ones. The comparisons revealed that the new genome was unique. “Future studies need to be completed to find out if phages like this actually kill the bacteria they infect,” Zsak says. “Once we can identify this mechanism, we can design identical ways to attack and kill these pathogens.” Phages infect bacteria and then replicate, Seal explains. They do this by digesting the cell walls of bacteria. “We are interested in being able to clone the gene that expressesenzymes that digest the cell wall,”Seal says. “If we can express

LAURA FERGUSON (D2470-1)

Agricultural Research O April 2012 19

B$SULOLQGG 30 LYNN BETTS (NRCSIA99126) The core function of RUSLE2 is to estimate soil eroded by the impact of raindrops and by the flow of runoff water across land disturbed by plowing and tilling. Erosion caused by concentrated ephemeral gully flow in Long-Standing topographic swales is not currently included in RUSLE2 predictions but will be in the future.

factor derived from paper tables of figures Erosion for different parts of the country and dif- ferent soils. Proof of the worth of USLE’s humble pencil-and-paper calculation is Calculator that it is still embedded as the heart—and anchor—of the new, sophisticated model that houses it, although the tables of factors no longer exist. Its corefunction is to estimate soileroded by the impact of raindrops and by the flow st of runoff water across land disturbed by Enters 21 Century plowing and tilling. But functions have been added over the years, such as esti- mating how much plant residue can be removed from crop and pasture lands for ethanol production without exposing the soil to excessive erosion. And the equation continues to add capa- eth Dabney is busy tweaking a database, and scientific knowledge, makes bilities. It is now used to estimate erosion soonS-to-be-unveiled update of the Revised it an excellent example of computational wherever land is disturbed, whether by Universal Soil Loss Equation, Version science and technology. farming or ranching, pasture replanting, 2 (RUSLE2), which moves the original But RUSLE2 remains first and last what or nonfarm activities such as construction, equation ever further from its origins in the it was always designed to be: a management mining, or clearcutting and road building age of slide rules to the era of computing. tool that allows conservationists to make in forests. Dabney is research leader of the Water- better management decisions. Since the USLE moved into widespread shed Physical Processes Research Unit, This state-of-the-art technology traces use in the 1960s and 1970s, every conser- at the Agricultural Research Service’s its heritage to thepaper-and-pencil version vation plan written for farmers, ranchers, National Sedimentation Laboratory in ofUSLE,whichhas beenthe standardway and others by the USDA Natural Resources Oxford, Mississippi. to estimate soil erosion on farms for more Conservation Service (NRCS) has been RUSLE2 has retained the integrity of than four decades. It was first developed in based on soil-erosion calculations derived the original Universal Soil Loss Equation the late 1950s, before calculators, let alone from this equation. It is used by all 3,000 (USLE)—in greatly expanded form—and computers. In fact, at one point the equation NRCS field offices as well as the agency’s integrated an updated database with a com- came with plastic slide rules custom made state and area offices. puter model that reflects both the latest in for it. The USLE is now recognized as one Ken Renard, a hydraulic engineer who computer technology and scientific discov- of the most significant developments in soil came to ARS in 1957 and has been a eries abouterosion processes. Itis unlikely and water conservation history, worldwide. leader in the development of the USLE that there is a more powerful, proven, Standing the Test of Time and RUSLE even in retirement, saysthat practical computer model than RUSLE2. The USLE began as a fairly simple as “research made a lot of progress in more Its ability to predict daily erosion related equation that gave its answer in tons of realistically describing erosion fromfarm- to any human activity anywhere in the na- soil lost per acre per year by multiplying ing operations,” the need to update USLE tion, based on a host of conditions, through five factors, with the numbers for each technology became clear. a combination of simulation model, vast

20 Agricultural Research O April 2012

B$SULOLQGG 30 The result was RUSLE1, which started growth. The model Dabney is working on that although RUSLE2 is still missing these moving into use in 1992; it was the first takes that into account. gullies, Dabney is moving in the direction wholesale reworking of the erosion-pre- A New Way To View Erosion: But Ephem- of solving that. dicting technique using digital computer eral Gullies Still Elude Ferruzzi says NRCS can use this new technology. Dabney also added a way to model the modeling technique to pinpoint where The Improvements Continue erosive flow of water on an entire hill permanent gullies might form and suggest Spearheaded by now-retired ARS slope as one block, from top to bottom, grassed waterways or other conservation agricultural engineer George Foster, missing—for now—only the topographic measures there to prevent gully formation. RUSLE2 is actually a combination of swales where water runs intermittently A Crown Jewel observation- and process-based science, when it rains heavily and forms ephemeral Yoder callsthe extensive NRCS database incorporating its original roots in collected gullies. The current version also predicts a “crown jewel of RUSLE2.” Federally field data with the latest in computer where the eroded soil will collect after it supported data collection began with 10 models that can simulate processes leaves the hillside, something particularly experiment stations in 1929, located such as erosion, says Giulio Ferruzzi, a important to construction planners. strategically to represent as many crops conservationagronomist at the NRCS Renard says that ephemeral gullies often and climate and management conditions West National Technology Support Center cause the most erosion on farms. They’re as possible. NRCS has since built it up to in Portland, Oregon. Ferruzzi forwards all particularly insidious because they are include every state in the nation plus the of the agency’s requests for changes to the obliterated by tillage, but reappear during territory of Puerto Rico and the Pacific RUSLE2 to Dabney and Daniel Yoder, a a wet spell the next season. Renard says protectorates. professor at the University of Tennessee at Knoxville. At the timeRUSLE1 moved into use, the Ephemeral gullies like this one in a task of improving its computer programs central Iowa field fell to Yoder, who, while a graduate student, often cause much worked for ARS as an agricultural engineer erosion on farms. at the National Soil Erosion Laboratory These gullies seem to disappear in West Lafayette, Indiana, at Purdue after tillage but University, the birthplace of the equation. then reappear Now, 20 years later, Yoder is still improv- during rain events. ing the RUSLE2 version, first developed in 2001. RUSLE2 brought the equation into the 21st century, making it a much more powerfultool, yet much more user-friendly, with graphics, including icons for things like bulldozers and tractors. Yoder says, “I take Dabney’s science ideas and translate them into computer code. But the process is give-and-take between me, Dabney and his ARS colleagues, and NRCS.”He saystheir recent work on improvements to RUSLE2 involved better prediction of crop residue. Ferruzzi adds that Dabney for the first time made it possible for NRCS to help ranchers more accurately learn the effects of cattle grazing on erosion. The update about to be unveiled simulates the natural life and death of pasture plants as well as the effects of the different eating habits of grazing animals. Ferruzzi explains that when cows are let into a pasture for a short time, they tend to eat everything in sight by “mob grazing.”But when cows are put in a pasture for a season, they have time to be more selective, eating only the freshest

LYNN BETTS (NRCSIA99140)

Agricultural Research O April 2012 21

B$SULOLQGG 30 ARS NATIONAL SEDIMENTATION LABORATORY (D2464-1)

Spearheaded by now- retired ARS engineer George Foster, seen here in the late 1960s or early 1970s, RUSLE2 is a combination of observation- and process-based science, incorporating itsoriginal roots in extensive field data into a state-of-the- art computer-based conservation tool.

Dabney agrees, saying that the NRCS management zones. The scenarios replace The database contains 20 key soil prop- database is exhaustive, with 30,000 USLE’s key crop management factor. erties that affect soil erosion for all soils combinations of vegetation, farming “RUSLE2 now creates these scenarios. It in the United States. It also contains the and ranching operations, and residue goes way beyond USLE and RUSLE1,” climate for all counties, the precipitation types. “NRCS developed this database says Lightle, who continues to work part- ranges across the country, and hundreds at regional centers in Nebraska, Oregon, time on RUSLE2 in retirement. Linda of tillage options. RUSLE2 provides daily South Carolina, and Texas, and it has Scheffe,attheNRCS National Soil Survey information, unlike USLE, which provided been in use for 10 years now,”Dabney Center in Lincoln, Nebraska, is the current information on a yearly basis. says. NRCS has also developed maps for RUSLE2 database manager. Lightle says that thisdatabase is the most RUSLE2 that divide the country into crop sought-after feature of RUSLE and is be- management zones. The maps disregard ing used in other models. Currently, users political boundaries, instead delineating have to download soil data from the NRCS zones based on common crops and 586/(FDQ National Soil Information System, the management practices. agency’s official soil database. But Lightle Dabney says that it is this very database RSHUDWHDFFXUDWHO\ says that in the near future RUSLE2 will that keeps RUSLE2 from coming unteth- DQ\ZKHUHLQWKH be live on the Internet, enabling access to ered from reality, as pure process models that data without downloading.—By Don do when they operate outside of known 8QLWHG6WDWHV³ Comis, formerly with ARS. territory. RUSLE2 can operate accurately This research is part of Climate Change, anywhere in the United States—and prob- DQGSUREDEO\WKH Soils, and Emissions (#212) and Water ably the world—because it is grounded ZRUOG³EHFDXVH Availability and Watershed Management by the vast database collected from field (#211), two ARS national programs de- measurements. LWLV JURXQGHGE\ scribed at www.nps.ars.usda.gov. Dave Lightle, former NRCS national WKHYDVWGDWDEDVH To reach scientists mentioned in this database manager for RUSLE2, worked story, contact Robert Sowers, USDA-ARS for 10 years to produce the database, with FROOHFWHGIURP Information Staff, 5601 Sunnyside Ave., the different combinations of manage- ӾHOGPHDVXUHPHQWV Beltsville, MD 20705-5129; (301) 504- ment scenarios broken down by the crop 1651, [email protected].*

22 Agricultural Research O April 2012

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Agricultural Research O April 2012 23

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