Living with both bad and beneficial Microbes...Andy Nyman 31 Ruminants and Their Rumen Microbes...

Vol. 58, No. 4, Fall 2015Vol. 58, No. 4, Fall 2015Vol. 58, No. 4, Fall 2015

Living with both bad and beneficial

Microbes

West Nile Viruspage 10

GMO Safetypage 18

Healthy Human Gutpage 20

EDITORIAL BOARD: John S. Russin, Chairman Linda Foster Benedict Michael Blazier Rick Bogren Melissa Cater Glen T. Gentry Kurt M. Guidry Dustin Harrell Claudia Husseneder Kathy Kramer Megan La Peyre

EDITOR: Linda Foster BenedictASSOCIATE EDITOR: Rick BogrenDESIGNER: Kathy Kramer CONTRIBUTORS: Tobie Blanchard, Elma Sue McCallum, Olivia McClure and Johnny MorganWEB DESIGN: Ronda Clark and Kathy Kramer

Louisiana Agriculture is published quarterly

by the Louisiana Agricultural Experiment Station. Subscriptions are free. You may also subscribe to a Web version of the magazine, which is available at www.LSUAgCenter.com. Please go to the “Louisiana Agriculture Magazine” site if you would like to receive an email notification when a new issue is online. If you would like to download the magazine to your e-reader, go to the magazine’s website, choose the correct format, and follow the directions on your mobile device. For more information or to subscribe, please contact: Linda Foster Benedict, Editor Louisiana Agriculture 115 Knapp Hall 110 LSU Union Square Baton Rouge, LA 70803 (225) 578-2263 [email protected]

William B. Richardson, LSU Vice President for AgricultureLouisiana State University Agricultural Center

Louisiana Agricultural Experiment StationLouisiana Cooperative Extension Service

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The mention of a pesticide or use of a trade name for any product is intended only as a report of research and does not constitute an endorsement or recommendation by the Louisiana Agricultural Experiment Station, nor does it imply that a mentioned product is superior to other products of a similar nature not mentioned. Uses of pesticides discussed here have not necessarily been approved by governmental regulatory agencies. Information on approved uses normally appears on the manufacturer’s label.

2 Louisiana Agriculture, Fall 2015

Microbes: Good, Bad and Overall FascinatingClaudia Husseneder

They go by many names – germs, bugs, microbes and microorganisms. The tiny organisms, which are usually invisible to the naked eye, include bacteria and archaea (both single-cell organisms that lack cell nuclei), protists (single cell algae, slime molds and protozoa that contain nuclei) and fungi. Viruses are considered “organisms at the edge of life” because they do not fit the classical definition of life. Viruses possess genetic material and reproduce; however, they do so by hijacking the metabolism of a host cell. Nevertheless, they are important plant and animal disease agents, and the viruses infecting bacteria (bacteriophages) are increasingly touted as tools for manipulating bacteria communities without the use of antibiotics. Therefore, viruses deserve their place among the microbes featured in this issue of Louisiana Agriculture.

Microbes are labelled with many superlatives. They are the tiniest and simplest organisms, yet the oldest life form on this planet. If the Earth were 24 hours old, microbes would have appeared at 5 a.m., dinosaurs at 10 p.m. and humans just seconds before midnight. Microbes had sole dominion over the Earth for about 3 billion years, and they are now found almost everywhere. They have successfully conquered every habitat from 7 miles deep in the ocean to 40 miles high in the atmosphere and from hot geysers to the Antarctic ice. Microbes are survivalists. They thrive in the vacuum of outer space and live through nuclear blasts; a few are resistant to almost every known antibiotic. They are Earth’s most abundant organisms. For every human on this planet, there are 10 million trillion microbes. The human gut alone contains 10 times more bacteria than cells in the human body.

Most people regard microbes as something negative to recoil from. However, less than 5 percent of microbes cause disease, and most play vital roles in many processes of life. Simply put, humans cannot live without microbes, not only because they are everywhere, but also because they are beneficial.

The LSU AgCenter is engaged in microbial research and diagnostics to combat disease, increase agriculture productivity and improve the health of our citizens and our environment. Because pathogenic microbes might pose a risk of infection, strict safeguards are in place for microbial research and application. Two committees oversee these activities. The Inter-Institutional Biological and Recombinant DNA Safety Committee follows federal guidelines for review and approval of all teaching, diagnostic, research and extension activities that involve potentially hazardous biological materials. The Institutional Review Board reviews research with human subjects to ensure their protection.

Pathogenic viruses, bacteria, protozoa and fungi are the cause of many infectious diseases of humans, animals and plants. This Louisiana Agriculture focus issue features articles on the impact and fight against microbial diseases that infect Louisiana’s crops and livestock and sometimes its citizens. The issue includes diagnostics and treatment of emerging plant diseases, fish disease, and disease threatening cattle and deer as well as detection and prevention of food-borne diseases. Special attention has been given to insects that are vectors for transmitting microbial diseases in plants, animals and humans. Fortunately, microorganisms also provide innovative and safe solutions for pest control. Microbes can be used as Trojan horses to deliver insecticidal and antimicrobial peptides. Proteins derived from bacteria – such as the Bacillus thuringiensis (Bt) toxin – are frequently used in crop protection and mosquito larvicide.

Despite the fear that microbial pathogens instill in us, it is important to keep in mind that most microbes play beneficial roles and that life without them is impossible. Microbes are responsible for nutrient cycling in the ecosystem; they are decomposers and fixate nitrogen from the air to make it available for plants. Microbes support fertile soil conditions and create compost. Microbial fermentation is used in food and beverage production – baking bread, making pickles and brewing beer, for example. Oxidative capabilities of microbes are harnessed for treatment of sewage and for oil spill cleanup. Microbes are the workhorses in energy production, turning agricultural and urban waste into biogas and biofuel; they are used in biochemical production, making organic acids and bioactive molecules like enzymes and vitamins. Microbes are genetic tools and expression systems to mass produce proteins. Last, but not least, almost all plants and animals, including humans, rely on microbial symbionts that live in tight association with their hosts to aid them in fending off disease, supplementing nutrition and facilitating energy metabolism.

Microbes have a powerful story to tell. Let’s put these tiny organisms under the microscope (pun intended) and examine their contributions to our world as the good, the bad and the overall fascinating.

Claudia Husseneder is a professor in the Department of Entomology and the lead scientist for this issue of Louisiana Agriculture.

Louisiana Agriculture, Fall 2015 3

2 Microbes: Good, Bad and Overall FascinatingClaudia Husseneder

4 AgCenter News6 College of Ag News8 Termite Gut Microbes: Tools and Targets for Control

Claudia Husseneder, Chinmay V. Tikhe, Lane Foil and Chris Gissendanner

10 Mosquitoes and West Nile VirusKristen Healy, Emily Boothe and Nicholas DeLisi

12 Norovirus and Oysters in LouisianaNaim Montazeri, Morgan Maite and Marlene Janes

14 Studying Rice-Microbe-Insect Interactions to Increase Rice ProductionLina Bernaola and Michael Stout

16 On-farm Food Safety Research Helps Louisiana Growers Comply with New Law Achyut Adhikari

18 Bt technology: A Major Advancement in Insect Pest ControlDavid Kerns

19 Internal Regulations and Safeguards for Biological MaterialsKenneth R. Bondioli

19 Protection for Human SubjectsMichael J. Keenan

20 Resistant Starch Fermentation and Human HealthMichael J. Keenan

21 Scientist Helps Ensure Safety for LSU AgCenter Food Incubator ProductsOlivia McClure

22 Plant Pathogens Threaten the Louisiana Plant WorldLawrence E. Datnoff

24 Linking Soil Microbes and Ecosystem FunctionsLisa Fultz

25 Use of Microbes As Expression SystemsTed Gauthier

26 Plant Diagnostic Center Serves LouisianaRaj Singh

27 Rose Disease Found for First Time in LouisianaJohnny Morgan

28 Studies on the Transmission of Insect-borne Viruses That Cause Hemorrhagic Disease in Deer, CattleLane Foil, Michael Becker, Willie Andrew Forbes, Glen T. Gentry, James M. LaCour, Stephanie R. Ringler and Jonathan L. Roberts

30 Use of Microbes in Oil Spill CleanupAndy Nyman

31 Ruminants and Their Rumen MicrobesGuillermo Scaglia

31 Using Microbes to Fight Disease in CatfishRon Thune

32 Pending FDA Rules Could Increase Demand for AgCenter-DevelopedCattle VaccineRick Bogren

33 Prevent Foodborne Illness at HomeWenqing Xu

34 AgCenter Scientists at the Forefront of Brucellosis EradicationSue D. Hagius and Philip H. Elzer

35 Using Microorganisms in the Manufacture of Dairy FoodsKayanush J. Aryana and Luis Vargas

Vol. 58, No. 4, Fall 2015Published Since 1957

ON THE COVER: Photo illustrations of microbes were provided by Ying Xiao, research associate in the Department of Biological Sciences, and courtesy of the LSU Socolofsky Microscopy Center, Shared Instrumentation Facility, Institute for Advanced Materials.


AgCenter News22 awarded Master Cattleman status

Graduates of the Louisiana Master Cattleman program were recognized on Oct. 8 during a field day at the Dean Lee Research and Extension Center in Alexandria. LSU AgCenter regional beef coordinator Vince Deshotel said 59 people have graduated in the past two years from the program in the LSU AgCenter Central Region. The program involves 10 sessions covering a wide range of material for beef production. From left, kneeling, Glen Rideau, Joe Doucet, Chris Douget, Tom Ardoin, AgCenter Central Regional Director Boyd Padgett, Scott Aymond, Danny Miller, Dwayne Landreneau; standing, Deshotel, Randy Beauboeuf, interim director of the AgCenter School of Animal Sciences Christine Navarre, Carol Bliss, Klaire Fontenot, Wesley Coffman, Russell Miller, Terry Latiolais, David Morris, Terry Ardoin and Robert Duncan. Not shown are Michael Fontenot, Shane Freeman, Tara Freeman, Ted Freeman and Jay Guidry. Photo by Bruce Schultz

New plants hot topic at Hammond field day

More than 335 people attended the Landscape Horticulture Field Day and the Southeast Louisiana Nursery Association Trade Show on Oct. 8 at the Hammond Research Station. LSU AgCenter horticulturist Yan Chen, at right, discusses different varieties of caladiums. Photo by Johnny Morgan

Facility renamed H. Rouse Caffey Rice Research Station

Family, friends and former co-workers gath-ered Nov. 4 for the renaming of the LSU AgCen-ter Rice Research Station for the late H. Rouse Caffey in recognition of his dedication to the rice industry and Louisiana agriculture.

Several facilities could have been chosen to honor Caffey’s name because of his work with numerous agricultural research facilities, said Bill Richardson, LSU vice president for agriculture and dean of the College of Agriculture, who suc-ceeded Caffey as LSU AgCenter chancellor. “The Rice Research Station was nearest and dearest to his heart,” he said.

Farmer Jackie Loewer, chairman of the Loui-siana Rice Research Board, said without the sta-tion, the rice industry would not exist in Louisi-ana today. “Without Rouse Caffey, it wouldn’t be the station it is today.”

Caffey, who died in 2012, retired from the LSU AgCenter in 1997 after serving 13 years as chan-cellor. He also was chancellor of LSU of Alexan-dria, vice chancellor of the LSU AgCenter, associ-ate director of the LSU Agricultural Experiment Station. He was director of the Rice Experiment Station from 1962 until 1970 and was rice re-search project leader in Mississippi. Bruce Schultz


AgCenter News

Sugarcane byproducts used in skin, bone tissue engineering

Louisiana sugar producers may one day have a new market for their crops.

LSU AgCenter researchers at the Audubon Sugar Institute are continuing a tissue engineer-ing study that began as a study by former grad-uate student Akanksha Kanitkar. The study in-volves making skin and bone tissue scaffolds from aconitic acid, cinnamic acid and glycerol – all byproducts of sugarcane processing.

According to Giovanna Aita, an associate professor who directed Kanitkar’s work, the study involves creating nontoxic, biodegrad-able polyesters from molasses and bagasse.

“This would be not only profitable for the sugarcane industry as a means of value addi-tion by the use of its byproducts, but it also un-folds a path for generating novel biomaterials for tissue engineering applications,” Aita said.

Scaffolds are structures made from the polyesters that scientists use to create new tissues.

“We are studying the polyesters for their mechanical properties and porosity, as well as their ability to support stem cell growth,” Aita said.

Scaffolds made from these polyesters de-grade at a rate that is favorable for new skin growth. This knowledge could be used for cre-ating skin tissue to use in wound repair. A. Denise Attaway

New multipurpose building opens at 4-H campAfter years of wishing, planning and fund-

raising, the new $1.2 million multipurpose pavil-ion at the LSU AgCenter Grant Walker 4-H Edu-cational Center became reality on Oct. 30 with a dedication and ribbon cutting.

The building is named after Ellis S. Martin, father of Jonathan Martin, chief executive offi-cer of RoyOMartin. The RoyOMartin company provided much of the funding for the project.

The 10,000-square-foot facility provides in-door space for 4-H campers to gather and line up for lunch during inclement weather as well as be available for other activities.

The project got off the ground after former state Sen. Randy Ewing took charge of fundrais-ing from private sources. Ewing said 137 con-tributors stepped up to offer funding and in-kind services. Bruce Schultz

Scientists try to slow spread of emerald ash borer

Efforts are underway in north Louisiana to slow the spread of an invasive species that threatens to destroy native ash trees. The trees play an important part in bottomland ecosys-tems and also have an economic value to the timber industry.

“The emerald ash borer was detected for the first time in northern Louisiana in February 2015,” said LSU AgCenter entomologist Rodri-go Diaz. “It is a beetle native to China that has decimated ash trees in the northeastern Unit-ed States within the past 15 years and has been spreading and moving south.”

Diaz said the emerald ash borer kills ash trees by digging tunnels below the bark, cut-ting the flow of sap throughout the tree. In six to seven years the tree will die, he said.

According to Wood Johnson, an entomol-ogist with the U.S. Forest Service, emerald ash borer adults have been collected in Claiborne, Bossier and Webster parishes. It has only been found in trees within Webster Parish.

The LSU AgCenter, the U.S. Forest Service, the Louisiana Department of Agriculture and Forestry, and the Animal and the Plant Health Inspection Service have collaborated to get a biocontrol effort off the ground in north Louisiana.

To help slow the spread of the borer, the entomologists are releasing three species of wasps, with each targeting different growth stages of the borer.

“We obtained a release permit for the par-asitoids from the USDA to use as a tool to man-age the emerald ash borer,” Diaz said.

Much of the information on the insect

comes from research conducted primarily in northern climates. By monitoring the borer and the parasitoid wasps in north Louisiana, the en-tomologists hope to learn more about both the borers and the wasps in the South. Brandy Orlando

LSU AgCenter entomologist Rodrigo Diaz releases a sample of parasitoid wasps onto an infected ash tree in Shongaloo, Louisiana. Photo by Brandy Orlando

Jonathan Martin, chief executive officer of RoyOMartin, cuts the ribbon to the Ellis S. Martin Multipurpose Pavilion at the LSU AgCenter Grant Walker 4-H Educational Center during the dedication ceremony Oct. 30. Left to right are Louisiana 4-H Foundation board chairman Charles Dill; Martin; former state Sen. Randy Ewing, and Patrick Tuck, 4-H Foundation executive director. Photo by Bruce Schultz

The multipurpose pavilion provides 10,000 square feet of space. Panorama photo by Bruce Schultz


College of Ag NewsStudents hope to improve agriculture in their home countries

LSU AgCenter International Programs is helping seven international graduate students earn their doctorates so they can return home with hopes of sparking much-needed change in farming practices and policies.

The students, who have worked as univer-sity lecturers and government employees, are part of the Borlaug Higher Education for Agri-cultural Research and Development (BHEARD) program. It is funded by the U.S. Agency for In-ternational Development (USAID) and man-aged by Michigan State University.

“It is an honor for the LSU AgCenter to have been selected as the host training institution for the BHEARD scholars,” said David Picha, di-rector of AgCenter International Programs.

The students will complete coursework on LSU’s campus and then return to their home countries to do a research project under their faculty adviser’s supervision. LSU will grant their degrees.

“BHEARD has given me an opportunity to be exposed to a high-level environment, build

my skills and research,” said Chunala Njombwa, a former livestock researcher at the Lunyang-wa Agricultural Research Station in Malawi. “By coming here, we will build partnerships. Now I know these guys who want to come up with projects that will help small-scale farmers in our countries.”

Though the students are from five coun-tries and work in different fields of expertise, they point to similar issues hindering agricul-ture in the developing world. Food security is a common theme. But unlike Americans may assume, it is more a problem of quality than quantity.

“As much as you want to make food af-fordable and accessible, safety and quality are equally important things,” said Bennett Dzan-du, a former University of Ghana teaching as-sistant studying food science at LSU. “The issue is that in Africa and many places, it is rather the opposite way. Quantity and cheapness is at the expense of quality and safety of the ones con-suming the food.” Olivia McClure

LSU AgCenter International Programs is helping seven students pursue doctorates at LSU. Front row from left, Murshida Khan, of Bangladesh, and Fausta Marie Dutuze, of Rwanda. Back row from left, Chunala Njombwa, of Malawi; Bennett Dzandu, of Ghana; Fydess Khundi, of Malawi; Emmanuel Kyereh, of Ghana; and Susan Karimiha, AgCenter International Programs coordinator. Another student, Sarah Kagoya, of Uganda, is not pictured. Photo by Olivia McClure

Slovakian student promotes study abroad

Natália Antošová, a College of Agriculture graduate student in agricultural economics from Slovakia, wants to help LSU students have a similar experience to the one she is having.

The college has partnered with the Slovak University of Agriculture (SUA) in Nitra, Slova-kia, for student and faculty exchanges and re-search collaboration. Faculty at SUA recom-mended Antošová for the exchange program. This is her first time in the U.S.

“School is demanding. Studies are deeper and move at a quick pace,” Antošová said.

Being away from home and family commit-ments allows her to focus on school more, An-tošová said. But that doesn’t mean she spends all of her time studying. In her short time at LSU, she has found ways to become involved in campus life, including taking part in the LSU homecoming parade with the LSU Internation-al Student Association.

As part of the exchange, Antošová is work-ing for the LSU AgCenter International Pro-grams department as an international relations student adviser.

“I bring information to students about op-portunities to travel and study abroad and or-ganize events, and promote study abroad in every way,” she said. Tobie Blanchard

Natália Antošová, an LSU graduate student from Slovakia, is working with LSU AgCenter International Programs to help promote study abroad opportunities for LSU students. Photo by Tobie Blanchard

Salassi new head of ag economics departmentThe LSU AgCenter and LSU College of Agriculture have

named Michael Salassi head of the Department of Agri-cultural Economics and Agribusiness. Salassi has served on the faculty of the department for 21 years and is the J. Nelson Fairbanks Endowed Professor for Agricultural Economics.

He replaces Gail Cramer, who retired in July. Salassi received bachelor’s and master’s degrees from

LSU and a doctorate from Mississippi State University. He worked for the U.S. Department of Agriculture’s Economic

Research Service in Washington, D.C., for nine years before returning to LSU as an associate professor in 1994. He be-came a full professor in 2002.

Salassi also served as the assistant director of the Lou-isiana Agricultural Experiment Station, the AgCenter’s re-search division. His main focus during his career has been production economics and farm management. Tobie Blanchard

Michael Salassi


College of Ag News

‘Cocktails and Cuisine’ raises $20,000 for scholarships

Casey Kenny, a freshman in the LSU College of Ag-riculture, is a recipient of the prestigious Penelope W. and E. Roe Stamps IV Leadership Scholarship. Kenny plans to use some of the scholarship money to fund research on feline viruses. Photo by Tobie Blanchard

Stamps scholarship goes to ag student

Casey Kenny considers cats her first love. The freshman in the LSU College of Agriculture wants to work with small animals, exotics and wildlife as a veterinarian. While in college she plans to search for a cure for feline leukemia and feline immunodeficiency virus.

Kenny is one of five LSU freshmen to receive the prestigious Penelope W. and E. Roe Stamps IV Leadership Scholarship. With the scholarship comes full cost of attendance for four years, with up to $14,000 for enrichment opportuni-ties, such as research.

Kenny plans to use the money to finance her research on feline viruses.

“I’ve seen these viruses, and they are terri-ble,” Kenny said. “I hate that so many cats are suffering from these diseases, which may have cures that have not been explored.”

During high school, Kenny began shadow-ing a veterinarian in her hometown of Mont-gomery, New York. Later, she was hired to work in the clinic. She said feral cats would come into the clinic with feline leukemia and feline immu-nodeficiency virus. With no cure, they would just have to treat the symptoms of the virus. She said the viruses are easily spread among cats.

Kenny’s grades and scores could have taken her anywhere, but she said she was looking for a big state school. She visited LSU and was in-vited to apply for the Stamps scholarship. Earn-ing the award made her decision easy.

“I really love LSU. Life is totally different here,” she said. Tobie Blanchard

Nine students receive research grantsEvery day for six weeks, Ariel Bergeron went

to the LSU AgCenter poultry research facility to feed and water quail. Bergeron, a senior major-ing in animal sciences, is studying nutrition re-quirements of quail less than six weeks old.

The LSU College of Agriculture gave grants to Bergeron and eight other undergraduate stu-dents to pursue research in their fields of study.

Bergeron and her faculty adviser, There-sia Lavergne, a poultry specialist, chose to look at this subject because not much research has been done on nutrition requirements of quail in the past 30 years.

“Bobwhite quail are a popular gamebird in Louisiana, but they are having trouble surviving in the wild,” Bergeron said. “More people will be raising quail, so we want to know what the op-timal diet is.”

Research projects help prepare students like Bergeron for graduate school and careers that involve research, Lavergne said.

Other College of Agriculture research grants went to the following students: Ryan Ardoin is studying consumer perception and purchase intent of low-sodium mayonnaise products with Witoon Prinyawiwatkul, a food sciences professor. Katie Bowes is looking at the effects of salini-ty in survival, growth and biomass of the aquat-ic plant Ruppa maritima with Megan Lapeyre, an adjunct professor in the School of Renewable Natural Resources.

Brittany Craft is examining the effects of par-ticipating in a culinary skill-building program on high school students’ willingness to consume fruits, vegetables and whole-grain foods with Georgianna Tuuri, an associate professor in the School of Nutrition and Food Sciences. Haley Hutchins plans to characterize the diver-sity of the fungi Colletotrichum associated with Louisiana plants with Vinson Doyle, assistant professor in the Department of Plant Pathology and Crop Physiology. Samantha Lanjewar wants to determine if sev-en specific genes, used to test various perfor-mance levels of cattle, can be successfully am-plified in a type of cell called a fibroblast. She is working with Kenneth Bondioli, professor in the School of Animal Sciences. Scarlett Swindler is working with food scientist Marlene Janes to look for rapid and reliable de-tection methods of the human norovirus in ma-rine waters. Carly Thaxton aims to assess dietary intake of pregnant women, while also looking at fatty acid levels by analyzing the mother’s red blood cells, with Carol Lammi-Keefe, a professor in the School of Nutrition and Food Sciences. Colleen Walsh is conducting research on an in-vasive species, Daphnia lumholtz, commonly known as water fleas, with William Kelso, a pro-fessor in the School of Renewable Natural Re-sources. Tobie Blanchard

More than 130 people attended the LSU College of Agriculture’s 2nd annual Cocktails and Cuisine Benefiting Scholarships, which was Oct. 16 at LaHouse on LSU’s campus. Attendees included (from left to right) Paula and Harold Lambert, and Gene and Sheila Reagan. The evening featured a silent auction, a jazz trio, and food from tenants of the LSU AgCenter Food Incubator. The event raised more than $20,000 for scholarships. Sponsors included ZEN-NOH Grain, ConAgra Foods/Lamb Weston, Horizon Ag LLC, Gowan Company, Bracy’s Nursery LLC, Louisiana Agricultural Consultants Association, East Iberville Industry Neighbor Companies, Louisiana Agribusiness Council, and Louisiana Association of Conservation Districts. Photo by Tobie Blanchard


Termite Gut MicrobesTools and Targets for ControlClaudia Husseneder, Chinmay V. Tikhe, Lane Foil and Chris Gissendanner

Termite colonies are often called “super-organisms” for several reasons. First of all, termites are social insects that live in large and densely populated colonies with specialized castes, including workers, soldiers and reproductives. These castes all perform different tasks. The superorganism concept then extends into the microbial world of the termite gut, which is sometimes referred to as the world’s smallest and most efficient biore-actor. Termites live exclusively on plant dry matter, such as wood, which lacks essential nutrients and is difficult to digest. Therefore, termites rely on microbial symbionts to aid in digestion and energy production in the gut of their workers, which are responsible for for-aging, digesting the food and feeding other members of the colony via regurgitation and anal excretion of fluids.

In subterranean termites, such as the Formosan subterranean termite, which is found in Louisiana and the Southeastern United States, the hind gut of workers is spe-cialized to harbor microbes from the three categories, or domains, of life – archaea, bac-teria and eukaryota.

Archaea were originally classified as bac-teria but were recently put into a domain of their own because of unique properties that make them different from both bacteria and eukaryota. Most archaea in the termite gut are methane producers, making termites the second largest source of atmospheric methane on the planet after the ruminants.

Research at the LSU AgCenter has identi-fied more than 200 different bacteria species residing in the gut of Formosan subterranean termites, some of them unique and specific to termites. The bacteria community supports important functions in regard to nutrition

and energy production. For example, bacteria produce and recycle nitrogen as a source for protein synthesis. They synthesize vitamins and reduce hydrogen to create energy-pro-ducing byproducts that the termites can use.

The bacteria scavenge oxygen in the ter-mite gut to provide the anaerobic environment required by the protists, which are eukaryota organisms. The three species of protists in the gut of Formosan subterranean termites are the most valuable players when it comes to digestion of wood. Without these protists in their guts, termites starve to death (Figure 1).

This need for termites to have obliga-tory microbial symbionts in their guts gave researchers at the LSU AgCenter and the University of Louisiana at Monroe a new idea for controlling termites. The researchers use bacteria and protists as tools and targets, rather than chemical insecticides, to kill termites.

Because the diverse bacteria community in the termite gut is of vital importance to the health of the termite colony, bacteria make a good target for aiming at termite control. Bacteriophages, also called phages, are viruses that kill bacteria. Phages are highly host-spe-cific and do not infect cells of plants, animals or humans. Thus, using a phage cocktail to disrupt the bacteria community in the ter-mite gut might be a feasible option for ter-mite management, either as a stand-alone or in conjunction with chemical insecticides.

Because nothing was known about bacte-riophages and their roles in the termite gut, researchers from the LSU AgCenter and the University of Louisiana at Monroe started to isolate and describe phages from the gut of Formosan subterranean workers (Figure 2). To date, the full genome of three phages has been sequenced. The host specificity of those phages

ranges from narrow, in which they infect only a single species of bacteria, to fairly broad, in which they infect several closely related spe-cies. The phages include lytic phages that infect and kill their host bacterium immediately and temperate phages that can integrate into the bacteria genome and stay dormant for a while until they go into the lytic phase. More phages are currently being tested for their efficacy against termite gut bacteria. Experiments are underway to provide proof of concept that natural phages can disrupt the termites’ bac-terial community and, thus, weaken or even kill a termite colony. In addition, researchers are planning to engineer phages to produce targeted antimicrobial peptides.

AgCenter researchers already have pro-vided proof of concept that genetically engi-neered microbes can serve as Trojan horses to express and spread gene products in the gut of Formosan subterranean termites without risking detection and elimination by the ter-mites’ natural defenses. Enterobacter cloacae, a bacterium common in the gut of many organ-isms, was isolated from Formosan termites and engineered to express green fluorescent pro-tein, a marker that could be easily traced visu-ally by its bright green fluorescence. Workers readily ingested the engineered bacteria, and the bacteria survived for six weeks and longer in their guts. Workers also transferred the bacteria to other workers and soldiers, which helps to spread the gene product throughout a termite colony.

The next step involved the search for a targeted toxin that specifically kills the cel-lulose-digesting protists in the termite gut. Researchers at the AgCenter already had experiences with lytic peptides as antimicro-bial agents. Lytic peptides are a natural part

Figure 1. The three species of cellulose-digesting protists in the gut of Formosan subterranean termite workers. Loss of these protists leads to the death of the termite colony by starvation.

Pseudotrichonympha Holomastigotoides Spirotrichonympha


of the immune system and kill protists and bacteria but have no negative effect on cells of plants or animals including humans. Research showed that minute concentrations of the lytic peptide Hecate kill all protists in the gut of Formosan termite workers when injected into the hindgut by way of a tiny enema (Figure 3). Unfortunately, feeding termites with lytic peptides was not an option because the pep-tides were broken down in the digestive tract and lost their lytic function. Delivery by a genetically engineered microbial Trojan horse was promising not only to spread the toxin, but also protect it from digestion.

To make the lytic peptide more specific to the target protists and protect nontarget organ-isms from the lytic peptide action, a ligand was designed that binds the lytic peptide to pro-tists but not to other cells. Freeze-dried yeast (Kluyveromyces lactis), which was engineered to express lytic peptide coupled to the ligand, was incorporated into cellulose bait discs and

fed to Formosan termites in the laboratory. Workers readily ingested the yeast and spread it to their colony mates (Figure 4). The yeast expressed and secreted ligand-lytic peptide into the termite gut. The ligand bound to the cellulose-digesting protists in the gut, and the lytic peptide killed them. All three species of protists in the gut of Formosan termites were killed within three weeks of first ingestion of the yeast, and termite lab colonies died within two weeks after. The comparatively slow-act-ing system is necessary to guarantee spread of the Trojan horse and its payload throughout the entire colony. Current research is testing termite-specific gut bacteria and bacterio-phages, instead of ubiquitous yeast, as Trojan horses to deliver protist-killing ligand-lytic peptides into termite colonies to increase environmental safety.

Research into the termite gut model shows that microbes (genetically engineered Trojan horses or natural bacteriophages) can be used

Figure 2. Electron microscope images of two novel bacteriophages isolated from the gut of Formosan termites.

to alter the microbial community in the gut. In the case of termites, such microbiome engi-neering has the ultimate goal of pest control. Similar approaches can be used in the future to strategically increase beneficial microbes and reduce pathogens in the gut of organisms, including humans.

Claudia Husseneder is a professor, and Lane Foil is Pennington Regents Chair for Wildlife Research in the Department of Entomology. Chinmay Tikhe is a Ph.D. student in Husseneder’s lab. Chris Gissendanner is an associate professor at the University of Louisiana at Monroe School of Pharmacy.

Figure 4. Yeast expressing ligand-lytic peptide as Trojan horse for termite control.

Figure 3. Injection of peptide solution via an enema into a termite worker immobilized in a pipette tip.


Mosquitoes and West Nile VirusBiology, behavior and insecticide susceptibility of Culex quinquefasciatus, the primary vector in LouisianaKristen Healy, Emily Boothe and Nicholas DeLisi

West Nile virus is considered the most widespread arbovirus (viruses transmitted by insects and related arthropods) in the world. The virus is transmitted to humans and animals by the bite of an infected mosquito. About 80 percent of the humans that become infected do not develop symptoms, whereas 20 percent develop flu-like symptoms. Less than 1 percent develop more serious neuro-logical complications, such as encephalitis, a disease that causes swelling of the brain.

Although it f irst appeared in the United States in 1999, West Nile virus still remains an important infectious dis-ease. According to the Centers for Disease Control and Prevention (CDC), West Nile virus has contributed to more than 41,762 cases of illness and 1,765 deaths in the United States since 1999. In Louisiana alone, there have been 1,575 cases reported to the CDC between 1999 and 2014. While there is the potential for year-round activity, most West Nile virus cases occur between July and October. Therefore, personal pro-tection from mosquitoes is crucial every summer and fall.

One of the main factors that has con-tributed to the success of West Nile virus in the United States is that mosquitoes cycle the virus in bird populations. Birds that do not become ill or die from the infection and that have high levels of virus aid in the cycle of the virus. In addition, mosquito species that are suitable vectors contribute to the complexity of this virus in Louisiana.

Southern house mosquito There are more than 3,000 species of

mosquitoes in the world, and only a few are potential vectors for West Nile virus. The Southern house mosquito (Culex quin-quefasciatus) is the most important vector for West Nile virus in Louisiana because it cycles the virus in birds and can occasion-ally transmit the virus to humans. Because of its importance in the cycle of West Nile virus in Louisiana, the Southern house mosquito is the main target for mosquito control districts in Louisiana. Mosquito districts across Louisiana will collect samples of mosquitoes, identify them by species, and then submit them for West Nile virus testing (Figure 1). In response

to West Nile virus activity in mosquitoes, control programs will employ different methodologies to control mosquitoes that can transmit the virus. To determine the effect of control on virus activity and mos-quito populations, LSU AgCenter scientists are developing new tools to assess control efficacy in the state. This includes develop-ing new trap designs to collect host-seeking house mosquitoes, understanding the biol-ogy of important vector mosquito species (Figure 2), and evaluating pesticide suscep-tibility in the state for products designed for larval control and adult mosquito control.

The gold standard for collecting house mosquitoes in the United States is the gravid trap (Figure 3). This trap functions by collecting mosquitoes ready to lay eggs in water (gravid females). While gravid traps have historically collected large number of house mosquitoes, this type of surveillance represents the population of gravid females within an area rather than the host-seek-ing population that poses the greatest risk to humans and West Nile virus infection. The host-seeking mosquitoes are seeking a blood meal.

Figure 1. A collection of mosquitoes being sorted by species. In Louisiana, mosquito control programs will sort mosquitoes and then submit them for testing for West Nile virus. Photo by Kristen Healy

Figure 2. Nicholas DeLisi, a graduate student, determines the temperature and pH of a container habitat being used by mosquito larvae. Photo by Kristen Healy

Figure 3. A gravid trap collects mosquitoes that are looking for a place to lay eggs. Mosquitoes locate the trap by the “stinky water,” which is attractive to egg-laying mosquitoes. Photo by Kristen Healy


To find a better trap for host-seeking mosquitoes, LSU AgCenter scientists are conducting studies within three parishes in Louisiana – St. Tammany, Tangipahoa and East Baton Rouge – to evaluate different trap designs. This has included a longitu-dinal study to compare traps throughout the entire season to make comparisons of gravid and host-seeking populations. AgCenter researchers are discovering that standard mosquito traps can be simply modified to collect more host-seeking mosquitoes by removing the lightbulb and baiting them with dry ice. Although the data are still being evaluated, this would be a useful discovery for mosquito control districts wishing to evaluate host-seeking house mosquito populations to evaluate different levels of risk for West Nile virus.

Mosquito control as a tool to prevent disease transmission

The best defense against transmission of West Nile virus comes in the form of mosquito control. Since the early 1900s, mosquito control has been built around the concept of integrated mosquito man-agement. This system involves using multiple control strategies, such as larval

control with mosquito-specific bacteria, reducing standing water, monitoring of mosquito populations, evaluating mos-quitoes and birds for viruses, and adult mosquito control using pesticides. Most people are not aware of the many compo-nents of integrated mosquito management that go on in Louisiana because they are most familiar with spray trucks. Because mosquito control does such a good job at reducing mosquitoes, most people do not realize how deleterious mosquitoes can be to human and animal populations. Before modern mosquito control, tens of thou-sands of individuals in Louisiana lost their lives to yellow fever and malaria, which are both caused by mosquito-borne pathogens. Thankfully, Louisiana has many parish-wide mosquito control districts conducting integrated mosquito management. A loss of mosquito control programs could result in dramatic increases in both nuisance and vector mosquito species.

One of the goals of LSU AgCenter research is to help mosquito programs ensure that their control methodology is effective. Fortunately, mosquito control programs have a selection of products that they can use to control mosquitoes. By

evaluating if a product is effective, they can determine if they need to switch to a product with a different mode of action. The efficacy of a product can easily be evaluated in the lab by conducting bottle bioassays for adult mosquitoes (Figure 4) or cup bioassays for the larvae (Figure 5). In these types of tests, scientists place mosqui-toes in vials with known concentrations of pesticides and determine the percent dying or time to death. If there is variation from standard susceptible populations, the sci-entists then evaluate the potential causes by doing more in-depth studies.

Figure 4. Emily Boothe, a research associate, conducts bottle bioassays to evaluate the efficacy of pesticides in adult mosquito populations. Photo by Kristen Healy

Figure 5. Researchers evaluate the efficacy of different mosquito control products that target the immature mosquitoes, which are called larvae. Photo by Kristen Healy

Dead adult mosquitoes. Photo by Emily Boothe

Kristen Healy is an assistant professor; Emily Boothe is a research associate; and Nicholas DeLisi is a graduate assistant, all in the Department of Entomology


Norovirus and Oysters in LouisianaNaim Montazeri, Morgan Maite and Marlene Janes

At some point in your life you have become ill with what is referred to as stomach flu. Stomach flu is caused by norovirus and is the foremost common cause of gastrointestinal tract infections in humans, resulting in nausea, vomiting, diar-rhea and low-grade fever. As a result, norovi-rus and many other similar viruses, such as enterovirus and hepatitis A virus, that cause intestinal disease are called enteric viruses. Infected individuals can shed millions of viruses in their feces or vomit. Annually, 15 million people get sick in the United States with norovirus; therefore, it can be found in large amounts in human sewage.

You may be exposed to the viruses through contaminated surfaces or foods. Ingesting the pathogen by touching con-taminated hands to the mouth or eating contaminated food can make you sick, and that’s how norovirus is spread. To the best of our knowledge, these viruses do not grow in foods; they are merely transmitted to food, where they linger for an extended period of time.

A sick food handler can transfer the virus to your food. Fresh produce grown using sewage-contaminated irrigation water can harbor enteric viruses. Filter-feeding mol-luscan shellfish such as oysters exposed to sewage-contaminated waters can actively concentrate viruses. If the oyster is con-sumed raw, the virus can be transmitted to humans and cause illness. These viruses enter source waterways through the direct or indirect discharge of treated and untreated human and animal waste into surface waters.

Sewage is one of the major contamina-tion sources for norovirus and other enteric pathogens. Regulatory agencies are using some bacteria and viruses of natural micro-bial f lora of human and warm-blooded animal guts – such as fecal coliforms, Escherichia coli, enterococci and coliphages – to assess the level of fecal pollution in environmental waters or food. Currently, the Louisiana Department of Health and Hospitals Molluscan Shellfish Program uses microbial indicators of fecal contamination as the determining factor in closing mollus-

can shellfish harvesting areas due to virus contamination. Furthermore, several out-breaks of norovirus have been traced to con-sumption of oysters obtained from Louisiana sites. On April 28 and 29, 2012, 14 people became ill with norovirus after consuming oysters at a restaurant in New Orleans. The oysters were traced back to an oyster harvest-ing area off the Louisiana coast. As a result, LDHH closed the harvesting area. In January 2013, LDHH closed another area due to sus-pected norovirus contamination of oysters. Samples of harvesting water or oysters are not directly tested for the presence of viral contamination during suspected outbreaks. These outbreaks further highlight the lim-ited data on virus occurrence in Louisiana harvest water and oysters.

AgCenter scientists investigated the occurrence of norovirus and microbial indi-cators of fecal contamination in the Eastern oysters (Crassostrea virginica) and water from commercial harvesting areas along the Louisiana Gulf Coast from January to November 2013. The sampling locations were among the most active commercial oyster-harvesting areas and remained open during the sampling period.

Microbial fecal indicators were detected but at levels too low to be of public health concern. Even with low levels of fecal con-tamination in the open areas were oysters and harvesting water were collected, norovi-rus was detected in only one oyster sample. In general, the level of microbial indicators was too low to draw a conclusion of their effectiveness in predicting the pathogens. However, norovirus can be present even when water and oysters are considered bacte-riologically safe. Although this study showed low concentrations of noroviruses in oysters, whether this can be a health concern needs to be further investigated because little is known regarding the risks associated with the concentration of norovirus in oysters.

An outbreak of norovirus occurred January 2013 in Cameron Parish, Louisiana. The individuals who had become ill had eaten oysters collected from the Louisiana Gulf Coast. A stool specimen from an Naim Montazeri and the first haul of oysters for the day. Photo by Liu Da


infected individual associated with the outbreak and the suspected oysters were analyzed. The norovirus strain in the stool belonged to one of the most widespread norovirus genotypes; however, the oysters were negative and could not be linked to the outbreak.

AgCenter scientists also looked at a municipal secondary wastewater treat-ment plant on the Mississippi River in New Orleans as a potential source of environ-mental contamination. For one year they collected monthly wastewater samples from the treatment plant both before treatment (influent) and after secondary treatment and chlorine disinfection (effluent). Enteric viruses were present in both influent and eff luent wasters year-round. Microbial loads in the influent indicate the presence of the pathogens, reflecting what is hap-pening within a population. They found that changes in concentrations of enteric viruses may not be in accordance with the expected epidemiological trends, and some strains circulating among the population have been overlooked from epidemiologi-cal standpoints. These results showed that enteric viral pathogens are more resistant than microbial indicators to the secondary treatment of wastewater and chlorine disin-fection (Figures 1 and 2). Furthermore, fecal coliforms, E. coli and enterococci commonly present in the sewage water had no remark-able changes over time. Coliphages (viruses infecting E. coli) were the only indicators that concentrations were similar to noroviruses, indicating that coliphages have the poten-tial to be used to assess the efficacy of the

treatment and disinfection of noroviruses in sewage.

Overall, this study shed light on the safety of Louisiana oysters and showed that noro-virus might occur in the oyster even when they are bacteriologically safe. This study also found that raw and even secondary treated sewage water can harbor high den-sities of the pathogenic viruses, which even-tually enter the environmental waters. A proper system to manage oyster harvesting areas using more effective criteria or even direct monitoring of enteric viruses is rec-ommended to reduce the risk of outbreaks.

The LSU AgCenter research team heading out for a day of harvesting oysters. Left to right are Naim Montazeri, Marlene Janes, Jen Shields, Morgan Maite and Jody Donewar, the captain. Photo by Liu Da

Marlene Janes, standing in center, and Naim Montazeri, left, harvest oysters from the Gulf of Mexico for sampling. Also in the boat are Jody Donewar, the captain, and Jen Shields. Photo by Liu Da

Marlene Janes is a professor and Morgan Maite is a graduate assistant in the School of Nutrition and Food Sciences. Naim Montazeri graduated from the School of Nutrition and Food Sciences, with a Ph. D. in May 2015 and is currently working with the NoroCore project as a post-doctoral researcher at North Carolina State University.

Acknowledgments: This research was funded through NoroCORE, an Agriculture and Food Research Initiative from the U.S. Department of Agriculture National Institute of Food and Agriculture.

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Studying Rice-Microbe-Insect Interactions to Increase Rice ProductionLina Bernaola and Michael Stout

Global needs for food and fiber are expected to increase dramatically over the next few decades, with some experts predicting that food production will need to double by 2050 to meet growing demand. Some of the biggest obstacles to increasing crop production are the challenges presented by biotic and abiotic stressors such as nutrient deficiencies and insect pests. One key to overcoming these obstacles is to better understand the interactions of crop plants with the great variety of organisms they encounter both above and below the soil surface.

Soil microbes represent an important but often neglected component of the soil environment. Over the past decade they have become an increasingly important subject of innovative research in agriculture because understanding the interac-tions of soil microbes with plants has the potential to lead to novel methods for increasing plant yields and productiv-ity under stressful environments. The LSU AgCenter rice entomology program conducts studies to develop cost-effec-tive management programs for insect pests that attack rice in Louisiana. This research program led to interest in the potential of soil microbes to increase nutrient availability, enhance crop growth and protect against insect and disease pests. The research focus in this area is to understand how soil microbes influence the mechanisms and processes that allow rice to resist or tolerate attacks by insect pests.

Soil microbes include fungi, bacteria and viruses. The idea that these microbes can harm crops is a familiar one. Many “good” soil microbes, however, have positive influences on agricultural production. Among the most abundant and common of these good microbes are the arbuscular mycor-rhizae fungi (AMF). AMF are ubiquitous, soil-dwelling fungi that form symbiotic relationships with many plant root systems; in fact, as many as 80 percent of plant species are thought to form associations with mycorrhizae. The

plant “partners” in these symbiotic associations supply pho-tosynthetically fixed carbon to the AMF, and in exchange, the AMF use this carbon to grow mycelial networks that allow plant root systems to expand more in the soil and efficiently absorb water and nutrients. Plant-AMF associ-ations generally show positive effects on plants in natural and agricultural ecosystems. In agriculture, these effects include plant growth promotion through enhanced uptake of essential nutrients, such as nitrogen and phosphorus. This may translate to higher yields in plants colonized by AMF.

Much past research with AMF has focused on the direct effects of AMF on plants. Interestingly, however, coloniza-tion by AMF has also been shown to alter plant resistance to abiotic and biotic stresses. In particular, colonization of a plant by AMF can alter the interactions of the plant with other members of the ecological community, both above ground and below ground, such as plant-eating insects and plant pathogens. AMF colonization alters interactions with these other organisms by influencing plant charac-teristics such as plant biomass, nutrient content and plant chemical defenses.

Rice, like most other plant species, can form associations with AMF. Preliminary observations of rice roots from Louisiana and other Southern states show that rice is often naturally colonized by AMF in the field, at least before rice is flooded (Figure 1). The entomology program is conducting research to understand how interactions between rice and AMF influence interactions of rice with pests.

A four-year field study has determined the effects of AMF on infestations of the rice water weevil, the most important insect pest of rice in the United States. When rice was inoc-ulated with a commercially available mixture of six species of AMF, the fungi formed associations with the rice roots and made rice much more susceptible to infestation by rice

Figure 1. Rice root samples collected from Mississippi and Arkansas rice fields show natural colonization by mycorrhizae. “Vesicles” and “Hyphae” are fungal structures.

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water weevil larvae. That is, populations of weevil larvae were larger on AMF-inoculated roots. In addition, studies evaluated the effects of AMF on rice susceptibility to the fall armyworm, a sporadic insect pest of rice, and sheath blight, an important disease pest of rice. Data obtained so far show that, after inoculation with mycorrhizae, rice plants become more susceptible to both armyworms and sheath blight.

The effects of AMF on rice resistance to pests are depen-dent on soil type, as both field and greenhouse experiments have shown that rice plants inoculated with mycorrhizae supported higher densities of rice water weevil larvae and

higher relative growth rates of armyworms when grown in soil from Crowley, Louisiana, but not when grown in soil from Mamou, Louisiana (Figures 2 and 3). Root biomass of AMF-colonized rice plants was also greater than non-AMF plants in an experiment in Crowley but not Mamou. No effects of AMF colonization on yields were observed in either loca-tion. The effects on AMF colonization on pest infestations and root biomass, coupled with the lack of effect on yields, suggest that AMF colonization may increase rice tolerance to rice water weevils. Nutritional analyses of root and shoot tissues indicated only slight differences in the concentration of nutrients in AMF-colonized plants.

Although the fact that rice-AMF interactions can make rice more susceptible to pests is not directly useful for man-aging pests, the dramatic change in rice resistance caused by AMF colonization provides a unique window for studying the traits or characteristics that make rice plants more sus-ceptible to insect and pathogen attack. Studies include the effect of AMF on the growth of rice plants and the uptake of nutrients from the soil as well as changes in the biochemistry and physiology of rice plants that occur following AMF colo-nization. This research can lead to insights into what makes rice resistant to or tolerant of pests, and this knowledge will ultimately contribute to developing more resistant varieties to improve pest management programs in Louisiana rice.

Lina Bernaola, a doctoral student in the Department of Entomology, poses in her mycorrhizae field experiment at a research field site located in Evangeline Parish. Photo by Thais Freitas

Lina Bernaola is a graduate assistant and Michael Stout is the L.D. Newsom Professor in Integrated Pest Management in the Department of Entomology.

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Figure 2. Mean numbers of rice water weevil larvae per core sample in four years of field experiments conducted in two locations, Crowley and Mamou. An * indicates a significant difference between treatments.

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Figure 3. Relative growth rates of armyworm larvae fed for 10 days on rice leaves from mycorrhizae-inoculated and control plants. Plants were grown in soil from three sources: a greenhouse soil mix, soil from Crowley, and soil from Mamou. An * indicates a significant difference between treatments.


On-farm Food Safety Research Helps Louisiana Growers Comply with New Law Achyut Adhikari

The Food Safety Modernization Act (FSMA) is one of the sweeping reforms of U.S. food safety laws in more than 70 years. The main focus of the act is to reduce foodborne hazards by pre-venting microbial contamination during production, processing, handling and transportation of food rather than relying on correction after problems occur. Under FSMA, “FDA will have a legislative mandate to require comprehensive, science-based

preventive controls across the food supply.” FSMA requires new standards for grow-

ing, harvesting, packing and holding pro-duce for human consumption, also known as the “Produce Safety Rule.” The rule identifies five routes of on-farm microbial contamination – agricultural water, domes-ticated and wild animals, workers, biolog-ical soil amendments, and equipment and tools – and sets requirements to prevent or reduce the introduction of pathogens.

The potential introduction of foodborne pathogens during growing, harvesting and packing necessitates that producers understand the on-farm sources of harm-ful microorganisms and apply appropriate practices to reduce the risk of contamina-tion. FSMA requires fresh-produce growers to follow practices that minimizes the level of harmful microorganisms before they harvest or market their produce. The rule states the waiting period between irrigation and harvesting or during storage depends upon the microbial quality of irrigation water and the survival of generic E. coli on the edible portion of the crops.

One of the long-term goals of the LSU AgCenter is to strengthen the productiv-ity, profitability and competitiveness of Louisiana’s agriculture. Scientists focus on applied research that has immediate impact on the quality, safety and economic viability of Louisiana-based fresh produce. This is achieved by collaboration among scientists from several disciplines that bring expertise to address critical food safety issues along the production chain.

On-farm applied research contributes to the assessment of produce food safety and guides development of control strate-gies to mitigate the risks during pre- and Cantaloupe irrigated with UV-C light treated

irrigation water. Photo by Achyut AdhikariAchyut Adhikari works with students evaluating the efficacy of ultraviolet light for surface water treatments. Photo by Robert Williams

From left, Achyut Adhikari, Vijay Singh Chettri and Kathryn Fontenot perform microbial analysis of watermelon samples. Photo by Kathryn Parraga

Robert Williams, left, and Ron Strahan collect watermelon samples for a food safety research project. Photo by Achyut Adhikari


post-harvest processing. Current research efforts focus on irrigation water treatments, the fate and persistence of microorganisms on fresh produce and the development of science-based post-harvest processing tech-niques for food safety risk reduction.

Irrigation water is one of the important sources of pathogen contamination. Several methods currently are available for water treatment, such as chlorine, ozone and filtration. Not all are suitable for surface water treatment, however, because of the complexity, variability and content of sus-pended particles.

In one on-farm study, AgCenter research-ers evaluate the efficacy of ultraviolet light treatment on reducing the generic Escherichia coli levels from surface water used for irrigating cantaloupes. The prelimi-nary results indicate significant reduction of generic E. coli even at low doses. This means growers who treat their water with ultravi-olet light can harvest earlier because FSMA requires producers to apply a time interval between last irrigation and harvest using a microbial die-off rate based on the generic E. coli levels of irrigation water. In addition, producers will benefit from a water treat-ment process that does not leave any residue that adversely affects crop production or soil quality. An ultraviolet light treatment replaces use of chemical disinfectants and leaves no residue in the water.

Pecans are native to the lower Midwest and Midsouth. Native pecan areas have a long-standing tradition of double-crop-

ping the land by allowing cattle to graze in pecan groves. Cattle manure is a primary source of foodborne pathogens such as E. coli O157:H7, Salmonella spp. and Listeria monocytogenes. With the potential food safety risk associated with raw manure, Louisiana pecan growers may not be able to have this second source of income by grazing cattle in pecan groves.

However, a provision in the food safety act exempts a farm from the produce safety rule if the produce is processed with a “kill step.” An LSU AgCenter team of food safety and quality specialists, pecan specialists and economists is working on a research proj-ect to address this issue. The study aims to optimize proper antimicrobial intervention techniques during pecan processing that can be regarded as a kill step and increase com-petitiveness of Louisiana-grown pecans.

The fate and persistence of pathogens on the edible portion of the crops is one of the important requirements set by the produce safety rule. Several factors affect the survival of pathogens, such as temperature, humid-ity, UV exposure and type of crops. Previous studies in this area have been performed in laboratory settings, simulating agricul-ture environmental conditions. Because of several variables in an actual farming and processing environment, laboratory set-tings may not accurately predict microbial response and die-off rates that would be in actual environmental conditions.

AgCenter on-farm studies with water-melons and cantaloupes are focused on

examining the die-off rate of indicator organisms specific to Louisiana climate and weather conditions. Results from this applied research will help growers identify best growing practices and conditions to increase productivity and minimize food safety risks.

Critical knowledge gaps exist regarding the fate of pathogens in agricultural envi-ronments. Understanding the behavior of indicator organisms in on-farm settings will enable researchers to conduct pre- and post-harvest food safety risk assessments. The outcomes of this applied research on produce safety will enhance researcher and grower knowledge of the spread of food-borne hazards on fresh produce and will help in the development of preventive con-trol practices, allowing Louisiana growers to comply with FSMA requirements.

Achyut Adhikari is an assistant professor in the School of Nutrition and Food Sciences and is in charge of training and education about the Food Safety Modernization Act.

On-farm study with ultraviolet light treatment of irrigation water. Photo by Achyut Adhikari

Preparation of cantaloupe samples. Photo by Achyut Adhikari

Using diluent to dislodge microorganism from the surface of cantaloupe. Photo by Achyut Adhikari


Bt technologyA Major Advancement in Insect Pest ControlDavid Kerns

Managing insect pests has been an ongoing strug-gle since the birth of agriculture some 10,000 years ago. For the most part, farmers have had little recourse to mitigate damaging insect populations in their fields. However, shortly following World War II, the development of organic insecticides made it possible to effectively manage most insect pests affecting crop produc-tion. However, these tools came with a price. Insect pests exhibited the ability to adapt to the use of these insecticides and develop resistance. Additionally, these insecticides exhibit broad-spectrum activity, meaning they kill insects indiscriminately – both bad and good insects. By removing natural predators and parasitoids while removing the pest, secondary pest outbreaks and resurgence of target pests are often negative side effects. Lastly, some organic insecticides had potentially damaging effects on the environment. In response, over the past 20 years, agricultural researchers have worked to develop insecticides safe to the environment and more target-specific, thus minimizing the impact on predators, parasitoids and pollinators.

One of the greatest advancements in insect pest control has been through Bt technology. Bt refers to a soil-dwelling bacterium, Bacillus thuringiensis. These bacteria produce

proteins that bind to the stomach of certain insects, caus-ing ulcers that result in death. In the 1970s, a number of Bt strains were introduced as sprayable insecticides. These products are extremely safe to nontarget insects and ani-mals, and they are still used, although primarily in organic production systems. The problem with these products is that they have to be eaten by the insect to exhibit toxicity. Also, they rapidly degrade in the environment. Essentially, they just aren’t very effective unless used every few days. Because it is the protein and not necessarily the bacterium itself that affects the insect, researchers had the idea to remove the gene that codes the protein and insert it into the plant. Thus, the plant would essentially produce its own defense mechanism based on the Bt proteins. The beauty of this strategy is that it is nontoxic, constantly produced by the plant, and for the most part, highly effective. Because the plant had a foreign gene introduced into it, it was termed a genetically modified organism, also known as a GMO.

In 1996, the first GMO corn hybrids developed to combat corn-boring caterpillars were introduced in the United

States. These GMO corn hybrids were highly efficacious, so much so that corn borers are hard to find in most corn ecosystems these days. Since these early introductions, additional Bt genes have been introduced to corn. These additional proteins are there to help prevent insects from developing resistance to the Bt proteins and control other corn pests such as corn rootworm and fall armyworm.

Advances have been made in other crops as well. In 1996, a Bt cotton, was introduced that targeted tobacco bud-worm and pink bollworm. Up until that time, the tobacco budworm was a terrible pest of cotton that developed resis-tance to many of the commonly used insecticides. It was not uncommon for cotton to be treated with insecticides six to eight times for tobacco budworm. However, with Bt cotton no insecticide sprays targeting this pest have been necessary. Results were similar for pink bollworm. This does not mean other pests do not affect Bt cotton. Cotton bollworm and fall armyworm, although affected by the Bt protein, would still cause unacceptable injury. Since the initial introduction into cotton, additional Bt protein genes have been introduced – much like corn – that help alleviate single toxin weaknesses and for the prevention of resistance.

GMO crops that express Bt toxin have had huge impacts on the ability to manage insect pests in corn and cotton. Since their introduction, the reliance on insecticide sprays to manage pests has decreased dramatically. Insect preda-tors, parasitoids and pollinators are better protected, and outbreaks of secondary pests are much less common.

There are individuals in society who are critical of GMO crop products, some to an extreme. They contend that GMOs are detrimental because they are not natural; they claim GMOs cause everything from allergies to cancer. The truth of the matter is that reputable, peer-reviewed research has time after time demonstrated that there are no detrimental effects of GMO crop products on humans, animals and nontarget insects. The U.S. food supply has never been safer or more productive than it is today, and GMO crops have played a key role in that development.

David Kerns is an entomologist and associate professor at the Macon Ridge Research Station in Winnsboro and holds the Jack Hamilton Regents Chair in Cotton Production.

The truth of the matter is that reputable, peer-reviewed research has time after time demonstrated that there are no detrimental effects of GMO crop products on humans, animals and nontarget insects.


Internal Regulations and Safeguards for Biological MaterialsKenneth R. Bondioli

All teaching, diagnostic, research and extension activities performed by LSU AgCenter faculty, students and visitors that involve recombinant DNA or potentially hazardous biological materials must be reviewed and approved by the Inter-Institutional Biological and Recombinant DNA Safety Committee. This applies to all such activities which take place on LSU AgCenter land or facilities.

The committee is composed of faculty from many academic disciplines at LSU, nonscientific members and community representatives not affiliated with the university. The objective of the review is to ensure compliance with federal, state and local guidelines and regulations, ensure the safety of faculty, students, employees and visitors, and protect the community and environment from any potential harm arising from these activities.

A review is initiated by the individual responsible for the activity, who completes an online registration and submits it for review. Information in this registration includes the relevant training and experience of each individual involved, the physical location where the activity will be conducted, the specific biological materials involved, specific procedures that will be performed, and physical containment equipment and personal

protection equipment that will be used. Most importantly, the principal investigator is required to do a risk assessment of the potential hazards, if any, to individuals, agricultural crops or animals and identify steps, such as confinement, protective equipment and disposal methods, to be used to control these risks. This risk assessment leads to assignment of a Biological Safety Level that determines minimal practices necessary to protect the individuals performing the work, the community and the environment. Each activity is considered individually.

The final step before a proposal is approved is a physical inspection of the laboratory or facility where the work is to be performed to ensure that any specified physical containment equipment and personnel protection equipment are present and in working order. Once each year the investigator is asked to provide any updates such as personnel changes, and every three years a new registration must be completed and the review process repeated.

Kenneth R. Bondioli is chair of the Inter-Institutional Biological and Recombinant DNA Safety Committee and a professor in the School of Animal Sciences.

Protection for Human SubjectsMichael J. Keenan

Scientists and extension specialists at LSU AgCenter frequently work with human subjects in scientific studies or educational programs. The LSU AgCenter has an Institutional Review Board that reviews research with human subjects to ensure their protection. This review ensures:

■ Benefits of the research outweigh risks. ■ Subjects consent to voluntary participation. ■ Vulnerable populations – such as children, the elderly,

pregnant women and prisoners – are protected.

There are three possible types of review by the board. The first type is called “exempt” and is done by one member for six cate-gories of research types. For example, one category is for children in the normal educational setting, such as school or 4-H. The sec-ond type is called “expedited” and includes, for example, stud-ies with collection of blood samples. This review is also done by one member of the Institutional Review Board. These two catego-ries of review are only allowed if the risk is deemed minimal and not different from most everyday activities. The third type of re-view is by the full committee when the research is not exempt or expedited. The board will determine the category of risk to be

minimal, uncertain or more than minimal. All of these regulations have been established by the federal government. Also, no hu-man subject can be used to benefit another if there is risk for the research subject and no benefit for the research subject. For ex-ample, the testing of a new high-risk drug to regulate blood glu-cose would only be used in subjects that had very poor glucose regulation because they may benefit, and the benefit-to-risk ratio was beneficial for them.

For review of programs of LSU AgCenter extension specialists, there is a checklist to determine if the program is research or not. If the program is not research and approved as not research by the extension specialist’s supervisor, the board will not review the program. Any possible risk would then be in the purview of risk management and LSU AgCenter administration. This is the level of protection of human subjects in programs that are not consid-ered research. Thus, human subjects who participate in either sci-entific research studies or programs not deemed to be research are protected from any risk greater than their possible benefit.

Michael J. Keenan is a professor in the School of Nutrition and Food Science.


Resistant Starch Fermentation and Human HealthMichael J. Keenan

In 2003, LSU AgCenter researchers first became aware of the use of resistant starch to reduce body fat in rats. Resistant starch, which is not digested in the small intestine, is a fermentable fiber found in peas, beans, lentils and some grain products.

Since then, research conducted at the AgCenter has demonstrated many beneficial health effects of resistant starch in the diet of rodents. These include:

1) Reduced body fat when the control diet without resistant starch has the same energy as the diet with resistant starch. This is the effect of fermentation in which bacteria feed on the resistant starch.

2) Increased production of blood levels of hormones from the intestines that promote increased energy expenditure and other health parameters beyond the intestine.

3) Improved insulin sensitivity and blood glucose control.

4) A healthier large intestine demonstrated by measures of fermentation of resistant starch.

5) Increased gene expression for proteins that improve the function and structure of the large intestine.

6) Increased levels of a serum peptide, adiponectin, which prevent inflammation.

7) A healthier microbiota, which is the community of microbes, in the gastrointestinal tract.

LSU AgCenter research focuses on the study of the microbes in the gastrointestinal tract. These microbes consist of bacteria, bacteria-like organisms, viruses and single-celled organisms. Bacteria are the dominant entity. Some have described this community of microbes, or microbiota, as essentially another organ of the body because there are more bacterial cells in the gastroin-testinal tract than cells in the body. Thus, making the microbiota healthier would be good for overall health.

Initially, researchers demonstrated that fermenta-tion of resistant starch resulted in major changes to the bacterial populations in the large intestine of rats. However, this was fairly early in the development of the science of bioinformatics, which aids in identification of genetic material of bacterial genes, DNA. The next step in studying the effects of resistant starch on the microbiota occurred after the science of bioinformat-ics had advanced, and researchers could measure the different types of bacteria involved in fermentation of resistant starch.

Researchers then used an advanced technique called high throughput microbial DNA analysis to assess the entire bacterial community in the large intestine of a mouse model fed resistant starch. By this time bioin-formatics for the microbiota had advanced greatly. The microbiota composition of obese mice has been shown to have a different microbiota than lean mice.

One of the most recent projects involved the investi-gation of the microbiota in Zucker Diabetic Fatty rats. These are obese rats that possess a defective leptin receptor. Leptin is primarily produced in white fat and is a major signal to the brain for the level of body fat. The research group found that this rat model did ferment resistant starch and demonstrated what appears to be a healthier microbiota, even though these rats did not have reduced body fat, which is typical in other studies in rodents with sufficient leptin signaling.

Researchers have found that the composition of healthy microbiota is different among different organ-isms – human, pig or rodent – and is affected by differ-ent dietary fermentable fibers. Resistant starch is one type of fermentable fiber.

One further note is that AgCenter researchers have found that feeding resistant starch in a high fat diet, in which 42 percent of the dietary energy comes from fat, reduced the fermentation. Future studies will include microbiota analyses for rodents fed resistant starch in a high fat diet.

Michael J. Keenan is a professor and researcher in the School of Nutrition and Food Sciences.

Acknowledgements: The author would like to thank his research group – Roy Martin, formerly with the LSU AgCenter and now at U.S. Department of Agriculture Human Research Center in Davis, California; Diana Coulon, manager of the Rodent Bioassay Core Lab for the AgCenter and a veterinarian; Anne Raggio, research associate; Justin Guice, Ryan Page and Diana Obanda, graduate students; and Felicia Goldsmith, a former graduate student and now a post-doctoral researcher at the Pennington Biomedical Research Institute; and collaborators Marlene Janes, professor in the School of Nutrition and Food Sciences, and Claudia Husseneder, professor in the Department of Entomology; Maria Marco at the University of California at Davis; and David Welsh at the LSU Health Sciences Center in New Orleans.


Scientist Helps Ensure Safety for LSU AgCenter Food Incubator ProductsOlivia McClure

When food entrepreneurs first become tenants at the LSU AgCenter Food Incubator, they must begin transforming their recipe from a home kitchen version to something com-mercially viable. Often, that can mean scal-ing up the recipe to make larger quantities or replacing ingredients to make the product shelf stable.

But one of the most important early steps incubator tenants must take is ensuring their product is safe for consumers, said AgCenter food scientist Luis Espinoza.

“Food safety is all about preventing foodborne pathogens,” he said. “Your pro-cessing has to be designed to kill the target pathogen.”

After reviewing a tenant’s recipe and pro-cess, Espinoza determines which U.S. Food and Drug Administration product category it falls into. The different categories, which include everything from low-acid canned foods to dairy products, each have their own set of rules for processing.

The process depends on the prod-uct because every product is different, Espinoza said.

Pickled products, for example, must be thermally processed, he said. Those prod-ucts, along with any others that are cooked, have to reach a certain temperature that is maintained for a designated holding time. Next, samples are tested to verify there are no pathogens present.

Cooked products should be packed while they’re still hot — above 185 degrees — and gradually cooled down.

“If you cool it down before packing and it’s exposed to the environment, the air is full of food spoilage microorganisms,” Espinoza said. “Something’s going to get in there. While it’s still hot, they won’t develop.”

There is a well-established process for dairy products, which must reach and stay at a minimum of 145 degrees for 30 minutes in a pasteurizer.

But some products — like the new ones Food Incubator tenants are known for craft-ing — don’t neatly fit into the FDA’s catego-ries or existing rules. That’s when Espinoza starts doing trials in his lab to find ways to

eliminate dangerous pathogen populations and meet regulations.

The FDA and state Department of Health and Hospitals are kept in the loop through-out the process. Espinoza helps tenants write a process authority review letter — known as a “PA letter” — that includes a product description and processing instructions with weights, temperatures and pH levels. The FDA requires the letter for processors of low-acid and acidified foods, or those with acid added to them.

While the letter is a requirement, it also gets tenants in the habit of keeping docu-mentation of how they process their prod-ucts, Espinoza said.

“It will help you have a consistent quality and a safe product,” he said.

Sometimes, recipes have to be changed to ensure their safety. In those cases, it is especially important to help tenants understand food safety and why it matters, Espinoza said.

The AgCenter’s School of Nutrition and Food Sciences hosts a four-day Better Process Control School twice a year — in June and January. Attendees of the pro-gram are certified per FDA guidelines for supervising the production of low-acid and acidified foods. AgCenter experts teach the courses.

Olivia McClure is a graduate assistant with LSU AgCenter Communications.

Luis Espinoza tests product safety at the LSU AgCenter Food Incubator. Photo by Olivia McClure


Plant Pathogens Threaten the Louisiana Plant WorldLawrence E. Datnoff

The disease-conducive environment in Louisiana and the frequent incursions of new pathogens into the state make it clear that plant diseases are, and will continue to be, one of the most limiting factors in crop production. Plant pathologists will continue to address future threats with a comprehensive approach, including the development of resistant varieties and other methods of control. They will increase the use of molecular methods to aid in rapid, precise diagnosis and the use of GPS and other new technologies to monitor and predict spread of new diseases. The goal is to develop methods of disease management that are effective and environmentally friendly.

Plant diseases caused by microbes – including plant pathogenic fungi, bacteria, viruses and nematodes – have seriously limited the development and production of agronomic and horticul-tural crops in Louisiana. Although a number of control strategies have been developed, new disease threats continually appear. This can occur because of changes in crop varieties and production practices or because new plant pathogens are introduced into Louisiana from other states or countries. Each time a new pathogen arrives or a new disease outbreak occurs in Louisiana, LSU AgCenter scientists in the Department of Plant Pathology and Crop Physiology, operating the only program of its type in the state, provide leadership for gener-ating knowledge and applied solutions for managing these diseases. Following are examples of important pathogens causing continuing plant disease threats to agricultural production.

Brown Rust of SugarcaneBrown rust of sugarcane (Figure 1), caused by a fungus named

Puccinia melanocephala, has been in Louisiana since the late 1970s. The disease causes reddish-brown leaf lesions that result in reduced yield. Severe winters limit this pathogen because it must survive from season to season in living leaf tissue. In the early 2000s, a series of mild winters resulted in brown rust becoming the most important disease problem in what is the most valuable row crop in Louisiana. Rust fungi are highly adaptable plant pathogens that are able to overcome host plant resistance. Breeding for resistant varieties is the primary control measure for brown rust. However, resistance has been overcome in 11 of the last 13 varieties released, probably due to pathogenic vari-ability in the population of the pathogen. Therefore, LSU AgCenter plant pathologists developed a controlled conditions inoculation system to further evaluate resistance. Experiments proved that spe-cialization existed within the pathogen population to varieties. The test results also revealed that one variety that has remained resistant under cultivat

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