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Agnet Oct. 6/05
Iowa grain elevators turn away crops affected by alfotoxin
Chewing against SARS
Engineering broad-spectrum disease resistance
Dow Agrosciences, Sangamo biosciences announce research and commercial license agreement in plant agriculture
Athenix reports successful field trials with novel genes providing high-level tolerance to the herbicide glyphosate
A fly, new to North America, hunts down greenhouse pests
Food safety gets a facelift
Cargill to process Monsanto's low-linolenic soybeans
Application of a regeneration QTL gene to plant transformation
Sing transcriptional analysis to determine responses to phosphate deprivation
Delivering a deadly drop to locusts
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Iowa grain elevators turn away crops affected by alfotoxin
October 5, 2005
Associated Press
Amy Lorentzen
Farmers in about a dozen drought-stricken counties in eastern Iowa are, according to this story, suffering more woes as grain elevators turn away their crops because of a toxin encouraged by the dry weather.
Virgil Schmitt, a crop specialist for Iowa State University Extension, was cited as saying the problem is with aflatoxin, caused by a mold that grows in kernels damaged by insects or heat stress while they are still on the stalk, adding, "We had a very hot, dry summer here, and we did have the right conditions for the mold to enter the kernels."
The story notes that other areas of the country that suffered drought conditions also have recorded high aflatoxin levels, including parts of Texas .
In Iowa , the problem came to light mostly in the southeastern part of the state over the past three weeks as farmers harvested their fields and headed to the grain elevators. Some elevators have turned away loads of corn, and had some rejected by processors, extension officials said. No figures were available on how much grain was found to contain high levels of aflatoxin.
The Food and Drug Administration has set limits on how much aflatoxin can go into general trade at 20 parts per billion.
A spokeswoman for the Iowa Department of Agriculture was cited as saying the agency was testing for aflatoxin in 26 counties, but results weren’t expected until later this week. Schmitt said much of the grain the extension is seeing is measuring in at between just over 20 ppb to about 100 ppb.
Chewing against SARS
October 6, 2005
Checkbiotech
Shelley Jambresic
The rapid spread of Severe Acute Respiratory Syndrome, more commonly known as SARS, at end of 2002 – coupled with a mortality rate close to 20 percent – caused an instant demand for an effective vaccine in order to prevent another outbreak of the disease. In order to produce an effective and inexpensive vaccine, Dr. Koprowski and his team from the Thomas Jefferson University in Philadelphia , expressed the recombinant SARS antigen in transgenic plants.
SARS first appeared in 2002 in China , where it spread rapidly, reaching neighbouring areas as well as international countries. Even though no cases of SARS were reported in late 2004 and early 2005, no one is underestimating another future outbreak. “We understand that the disease is currently contained, but the threat remains strong,” Dr. Koprowski told to Checkbiotech.
SARS shows flue-like symptoms with moderate to high fevers, and about 10-20 percent of the infected individuals require mechanical ventilation. Depending of the age of the infected, the mortality lies between 6.8 percent (in people under the age of 60) and 55 percent (for patients over 60 years old).
The virus causing the disease is officially called the SARS coronavirus. Coronavirus is a genus of animal viruses, named after the “corona” - the ring-like structure formed by the atmosphere of the sun - due to a similar viral structure that can be seen from electron microscopy pictures. The ring-like structure is caused by different proteins on its surface, one of them being called spike protein (S protein).
Several researcher groups have showed that the S proteins are the major inducers of an immune response. Proteins that cause an immune response, such as S proteins, are known as good antigens, which can be used to make effective vaccines. Due to this insight, the S proteins are currently considered to be the best agents for production of a vaccine against the disease.
There are different types of vaccines, but all of them have the same aim - to protect an organism against an infection. For this purpose the body produces antibodies, which attach to the foreign substances, triggering other cells of the immune system to destroy the foreign invader.
There are five major types of antibodies of which IgG are the smallest ones. They are found in all body fluids and represent 75 to 80 percent of all the antibodies in the human organism. IgG are the only type of antibody which can cross the placenta and thus help protect the foetus. Another important group are IgA. They represent about 10 to 15 percent of all antibodies and protect body surfaces that are exposed to the outside.
Nowadays, vaccination by inoculation can be very expensive. That is why Dr. Koprowski, and his team from the Thomas Jefferson University in Philadelphia , worked towards an inexpensive, effective and safe vaccine. To satisfy these goals, Dr. Koprowski decided to use plants as a possible oral vaccine for SARS.
The first step was to express the S1 gene, which produces the S protein, in transgenic plants such as tomato and low-nicotine tobacco. More recently also other plants were modified. “We tested several crop plants such as lettuce, red beat, cabbage and some other plants from the Brassicaceae family,” Dr. Koprowski told to Checkbiotech.
For oral immunization, the researchers fed the transgenic plants to mice using different methods. With the first group, each mice received 500mg of dried tomato fruit over a period of four to five hours, while with the second group, 50mg of dry tobacco root material reconstituted in saline were given three times by gastric incubation at intervals of 2 weeks. As control, a separate group of mice was fed the same amount of wildtype (non-transgenic) plants.
Before and 10 days after each immunization, sera from each mouse were collected and tested for the presence of antibodies.
Whereas no increased levels of antibodies was found in mices fed with non-transgenic plants, the group fed with tomato fruit showed a significant increase of IgA level. Nevertheless the group exhibited no increase in the IgG level.
”This experiment was limited by the quantity of transgenic tomato fruit material,” Dr. Koprowski explains in the publication published in the PNAS .
The second mice group, which was fed parenterally with dried tobacco root material, showed no increase in level of S protein reactive antibodies. Yet after a booster dose of S protein, the group showed a significant increase of the IgG antibodies, likely to a secondary immune response achieved with commercially obtained S protein.
The different results might be explained by the higher amounts of antigen in the tomato fruits, or by the longer mucosal exposure to the antigen by the process of chewing compared to the parenteral feeding of the tobacco root material.
However, the required boosting after an immunization with the tobacco root material was not surprising, since similar observations were made in several earlier studies. “Necessity of boosting immunization may be due to the quantity and nature of expressed antigen as well as usage of adjuvants,” Dr. Koprowski and the team explained in their publication.
”Recently, we have tested another approach for oral immunization of mice,” Dr. Koprowski told Checkbiotech. Using the same transgenic lines and comparable doses as in the first experiment described, the mices were fed directly with three doses of tobacco roots within intervals of four weeks.
”We detected in the serum of these mice a distinct IgG immune response, after the third feeding without the further boost.”
Still, the researcher team have not yet infected the mice with higher levels of S protein reactive antibodies with SARS to see if they are now protected against future infections. “Due to safety issues, we don’t have a permission to carry out these kinds of experiments at the University premises,” Dr. Koprowski explained. “Therefore, we have contacted several labs that are authorized to work with the SARS virus and they agreed to test our material.”
Further research will also include testing with other plants as well as other animals. “We are going to evaluate a sufficient amount of plant material, which has a SARS antigen expression for oral feeding. This purely empirical study will be conducted in mice and pigs,” Dr. Koprowski explained.
When asked when a commercially available vaccine might be ready, Dr. Koprowski answered, “Development, testing and approval of new vaccines, is a lengthy process. We are trying to make the research and development stage as short as possible. Once we have a continuous supply of transgenic plants, with stabilized amounts of antigen expression, we will shift our efforts to extensive animal testing.” That might include many factors such as storage of the plant material, feeding protocols or stabilization of the antigens in the transgenic plant.
Shelley Jambresic is a Science Writer for Checkbiotech in Basel , Switzerland .
Engineering broad-spectrum disease resistance
October 6, 2005
ISB News Report
Santosh Misra
The global production of agricultural and food products is the world’s largest industry, with total revenues of ~$5 trillion (US) per year. Diseases caused by phytopathogens are economically important, resulting in multibillion-dollar losses annually, and it has been estimated that more than 25% of all crop plants worldwide are lost to fungal, bacterial, and viral diseases.
In the developing world, up to 40% of all crop destruction can be directly attributed to plant diseases, with occasional catastrophic, life-threatening losses. The increased reliance on chemical solutions to diseases has compromised environmental quality, engendered a negative impact with consumers, and resulted in a rise of fungicide resistant microorganisms.
Conventional breeding, though relatively successful in the development of disease resistant plant cultivars, has become increasingly difficult because of the limitations of resistance genes within usable gene pools. However, advances in plant genetic engineering have facilitated the integration of beneficial “designer” genes into plants. This technology has been successfully applied to generate plant resistance to herbicides, some insects, and occasionally some phytopathogens. Strategies to improve the health of crops through genetic engineering have included transgenic expression of plant, fungal, or bacterial hydrolytic enzymes, pathogenesis-related proteins, components of plant defense-response pathways, antimicrobial proteins, and peptides. Although food safety is of utmost importance and disease resistant plants are required, the commercial application of this technology is lagging. The protection offered in some cases is limited to specific diseases, which means other pathogens can thrive and plants must still be sprayed with fungicides.
To address this problem, we have developed a platform methodology of protecting plants from a broad-spectrum of bacterial and fungal diseases. Work is in progress to extend this spectrum to viruses and insects. The technology is based on engineering antimicrobial peptides in plants. Because of their wide spectrum of antimicrobial activity and, at low concentrations, lack of toxicity to eukaryotic cells, the antimicrobial peptides represent promising candidates for transgenic application in plants.
Candidate antimicrobial peptides
Recently-discovered antimicrobial peptides are a class of potent, natural antibiotics found widely in nature from insects to plants and animals1. Several related antimicrobial peptides have also been found in humans, where they are part of our early defense “innate” immune system. Depending on the source, these natural products vary greatly in their killing activity and spectrum, which extends from various types of bacteria to fungi, viruses, parasites, and even to such destructive pests as nematodes. The mechanism for killing microbial cells is multi-targeted and highly effective, and involves rapidly killing the microbe by drilling a molecular “hole” in its membrane, then inactivating and destroying its genome. Because the primary target of membrane-active, positively charged antimicrobial peptides is the cell membrane and not specific receptors or substrates, these peptides usually confer their activity against a broad spectrum of pathogenic microorganisms, and there is less probability of resistance arising by variation of its metabolic pathways.
Antimicrobial peptides of plant, insect, and amphibian origin have been expressed in transgenic plants, but only a few provide the target plants with any degree of broad-spectrum antimicrobial resistance. We have pioneered the development of unique, candidate target peptides (probiotics) with powerful broad-spectrum antimicrobial activities but with reduced cytotoxicity to plant and animal cells. This was achieved by using Insight II molecular modeling, and 2-D NMR structural determinations, combined with extensive screening against a variety of plant pathogens (collaboration: REW Hancock, UBC). These approaches have yielded three new target families of probiotics with fungal, bacterial, and viral disease resistance.
Shown below is a predicted model of temporin A and its modified analogue MsrA32. Using the Insight II (version 97.2) molecular modeling program, Homology (Molecular Simulations Inc., San Diego, USA), the temporin structure was drawn as an Éø-helix, based on the known Éø-helicity of temporin. The structure was then energy minimized using the Discover Program of Insight II.
Engineering select peptide / probiotics in plants
We used one of these peptides, MsrA1, to engineer broad-spectrum disease resistance in potato, including resistance to ‘late blight’, a disease of pandemic proportions2. Potato is an ideal crop for the introduction of disease-resistance technology due to its severe and growing problems with bacterial, fungal, and viral diseases and accompanying storage losses.
The probiotic-enhanced potatoes demonstrated a phenomenal degree of broad-spectrum disease resistance to pathogens, including late blight and pink rot, and a variety of post-harvest pests. Potato tubers stored for over 27 months remain in nearly pristine condition when compared to unprotected tubers. These results have profound implications not only for diseases in the field but also with respect to the mitigation of storage crop losses ($600B worldwide).
We have continued to test new peptides in potato and tobacco. Our recent data shows that MsrA3, a temporin analogue3, as well as MsrA2, derived from Dermaseptin4, and combinations thereof, provide a greater degree of disease resistance against a broad range of pathogens. When MsrA2 was used to inhibit the growth of agronomically important fungal pathogens, including Fusarium, Alternaria, Rhizoctonia, Phytophthora , and Pythium sp., its activity was far superior to the activities of other peptides tested. The estimated concentration of MsrA2 in leaf tissue was ~ 1 – 5 µg/g of fresh tissue, which, although not high, seems to be sufficient to protect the plants from the attack of pathogen(s)4. Constitutive expression of MsrA2 at this level is apparently non-toxic to transgenic plants, as no deleterious effects on the morphology or yield of plants and tubers could be seen.
Regulated transgene expression, whereby a promoter is specifically activated in response to pathogen invasion or pest attack, has distinct advantages for genetic engineering disease/pest resistant traits in plants. We used a truncated 823 bp downstream fragment of the win3.12 promoter from poplar and showed that the win3.12 promoter-regulated expression of the antimicrobial peptide was sufficient to confer resistance against F. solan i in transgenic tobacco.
This technology is considered a flexible platform, which can be extended to a host of economically important food and non-food crops. Equally important, plants can be used for cost-effective production of the peptides/probiotics destined for human pharmaceuticals and veterinary applications. We have extended this work to include canola, soybean, wheat, and even poplar, a model tree species.
Mycotoxins and food/feed safety
In addition to managing diseases, probiotic-enhanced plants can address the problem of food and feed safety. Phytopathogenic fungi not only cause a decrease in quantity and quality of crops, they often cause acute toxicity in animals and humans. Widely different genera of fungi ( Fusarium, Penicillium, Aspergillus, Claviceps, Stachybotrys sp. etc.) produce mycotoxins in a wide variety of grains and foods. Mycotoxins are highly stable and cannot be destroyed by boiling, pressing, or processing, and infested produce has to be destroyed. Toxicological manifestations are both acute and chronic, such as cancer, immunosuppression, mutagenicity, and estrogenicity, and gastrointestinal, urogenital, vascular, renal, and nervous disorders. These mycotoxins can also be metabolized by animals fed contaminated grains and passed into milk, eggs, and other organs, thus reentering the food chain. Mycotoxin contamination is a worldwide problem affecting staple crops such as corn and small grains, as well as tree nuts, peanuts, sorghum, and many others. Maize, wheat, barley, and rice are high-risk commodities, and it is estimated that approximately 16,000 tons of maize, 123,000 tons of wheat and barley, and 12,000 tons of rice are affected by mycotoxins in southeast Asia alone. According to FAO estimates, world losses of foodstuffs due to mycotoxins are in the range of 1 B tons per year. One strategy to control mycotoxin levels is by controlling the growth of fungi.
We are testing the ability of our peptide probiotics to control Fusarium Head Blight (FHB) of wheat and barley caused by Fusarium graminearum . FHB has emerged as one of the most serious and damaging diseases of small grains. Trichothecenes are the virulence factors produced by the fungus. We are introducing probiotic genes into wheat in order to increase the FHB defense mechanism in wheat spikes and hence reduce or prevent the initial infection.
Summary
Our current growing reliance on antimicrobial chemicals remains unabated and is compromised due to the rise of fungicide and bactericide resistant microorganisms. We believe that the widespread incorporation of probiotic-enhanced plants and crops will make a large contribution to the general reduction in chemical solutions to disease control, increase yields, and reduce losses during storage. Furthermore, the antimicrobial peptides do not readily lead to microbial resistance and would help stem the rise of antibiotic and fungicide resistant microorganisms directly threatening human and animal health. The importance of feed or food microbial contamination in the industry is expected to increase with time. The emergence of pathogen strains resistant to conventional antibiotics and pesticides currently in use warrants application of new approaches to the containment of pathogenic microbes and to enhance food safety.
References
1. Hancock REW Lehrer R (1998) Trends Biotechnol 16: 82–88 2. Osusky M et al. (2000) Nature Biotechnol 18: 1162–1166 3. Osusky M et al. (2004) Transgenic Research 13: 181-190 4. Osusky M et al. (2005) Theoretical Applied Genetics DOI:10.1007/s00122005-2056-y
Santosh Misra Department of Biochemistry Microbiology University of Victoria smisra@...
Dow Agrosciences, Sangamo biosciences announce research and commercial license agreement in plant agriculture
October 6, 2005
Dow AgroSciences
INDIANAPOLIS , IN and RICHMOND , CA - Dow AgroSciences LLC, a wholly owned subsidiary of The Dow Chemical Company (NYSE: DOW), and Sangamo BioSciences, Inc. (Nasdaq: SGMO) today announced the signing of a Research and Commercial License Agreement. The agreement provides Dow AgroSciences with access to Sangamo’s proprietary zinc finger DNA-binding protein (ZFP) technology for use in plants and plant cell cultures to develop products in areas including, on an exclusive basis, plant agriculture and industrial products, and, on a non-exclusive basis, animal health and biopharmaceutical products produced in plants. “Dow AgroSciences has a strong tradition of innovation and early adoption of new technologies. We pride ourselves on operating at the cutting edge of plant biotechnology in our mission to provide products that improve the quality and quantity of the earth’s food supply and contribute to improving the health and quality of life of the world’s growing population,” said Dan Kittle, vice president, Research and Development for Dow AgroSciences. “We believe that access to Sangamo’s ZFP technology will ensure an early and sustainable competitive advantage for our business. We also look forward to working with the public research sector and other companies to fully develop and apply this technology to plant crop improvement.” ”Dow AgroSciences is recognized as a world leader in innovative plant biotechnology,” said Edward Lanphier, Sangamo’s president and chief executive officer. “Sangamo has demonstrated that our ZFP technology provides a robust and broadly applicable approach for both gene regulation and gene modification in a wide range of organisms. Our business strategy has always been to maximize the commercial potential of this technology across all fields of use. We believe that the combination of our novel technology with Dow AgroSciences’ proven experience in development of agricultural biotech products will enable us to accomplish this goal in plant agriculture. In Dow AgroSciences, we have a partner that shares our vision and is capable of fully exploiting the applications of ZFP transcription factors (ZFP TFs™) and ZFP nucleases (ZFNs™) in plants.” ZFPs are the dominant class of naturally occurring transcription factors in organisms from yeast to humans. Transcription factors, which are found in the nucleus of every cell, bind to DNA to regulate gene expression. The ability to selectively control specific genes is emerging as a critical tool in modern biotechnology. Though there are many kinds of transcription factors, only ZFPs are amenable to engineering and precise targeting to a particular gene or genes of interest. By engineering ZFPs that recognize a specific DNA sequence Sangamo scientists have created ZFP TFs™ that can control gene expression and consequently, cell function. For example, Sangamo has demonstrated that plant oils can be improved using ZFP TFs™. Sangamo has also developed sequence-specific ZFNs™ for precision gene modification and targeted gene insertion. These technologies have the potential to play a major role in bringing new discoveries in genomics forward to the marketplace. According to a 2004 International Service for the Acquisition of Agri-biotech Applications (ISAAA) report, transgenic traits were planted on an estimated 200 million acres, or 29 percent of the global acres for soybean, cotton, maize, and canola. Phillips McDougall, international crop protection and agricultural biotechnology consultants, estimates the value of agricultural biotechnology in these crops for 2004 to be $4.7 billion. Both the acreage and the value of agricultural biotechnology are expected to grow. This increasing demand could be addressed by the use of Sangamo’s ZFN and ZFP technologies for combinations or stacks of multiple traits and new traits. Investments globally in genomics are also revealing large numbers of genes with the potential to substantially improve crop quality, expand crop uses and improve agronomic performance. About Dow AgroSciences, LLC Dow AgroSciences LLC, based in Indianapolis , Indiana , USA , is a global leader in providing pest management, biotechnology and crop products that improve the quality and quantity of the earth’s food supply and contribute to improving the health and quality of life of the world’s growing population. Dow AgroSciences has approximately 5,500 people in more than 50 countries dedicated to its business, and has worldwide sales of U.S. $3.4 billion. Dow AgroSciences is a wholly owned subsidiary of The Dow Chemical Company. For more information about Dow AgroSciences, visit www.dowagro.com . About Sangamo Sangamo BioSciences, Inc. is focused on the research and development of novel DNA-binding proteins for therapeutic gene regulation and modification. The most advanced ZFP Therapeutic™ development programs are currently in Phase I clinical trials for evaluation of safety in patients with diabetic neuropathy and peripheral artery disease. Other therapeutic development programs are focused on macular degeneration, ischemic heart disease, congestive heart failure, neuropathic pain, and infectious and monogenic diseases. Sangamo’s core competencies enable the engineering of a class of DNA-binding proteins known as zinc finger DNA-binding proteins (ZFPs). By engineering ZFPs that recognize a specific DNA sequence Sangamo has created ZFP transcription factors (ZFP TF™) that can control gene expression and, consequently, cell function. Sangamo is also developing sequence-specific ZFP Nucleases (ZFN™) for therapeutic gene modification as a treatment for a variety of monogenic diseases, such as sickle cell anemia, and for infectious diseases, such as HIV. For more information about Sangamo, visit the company’s web site at www.sangamo.com
Athenix reports successful field trials with novel genes providing high-level tolerance to the herbicide glyphosate
October 6, 2005
Athenix Corp.
NORTH CAROLINA - Athenix Corp., a leading biotechnology company developing novel products, technologies, and processes for agricultural and chemical applications, announced today it has completed successful field trials of transgenic plants containing proprietary glyphosate tolerance genes. Athenix has discovered a new class of genes that provide high levels of tolerance to glyphosate, a herbicide commonly used to kill weeds. Results from these field trials demonstrate that plants expressing these genes can withstand 16 times the amount of glyphosate recommended for currently available glyphosate tolerant corn. “These remarkably successful field trials mark a major product development milestone for Athenix. Herbicide tolerance is one of our key products. And, while important in its own right, when combined with our nematode and insect resistance technologies, these genes allow Athenix to provide the Crop Production Industry with the most advanced trait stack products,” said Mike Koziel, President and CEO of Athenix. ”Athenix has successfully demonstrated the efficacy of its proprietary genes in plants under field conditions. This breakthrough allows Athenix to move quickly towards the introduction of this trait into our other key crops,” added Nadine Carozzi, Vice President of Product Development of Athenix. Athenix has discovered the next-generation of glyphosate tolerance genes and has filed for broad patent protection on this new class of genes and the proteins they encode. The genes tested in the field this year represent the first in a newly discovered class of genes which encode enzymes capable of providing high levels of tolerance to the popular herbicide glyphosate. Crops tolerant to this herbicide have been rapidly adopted by growers worldwide and demand continues to grow. About Athenix Corp. Athenix is a leading biotechnology company that discovers and develops novel products, technologies, and processes for the agricultural and chemical businesses. Athenix is focused on developing enhanced plants, microbes, enzymes and processes with emphasis on two major market opportunities: 1) the discovery of genes and proteins for novel input traits such as insect resistance, nematode resistance, and herbicide tolerance, and their use to develop transgenic plants for the Crop Production Industry; and 2) the discovery of genes and proteins for use in the Chemical and Animal Feed Industries. For more information about Athenix, visit www.athenixcorp.com .
A fly, new to North America, hunts down greenhouse pests
October 6, 2005
ARS News Service
Agricultural Research Service, USDA
View this report online, plus any included photos or other images, at www.ars.usda.gov/is/pr
Insects in North American greenhouses had best beware: There’s a new predator among them.
Agricultural Research Service (ARS) scientists have helped Cornell University colleagues make the first-ever identification on this continent of the Old World hunter fly, Coenosia attenuata.
This winged predator is originally from Europe , where it’s also known as the “killer fly.” A member of the same insect family as the common housefly (Muscidae), the Old World hunter fly preys upon some of the insects that greenhouse keepers hate the most. These include fungus gnats, shore flies, leafminers, fruit flies, moth flies and some leafhoppers.
The Old World hunter fly’s presence here was confirmed in studies by Cornell graduate student Emily Sensenbach, under the direction of ecologist Steve Wraight of ARS’ Plant Protection Research Unit (PPRU) and associate professor John Sanderson. The PPRU is located on Cornell’s Ithaca , N.Y. , campus.
According to Wraight, this particular fly lives up to its name—and not just because it preys upon other flying insects. Apparently, it enjoys a challenge.
It sits, waits and only pursues prey that is in flight. When it catches its target, the fly punctures it with a daggerlike mouthpart and consumes the liquid inside. Its soil-dwelling larvae are also predatory, feeding mainly on larvae of other insects.
This fly was first noticed in the United States in 1999 in Onondaga County, N.Y. Wraight is not certain exactly how it got to the New World, but suspects that the horticulture industry played a role.
He added that the fly was seen in South America , southern Asia , Africa , the Canary Islands , New Guinea and Australia before being identified here.
According to Wraight, there is considerable potential for using hunter flies in biological control of insect pests.
Read more about the hunter fly research, which is funded through the U.S. Department of Agriculture’s Floriculture and Nursery Research Initiative, in the October issue of Agricultural Research magazine, available online at: http://www.ars.usda.gov/is/AR/archive/oct05/pests1005.htm
ARS is the USDA’s chief in-house scientific research agency.
Food safety gets a facelift
October 6, 2005
Guelph Mercury SPARKPlug
Alicia Roberts
Food safety practices must start at the source – that is, on the farm—to be truly effective. But such practices are tough for some horticultural producers. University of Guelph graduate student Ben Chapman, Department of Plant Agriculture, is working towards developing effective food safety strategies that are also easier for producers to implement. “We evaluated on-farm food safety programs for horticultural producers with an emphasis on greenhouse production,” says Chapman. “An effective food safety strategy will centre around bacteria. There’s a big stigma out there about pesticides being bad, but really, bacteria is what you need to look for.” Since 1990, more than 400 illness outbreaks have been linked to bacteria in produce in North America . Most of those outbreaks were made public in the United States , and only a small percentage were traced back to Canadian produce. But the low illness origin rates in Canada could be lulling Canadian producers into a false sense of security. Chapman wanted to identify the barriers that interfere with current on-farm food safety practices, and how food safety guidelines—such as those put forward by the Ontario Greenhouse Vegetable Growers (OGVG) -- could be improved. He interviewed horticultural producers from across the province, observed food safety practices and looked for possible contamination in tomato, cucumber and water samples from greenhouse operations. In particular, he tested for fecal matter contamination by looking for indicator bacteria. Chapman found minor traces of microbial contaminants, which he says demonstrates contamination can happen even at the production level. He also found that producers who deal with larger U.S. markets are more aware of food safety issues and strategies than smaller, locally based producers. As well, he found some producers understood that more can be done to ensure food safety, but were unclear about where to go for help. The costs associated with food safety practices were also a deterrent. OGVG responded to this sampling study by introducing a more strict food safety program. Chapman compared the OGVG food safety strategy to other food safety programs that incorporated third-party auditing, such as Primus Labs, Silliker Labs and the American Institute of Baking. And finally, he measured the strategy against food safety guidelines from the Canadian Horticultural Council, the United States Food and Drug Administration, and the United Fresh Fruit and Vegetable Association. He found the programs were very similar to each other. The difference was personnel. “What’s really lacking is an implementation aid, someone to help producers apply on-farm food safety practices and understand how to use them,” he says. Chapman says producers consider implementing an on-farm food safety program an expensive endeavour. Initial costs associated with starting the practices are comparatively low, but the budget begins to balloon when third party auditors and safety verification enters the picture. He says these expensive processes can help gain markets, but don’t guarantee a safe product. Chapman’s research has helped promote changes to the OGVG food safety guidelines. An implementation aid is now an included component of the start-up process for producers and OGVG has also linked third party auditing with grower, packer and marketer licenses for 2006 through 2008. “Most greenhouse producers are now up to speed about what they should be doing,” says Chapman. “It’s time to prove that the industry is aware of issues and demonstrate that we can take action.” Chapman’s research was supervised by Prof. Doug Powell, Department of Plant Agriculture, and sponsored by the Ontario Ministry of Agriculture, Food and Rural Affairs, and the OGVG.
Cargill to process Monsanto's low-linolenic soybeans
October 6, 2005
IFT Daily Newsletter
http://www.ift.org/cms/
Monsanto and Cargill, Inc. announced today that Cargill will be a participating processor of Monsanto's Vistive™ low-linolenic soybeans in 2006 and will continue to accelerate the marketing of VISTIVE oil for use by the food industry. According to the company, low-linolenic soybeans will reduce the need for partial hydrogenation of soybean oil, helping food companies reduce the presence of trans fatty acids (trans fats) in their products. Cargill is one of the initial Vistive processors for 2005, and it will continue in that capacity.
For the 2006 growing season, Cargill will be contracting with growers in Iowa for up to 150,000 acres of Vistive soybean production. Cargill will pay a premium to producers who grow the soybeans under contract, then it will crush and sell the processed soybean oil to food companies.
Application of a regeneration QTL gene to plant transformation
October 6, 2005
ISB News Report
Asuka Nishimura
Plant culture systems are vital to many areas of plant science and crop improvement, particularly in plant transformation. The ability of plants to regenerate is essential for establishing a successful plant culture system. However, not all plant species or varieties can regenerate easily. Generally, it is difficult to culture and regenerate agronomically important crops such as rice, wheat, and maize. In rice, an efficient culture system using mature seeds has been established for some research model varieties such as Nipponbare ( Japonica ) and Kasalath ( Indica ). Conversely, some leading varieties used for food production, such as Koshihikari ( Japonica ) in Japan and IR64 ( Indica ) in tropical countries, have low regeneration ability, which is a serious obstacle to production of transgenic plants. The ability to regenerate is mainly controlled by quantitative trait loci (QTLs); however, no specific gene has yet been identified and the molecular mechanisms of regulation are not well understood. Therefore, the challenge is to identify optimal culture conditions required for regeneration in varieties with low regeneration success. A potential solution to this problem is to identify the QTL genes associated with regeneration ability, and to transfer the high regeneration ability QTL gene(s) into low regeneration varieties. However, this method is especially laborious and gives no understanding of the molecular mechanisms of plant regeneration. Recently, we succeeded in isolating a rice regeneration QTL gene using a map-based cloning method1. Isolation of a rice QTL gene in Japonica rice
We first chose Kasalath ( Indica ) as a high-regenerative variety to cross with a low-regenerative variety, Koshihikari ( Japonica ). The resulting Koshihikari x Kasalath F1 plants were backcrossed with Koshihikari to produce 99 BC1F1 plants for which the genotypes of each plant were determined. Next, twenty BC1F2 seeds from each BC1F1 line were tested for regeneration ability and then subjected to QTL analysis. As a result, we found four putative QTLs located on chromosomes 1, 2, 3, and 6 in which Kasalath alleles were correlated with a positive effect on regeneration ability. The QTL located at around 45.4 cM on chromosome 1 had the largest effect and was designated Promoter of Shoot Regeneration 1 ( PSR1 ). Fine mapping analysis of ca. 3,800 BC3F2 seeds revealed that PSR1 lies within a 50.8-kb interval between two molecular markers. In this region, four putative genes were predicted. Complementation analyses with several fragments covering each candidate gene indicated that the PSR1 gene may be a putative ferredoxin-nitrite reductase ( NiR ) gene. We compared the NiR sequences of Koshihikari and Kasalath and found many polymorphisms, especially in the promoter regions. Therefore, we examined NiR expression in the callus using semi-quantitative RT-PCR. NiR expression was detected in both Koshihikari and Kasalath, but the expression level was much lower in Koshihikari. Immunoblot assays also showed that the NiR protein was expressed at a much higher level in Kasalath than in Koshihikari. Furthermore, enzymatic analyses revealed that NiR activity in extracts of Koshihikari calli was about 22-times lower than that of Kasalath. We also examined the relationship between the NiR activity in calli and regeneration ability in various kinds of Japonica rice varieties and found that the varieties with high regeneration ability always had high NiR activity, and the low regeneration varieties showed low NiR activity. This indicates that NiR activity is a determining factor for the regeneration ability of Japonica rice varieties. NiR catalyzes the reduction of nitrite to ammonium and is a key enzyme in nitrate assimilation. Nitrate is commonly used as a nitrogen source for plant cell culture but its metabolite, nitrite, has a toxic effect on plant cell growth. Thus, rapid metabolism of nitrite is crucial for plant cell growth and regeneration. In this context, it is reasonable that Kasalath calli, having higher NiR activity, can rapidly metabolize nitrite while Koshihikari calli cannot. We measured the accumulation of nitrite in callus culture media and found an appreciable accumulation of nitrite ions in the Koshihikari culture medium, but we were not able to detect nitrite in the Kasalath medium. It is likely, then, that nitrite assimilation is a critical process for rice plant regeneration. In Koshihikari, lower NiR activity was detected not only in calli, but also in normally grown root and leaf tissues. However, no growth defects were observed in Koshihikari. Therefore, it was thought that the nitrate concentration in the culture medium was not suitable for Koshihikari regeneration. We tried Koshihikari culture using several nitrogen conditions and could not find the best medium for Koshihikari regeneration. As shown in these experiments, QTL gene isolation can be an efficient method for understanding regeneration mechanism(s) and finding optimum culture condition(s). In our preliminary studies, it was shown that the key genes regulating regeneration ability in Indica rice varieties were different from those in Japonica rice. An Indica variety such as IR64 has high NiR activity but low regeneration ability. We are now using QTL gene isolation to resolve the low regeneration ability in Indica rice using a strategy similar to the one we used with Japonica rice. We think these results in rice will help improve the regeneration ability of other crops and elucidate the molecular mechanisms of plant regeneration. Application of NiR in transformation
Genes encoding antibiotic and herbicide resistance are widely used as selection markers in plant transformation. Recently, a positive selection system based on the E. coli phosphomannose isomerase (PMI) gene as a selection marker was developed in various plants, including rice2. These selection markers are exogenous to plants. Moreover, several methods have been reported to generate selection marker-free transgenic plants, including site-specific recombination and intrachromosomal recombination to remove the selection marker, co-transformation, and transposable elements to segregate the selection marker3. A transformation method in chloroplasts has been established without using an antibiotic resistance gene4. However, these methods are generally time-consuming and inefficient. We hypothesized that the NiR gene we identified could be used as a selection marker for gene transformation because the introduction of the Kasalath NiR genome sequence conferred regeneration ability on Koshihikari. If the Kasalath NiR gene is co-delivered with the gene(s) of interest into Koshihikari, only successfully transformed cells will regenerate. To test the efficiency of NiR as a selection marker, we constructed five vectors carrying the beta-glucuronidase gene ( GUS ) as a reporter: ü Kasalath NiR genome + 35S promoter:: GUS (plasmid 1); ü Kasalath NiR promoter:: NiR cDNA:: NiR terminator + 35S promoter:: GUS (plasmid 2); ü Rice Actin1 promoter:: NiR cDNA:: NiR terminator + 35S promoter GUS (plasmid 3); and ü two control vectors not containing NiR (which only have the 35S promoter: plasmid 4, and an empty vector: plasmid 5). These constructs were transformed into Koshihikari calli by Agrobacterium tumefaciens -mediated transformation by standard methods and cultured on a medium containing no chemicals for selection. Calli transformed with the three constructs carrying the Kasalath NiR (plasmid 1 – 3) formed many shoots that displayed GUS activity. On the other hand, calli transformed with constructs lacking Kasalath NiR (plasmid 4 and 5) formed either no shoots or a few non-transformed shoots. About 54% of calli transformed with Kasalath NiR as a selection marker regenerated into plants. Of these, more than 77% stained for GUS activity. These results indicate that the Kasalath NiR gene is useful as a selection marker for transformation of Koshihikari rice. We also attempted to adopt the Kasalath NiR selection system for high regenerative varieties. Because nitrite is toxic to plants, the addition of excess nitrite in the culture medium causes growth inhibition of calli regardless of their regeneration ability. For example, the high regeneration varieties, Nipponbare and Kasalath, cannot grow under high-nitrite conditions. However, cells that were transformed with the NiR overexpression construct (plasmid 3) grew normally and showed GUS activity. This demonstrates that NiR can be used as a selection marker for transformation even in rice plants with high regeneration ability. Future perspectives on new transgenic crops
A major advantage of using NiR for selection is that it is an endogenous rice gene that does not confer a selective advantage beyond that which may be found widely within the rice species. Unfortunately, it is likely that this NiR selection system cannot be applicable to all rice plants as described above. The regeneration ability of each rice plant would likely have been separately altered during evolution or the breeding process. However, we think that a similar selection system using endogenous genes is possible in any plant species having two varieties with differing regeneration ability—the QTL genes can be isolated and used in transformation. We have been developing a high-throughput QTL isolation system in rice. By using this system, we aim to isolate various rice QTL genes, including regeneration genes, and hope to find a gene that is applicable to other crops such as maize and wheat. Moreover, we have been isolating some promoters for callus-specific expression of selective markers, which would be important for preventing marker expression after regeneration. This expression control system could become standard for new transgenic crops. It would also be important to develop an efficient homologous recombination technique for removing the risks of unintentional effects, such as those caused by positional effect and copy number. Combining these systems would enable a transformation protocol that could ease some public concerns concerning genetically modified crops. References 1. Nishimura A et al . (2005) Isolation of a rice regeneration quantitative trait loci gene and its application to transformation systems. Proc Nat Acad Sci 102, 11940-11944 2. Datta K et al . (2003) Bioengineered ‘golden’ indica rice cultivars with beta-carotene metabolism in the endosperm with hygromycin and mannose selection systems. Plant Biotechnol J 1, 81-90 3. Ebinuma H et al . (2001) System for the removal of a selection marker and their combination with a positive marker. Plant Cell Rep 20, 383-392 4. Daniell H et al . (2001) Antibiotic-free chloroplast genetic engineering: an environmentally friendly approach. Trends in Plant Science 6, 237-239 Asuka Nishimura
Honda Research Institute Japan Co., Ltd.
Chiba , Japan
Sing transcriptional analysis to determine responses to phosphate deprivation
October 6, 2005
ISB News Report
M C Thibaud and L Nussaume
Phosphate (Pi) is an essential macronutriment required for plant growth and development. Its low availability to plants in many soils results not only from limiting amounts but also from its association with cations and organic compounds that create insoluble complexes. Thus, Pi has become one of the major plant nutrition problems limiting growth in both acidic and calcareous soils1. Nevertheless, applications of large quantities of fertilizers to correct this problem are not economically sustainable and also lead to environmental pollution. In most environmental conditions, plants must cope with limiting amounts of soluble Pi (5mM Pi compared to 500mM in controlled conditions). Significant changes in plant morphology and biochemical processes are associated with phosphate (Pi) deficiency1,2 and result in plant adaptation to this abiotic stress (Fig. 1). However, the molecular bases of these responses to Pi deficiency are not thoroughly elucidated. Therefore, efforts have been made to understand the molecular basis of plants responses to Pi deficiency and to identify Pi-responsive genes whose expression can be manipulated to enable plant growth in low Pi environments. A comprehensive survey of global gene expression in response to Pi deprivation was performed using an Arabidopsis thaliana whole genome Affymetrix ( http://www.affymetrix.com/index.affx ) gene chip (ATH1) to quantify the spatio-temporal variations in transcript abundance of 22,810 genes2. This analysis was corroborated by other techniques (RT-qPCR, northern blots, and production of transgenic plants) and revealed a coordinated induction and suppression of 612 and 254 Pi-responsive genes, respectively (more than a two-fold change). The functional classification of some of these genes indicated their involvement in various metabolic pathways, ion transport, signal transduction, transcriptional regulation, and other processes related to growth and development. Moreover, a time-course experiment permitted a global evaluation of genes that are regulated in response to short- (less than 12 hours), medium- (1 – 2 days), and long-term (10 days) Pi deprivation. In addition, leaf and root samples of the long-term experiment were analyzed separately to investigate specific spatial responses. During short-term Pi deficiency, 72 genes were induced, whereas only four genes were suppressed. These numbers increased significantly (291 genes induced, 34 genes suppressed) during medium-term Pi starvation (Fig. 2). At these two time points, 16% of the induced genes had overlapping expression, whereas only one gene was suppressed. Furthermore, the induction (91 genes) or suppression (22 genes) of some genes was only transient. This pattern of gene expression indicates a very rapid but transient change occurring even during short periods of Pi deficiency. Modulation in the expression of the Pi-responsive genes correlated with a decline of soluble Pi content during the early stages of Pi deficiency treatments (Fig. 1A). Long-term Pi deprivation resulted in the differential regulation of 732 genes of which 501 were induced (228 in roots and 404 in leaves) and 231 were suppressed (74 in roots and 169 in leaves). Expression of 26.1% of the induced and 4.8% of the suppressed genes overlapped in both leaves and roots. Nevertheless, most of the genes were specific for either roots or leaves, suggesting that different plant organs respond to Pi deficiency by activating distinct sets of genes. Comparison of the microarray data from all three time points showed the common induction of 48 genes and suppression of only one gene. These results are in agreement with results from smaller microarrays with Arabidopsis, rice, and white lupin, showing similar patterns of gene expression2,7,8,9. The differential expression of Pi-responsive genes is considered an adaptive response by plants to Pi deficiency that facilitates acquisition of sparingly available Pi and concurrent attenuation of some of the energy-requiring metabolic pathways. Identification of differentially-expressed genes revealed the coordinated activation and repression of genes involved in many biochemical pathways that are closely associated with plant responses to Pi deficiency. In addition to genes affecting general metabolic functions, this study highlights the induction2 of those genes related to uptake and transport of Pi (PHT1 family genes and other Pi transporters are induced rapidly and in both tissues) and other inorganic ions (sulfate); and to the Pi salvage systems (phosphatase, RNAse). Detailed analysis of Pi responsive genes also revealed that about 7% (44 genes) are involved in lipid biosynthetic pathways (Fig. 3) and only two genes were suppressed2. About 50% of the lipid-related genes were induced within two days of Pi deprivation. Induced genes largely represent those coding for enzymes involved in phospholipid degradation and galactolipid and sulfolipid synthesis. Interestingly, only a few of the genes coding for phospholipases C and D were induced during Pi deficiency. These results suggest a role for these genes in the lipid metabolic pathway during Pi deficiency. Genes involved in the subsequent utilization of diacylglycerol (DAG) to synthesize mono- and digalactosyldiacylglycerol (MGDG and DGDG) galactolipids were strongly up-regulated at early stages of Pi deprivation, which is consistent with previous data7. Genes coding for MGDG synthases (MGD2 and MGD3) were induced 4 – 10 fold during short-term Pi deprivation, whereas expression of DGD1 and DGD2, coding for DGDG synthases, was enhanced during medium- and long-term Pi deficiency, respectively. Furthermore, DGD1 and DGD2 exhibited differential regulation in roots and leaves. Similarly, the genes encoding UDP glucose-4-epimerase and UDP galactose-4-epimerase, which convert UDP-glucose to UDP-galactose (galactolipid precursor), were induced during medium- and long-term Pi deficiency. This could facilitate the production of galactose required for galactolipid synthesis. Comparatively, the genes coding for UDP-sulfoquinovose synthase and UDP-sulfoquinovosyl:DAG sulfoquinovosyltransferase exhibited early and sustained induction during Pi deficiency treatments. This was reflected by a four-fold increase in the level of sulfoquinovosyl diacylglycerol (SQDG) in P(-) leaves during long-term Pi deficiency (Fig. 3). Although SQDG is not considered essential for plant development, under Pi deficiency conditions it could possibly replace phosphatidylglycerol (PG) and may allow photosynthesis to continue despite a reduction in phospholipid content in the photosynthetic apparatus. These modulations of lipid biosynthetic pathways indicate a complex mechanism to replace membrane phospholipids with non-phosphorus galacto and sulfonyl lipids, which may have evolved to scavenge and conserve Pi in plants under Pi limiting conditions7. These results are in agreement with variations in phospholipid, sulfolipid, and glycosylglyceride content (ref. 2, Fig. 3). Alterations in lipid content became apparent within two days, whereby a decrease in PG and phosphatidylcholine (PC) was compensated by an increase of SQDG and DGDG. In leaves of plants grown in Pi-deficient medium, a reduction in levels of all phospholipids except diphosphatidylglycerol (DPG) was observed. Interestingly, in the P(-) roots, no significant difference was detected in any of the phospholipid species, including PC, but there was a substantial increase in the level of DGDG. This suggests that lipid composition is more sensitive to Pi deficiency in leaves than in roots, probably as a consequence of their high concentration in chloroplasts. Despite an early induction of MGD2 and MGD3, there was no significant increase in MGDG level, even during long-term Pi deficiency. This may be due to rapid conversion of MGDG into DGDG by DGD1 and DGD2, whose activities increased during long-term Pi deficiency. Furthermore, DGD1 and DGD2 exhibited differential regulation in roots and leaves. Microarray analysis revealed an early, sustained, and coordinated induction of a host of Pi-responsive genes involved in Pi acquisition, and conversion of organic phosphorus into available Pi. These experiments also indicate that Pi deprivation can be perceived at the molecular level as soon as Pi is withdrawn from the medium, or after some very short delay, suggesting that (i) the plant is able to sense a decrease of Pi concentration either in the medium or in cells, and (ii) some responses could be indirect. Moreover, among several members in gene families, specific expression was observed according to the duration of Pi starvation (hours, days) or the tissue (leaf, root). As developed in this paper, genes coding some of the isoenzymes involved in lipid metabolism are induced in plants grown in low Pi conditions. Genes coding phosphate transporters (PHT1 family), acid phosphatases, enzymes involved in the synthesis of anthocyanins, and flavonoids2 are differentially modulated by Pi starvation, suggesting specific roles for some family members. These genes could serve as potential candidates to decipher the components of Pi sensing mechanisms and to develop strategies to improve Pi efficiency in crops. Here we present a detailed analysis of the ‘integrated’ response of plants to Pi-starvation at the transcriptional level of the entire genome of Arabidopsis, correlated with biochemical processes. This analysis allowed a global view of the transcripts levels in low Pi conditions in plant metabolic pathways ( www.arabidopsis.org/tools/aracyc ) and in the regulation of gene expression2. The results not only enhance our knowledge about molecular processes associated with Pi deficiency but also facilitate the identification of key molecular determinants for improving Pi use by crop species. They provide a powerful background for investigating (i) Pi-signaling and signal transduction in plants exposed to Pi-depleted media, (ii) specificity of the response to Pi-starvation, and (iii) coordination between different levels of response. Acknowledgements This project was supported partly by a grant from CEA and PACA region and financial support of the various laboratories involved in this study. References 1. Raghothama KG. (1999) Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 665-693 2. Misson J et al. (2005) PNAS 102, 11934-11939 3. Wu P et al. (2003) Plant Physiol. 132, 1260-1271 4. Hammond JP et al. (2003) Plant Physiol. 132, 578-596 5. Uhde-Stone C et al. (2003) Plant Physiol. 131, 1064-1079 6. Wasaki J et al. (2003) Plant Cell Environ. 26: 1515-1523 7. Benning C Otha H. (2005) J. Biol. Chem. 280, 2397-2400 Marie Christine Thibaud and Laurent Nussaume Laboratoire de Biologie du Développement des Plantes UMR 6191 CEA/CNRS/Université Aix-Marseille II CEA, Cadarache, 13108, Saint Paul Lez Durance Cedex , France mcthibaud@...
Delivering a deadly drop to locusts
October 6, 2005
Commonwealth Scientific and Industrial Research Organisation
CSIRO scientists have successfully utilised a rare Australian native fungus – Metarhizium – to produce an environmentally friendly 'bioinsecticide' spray, Green Guard®, which has proven effective in controlling one of the world's major agricultural scourges, plague locusts. Green Guard® has already been used in Australia to control locust outbreaks, but only under a special licence. Now it will be available to all rural producers. CSIRO recently signed a commercial agreement with the agricultural biotechnology firm, Becker Underwood Pty Ltd and soon Green Guard® will be available worldwide. Managing Director of Becker Underwood Australia, Mr Richard Waterworth, said that Green Guard® had now been granted full registration by the Australian Pesticides and Veterinary Medicines Authority and will be made available to farmers through agricultural resellers and government bodies involved in locust control such as the Australian Plague Locust Commission and the NSW Rural Lands Protection Boards. 'We have also had promising discussions with groups around the world and will be pursuing these,” Mr Waterworth said. ”Our first aim is registration of Green Guard® in China. Africa, Mexico, Canada, USA and South America will be targeted in the longer term.” It took Dr Richard Milner and his team at CSIRO Entomology a decade to produce a usable product. “When Metarhizium was discovered it didn't seem to be a likely candidate for controlling plague locusts,” Dr Milner said. “Locusts like it dry and the fungus likes it moist. However, the need for a 'green' alternative to insecticides for locust control encouraged us to persevere.” Metarizhium spores infect locusts by literally boring into their cuticle (skin). Once inside they use up water and nutrients and grow tiny tubes which eventually kill the insect. Early attempts to produce a water-based spray failed and Dr Milner's team spent years developing a mix of vegetable and mineral oils in which Metarhizium spores can be delivered with maximum efficiency. “In an oil suspension, Metarhizium can be sprayed under very hot conditions and won't dry out,” Dr Milner said. “In fact, the fungus will infect and kill locusts in conditions where we would not normally expect it to be active,” he said. After spraying, Green Guard® does not persist in the environment for more than two to four weeks and, while it is effective against a wide range of grasshoppers and locusts, it does not affect even close relatives like crickets. Aquatic life and birds are also safe. The research was conducted by CSIRO Entomology with assistance from the Australian Plague Locust Commission (APLC), the Queensland Department of Natural Resources and NSW Agriculture.
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