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Plant Biotechnology:
Current and Potential Impact
For Improving Pest Management
In U.S. Agriculture
An Analysis of 40 Case Studies
National Center for Food and Agricultural Policy Financial Support for this study was provided by the Rockefeller Foundation, Monsanto, The Biotechnology Industry Organization, The Council for Biotechnology Information, The Grocery Manufacturers of America and CropLife America. 21. GRAPE
Bacterial Resistant
Production California is number one in the U.S. in grape acreage and production for wine, table and raisin grapes [1]. In 1999, the state had 424,000 wine grape acres producing a crop of 2.7 million tons valued at $1.56 billion, 87,000 table grape acres producing a crop of 0.8 million tons worth $417 million, and 279,000 raisin grape acres producing a crop of 2.1 million tons valued at $764 million. California wine grapes are the basis for the state’s $33 billion wine economy, which produces more than 90% of all U.S. wine [2]. Xylella fastidiosa is a plant pathogenic bacterium that causes disease in a variety of plants [2]. There are strains that cause leaf scorch in almonds, oleander scorch in oleander, variegated chlorosis in citrus, and Pierce’s disease (PD) in grapevines. The bacterium is vectored by sharpshooters, piercing-sucking insects that feed on the xylem, the water conducting tissue of the plant. Once introduced into a host plant, the bacterium lives and reproduces in the xylem, eventually clogging the tissue. In grapes, the bacteria multiply rapidly, spread systemically and can reach concentrations of billions of live bacterial cells per gram of plant tissue [23]. The plant is deprived of water and nutrients, and dies. Pierce’s disease is found in all southern states, and is the primary factor preventing European grape production in the southeastern U.S [3]. Symptoms of PD in grapes mimic those of drought: infected leaves and twigs turn brown and dry up, fruit wilts and dries up, and canes and roots may also die back [4]. A vine will be killed by PD within two years of infection [2]. Some grape varieties have some tolerance to PD and decline more slowly after infection is initiated. These include Riesling, Chenin Blanc, and Thompson Seedless. Pinot Noir and Chardonnay are very susceptible to PD. A one- year-old Chardonnay or Pinot Noir vine can die the year it becomes infected. Ten-year old Chenin Blanc or Ruby Cabernet vines can live with chronic PD infections for several years although they will not bear a full crop [23]. In Temulca, the cost of replanting a vineyard is $16,000 per acre, over five years, not including the cost of lost production [5]. History of Pierce’s Disease in California The bacterium that causes PD in grapes has been in California for more than a century [3]. The disease was extensively studied by USDA’s Newton B. Pierce and subsequently the disease was named for him. The first outbreak destroyed 35,000 acres of grapes and closed 50 wineries in the Los Angeles Basin in the late 1800’s. In the 1930’s another major outbreak devastated vineyards in the Central Valley. Since then, occasional and localized outbreaks have occurred in the Central Coast growing region and in isolated vineyards in the Central Valley. Although annual statewide losses to PD have been minimal, outbreaks are devastating for the vineyards in which they occur. In the Central Valley, there are two insects present that vector PD: the green sharpshooter and the red-headed sharpshooter [3]. Both are primarily grass feeders and rarely feed on grapes. By reducing weedy grass stands in and around vineyards, PD spread by these In coastal production areas, the insect vector of PD is the blue-green sharpshooter [3]. The blue-green sharpshooter has a wider host range than its Central Valley relatives, preferring woody perennials such as trees and shrubs that line waterways. When riparian vegetation dries up and grape leaves start to expand, the sharpshooters will move into vineyards to feed. Removing riparian vegetation is not possible, but because the blue- green sharpshooter has only one generation in most areas, well-timed insecticide sprays greatly reduce their levels, and therefore reduce PD spread. Not planting vineyards close to waterways also helps reduce PD incidence in the vines. In areas where PD is known to be a problem, other measures can be taken to manage it. Varieties, which are slower to show symptoms, can be planted in order to prolong production before replanting is necessary [3]. Because the three established vectors feed on the new canes that emerge each year, new infections are usually removed during normal winter vine pruning [6]. This prevents chronic infections that carry over into subsequent seasons. Without chronic infections, sources of PD (i.e. infected plants that sharpshooters will feed on) remain outside the vineyards. Because the three PD vectors are weak flyers, PD infections tend to remain along vineyard borders without progressing By 1990, a new sharpshooter, the glassy-winged sharpshooter (GWSS) had arrived in California [2]. It was most likely introduced as eggs on nursery stock from the Southeastern U.S. The sharpshooter excretes water droplets after filtering out minerals and amino acids. They have to filter out so much water to get adequate nutrition that the GWSS produces a fairly sizable droplet of water about every three minutes. By autumn the leftover salts from water dripped and evaporated make infested trees look like they have been whitewashed. The GWSS also vectors PD. Compared to the three PD- vectoring sharpshooters already present in California, the GWSS is bigger (0.5 inch), a stronger flyer (sometimes covering up to a half-mile radius, five times further than the other sharpshooters, so it can move deeply into vineyards), and a more voracious feeder. The California Department of Food and Agriculture website [7] lists more than 1000 host plants for GWSS, including horticultural and agricultural plants, wild plants and weeds. Such a large host range makes it easy for the GWSS to spread into new areas and proliferate, and makes management of the ubiquitous vector difficult. The miles of oleander and other plants lining the medians and shoulders of California’s highways are Like other sharpshooters, the GWSS feed by piercing plant stems and sucking water and nutrients out of the xylem. Unlike the other PD-vectoring sharpshooters, the GWSS tends to feed lower on the vine and can even penetrate and infect the old wood. This wood cannot be pruned out if infected, so infections introduced by the GWSS are more likely to become chronic more quickly. Chronic infections mean quicker decline and death of a vine. They also mean the infected vine can serve as a source for further spread within the vineyard [6]. Infections are no longer only associated with vineyard borders. With the GWSS, Pierce’s disease spreads faster, further and cannot be pruned out of infected vines. This vector/disease combination is a very serious threat to all grapes in California. Because of its wide host range and its ability to vector other strains of Xylella fastidiosa should they be introduced into the U.S., the GWSS potentially poses a serious threat to other agronomic crops such as alfalfa, almonds and citrus. Currently, however, the biggest impact of the GWSS is due to its spread of PD in grapes. The GWSS was first detected in 1996 in Orange and Ventura counties, and has since been detected in at least 11 counties [2, 8], with established infestations concentrated in the southern part of the state. Currently the GWSS has been found as far north as Chico, Contra Costa, and Sacramento counties, thought to have been transported on horticultural crops used for landscaping new development complexes. Within two years of its arrival into the Temecula Valley region of Riverside County in1999, the GWSS and the PD it vectors have caused an estimated $12 million in damage as 25% of the area’s grapevines have been removed [2]. More than 1000 acres of premium wine grapevines in the north coast and another 300 acres in Riverside county have been killed by the disease. Since the detection of the GWSS in California and its escalation of the threat of PD, almost $50 million has been raised from federal, state and local governments and commodity groups to research and manage the GWSS and PD [2]. In the fall of 1999, The Pierce’s Disease Research and Emergency Response Task Force was established to identify areas of research and strategies for fighting the crisis. There is no cure for Pierce’s disease. The best control for it is prevention [9]. The biology of the GWSS – its mobility, wide host range, and ability to feed on old wood – makes managing the plant disease by managing the vector even more complex than is usually the case with insect-vectored plant diseases. In order to stem the spread of the GWSS, the California Department of Food and Agriculture inspects nursery stock and bulk grape shipments [2]. Statewide monitoring programs have been set up to detect and track spread of the GWSS, and extensive training programs are provided for growers, nursery workers and others to identify the GWSS and PD symptoms. In areas where the GWSS is already established, measures are being taken, under the coordinated efforts of county agricultural commissioners, California Department of Food and Agriculture, and the California Department of Pesticide Regulation, to reduce their populations [7]. Parasitic wasps that kill GWSS eggs are being released, vegetation that harbors PD and GWSS is being selectively replaced with vegetation that does not, and insecticides are being applied. Monitoring programs have found a high correlation between a vineyard’s proximity to riparian vegetation or citrus orchards and the severity of PD infection [10]. Insecticide applications to stem GWSS activity therefore are not limited to vineyards, but are used on neighboring citrus plantings as well as non- The University of California IPM Guidelines list two insecticides for GWSS control on grapes: imidacloprid and dimethoate [9]. Carbaryl applications have been used on non- agricultural vegetation [12]. A limitation of biological and chemical control of a disease vector, however, is that most probably control needs to be 100% to stop the spread of the disease. In this case, feeding deterrence may be of greater value than toxicity when controlling the GWSS and PD. Studies show imidacloprid reduces GWSS numbers and also appears to deter them from feeding on vines [10]. The GWSS can detect a systemic insecticide (such as imidacloprid) in the xylem when it first starts to feed and are deterred from further feeding. Whether or not this first feeding is sufficient to transport the Applications of kaolin, a fine clay, also seem to discourage GWSS feeding [11]. Kaolin works by creating a protective plant coating to prevent feeding and also sticks to the GWSS, acting as an irritant and deterrent. When a sharpshooter finds a vineyard treated with kaolin it most likely will move back into citrus or the hedgerow area without feeding [11]. Kaolin is approved for organic use in California because it is a naturally occurring mineral. Table 21.1 and Table 21.2 delineate the per acre costs and use amounts of imidacloprid and kaolin treatments for GWSS control. In the meantime, research by private industry and the University of California is ongoing to find ways other than insecticide applications to slow down GWSS and PD. Projects include investigating the use of barriers and trap crops against GWSS, and winter pruning and injecting vines with micronutrients or beneficial bacteria against PD [2, 13]. The broad-spectrum antibiotic tetracycline is also being tested. Eradication and control efforts such as monitoring and inspection programs, vegetation modification, biological control programs, and insecticide applications are slowing the spread of GWSS and PD, providing temporary benefits and more time [2]. What is needed are long term solutions, such as a way to prevent the GWSS from transmitting PD, a way to kill PD, or development of PD-resistant grapevines [14]. This is particularly true for the northern part of the state, where grape growers and communities have already voiced objections to insecticide-based GWSS control programs [15]. The wine industry, and therefore California’s grape industry, is highly dependent on varietal name recognition [16]. The best solution to PD is one that will not compromise the qualities of the grape varieties that are already grown. At the same time, the best solution to PD would be the development of new, disease-resistant varieties. Many researchers are looking to biotechnology as a solution that meets both those criteria. Biotechnology may allow the insertion of selected resistance genes into elite grapevine varieties without changing qualities of the berry or the wine they would produce. Biotechnology can also greatly reduce the time it takes to develop a new resistant variety because genetic techniques allow for testing of PD resistance on plantlets rather than The Pierce’s Disease Research and Emergency Response Task Force concluded in its 2000 report that “breeding resistance to the disease using genetic engineering and other biotechnology applications holds the greatest promise for eliminating Pierce’s disease in grapes” [14]. Accordingly, several research projects are underway studying the biology and behavior of GWSS and PD and the biology of grapevines for clues as to how to interrupt the disease cycle and protect plants [2, 14]. Wild and cultivated grapes from the Southeastern United States are of a different genus than the grapes in the west. The Southeastern grapes, muscadine grapes, have natural resistance to Pierce’s disease [17]. Breeding that natural resistance into the Vitis grapes of the west may also introduce other qualities that would negatively impact grape and wine production, such as the musky flavor of muscadines and the tendency of their fruit to drop when ripe rather than stay on the vine until harvest. Researchers at UC Davis and in Florida are studying the genetics of muscadine grapes in hopes of incorporating their resistance genes into California grapevines [18, 19]. Other UC researchers are looking for resistance strategies based on the biology of PD. It has been discovered that some plant diseases involve a particular type of enzyme that degrades plant cell walls. Pierce’s disease may involve such an enzyme. It has also been discovered that many plants, including pear, produce a protein, called PGIP, that inhibits this cell wall-degrading enzyme. One strategy that is being investigated for use against Pierce’s disease is to insert the pear gene for PGIP into grapes and test for its effects on Pierce’s disease development in the plant [16]. Another project has for the first time successfully transformed a commercial grape variety with a useful gene [24, 28]. The gene used was taken from the pupae of the giant silkworm moth, and it produces cecropin, a protein which attaches to the PD cell membrane, punches a hole in it, and kills the cell [27]. Cecropin is expressed at low levels throughout the transgenic grape plant. Researchers at the University of Florida have transformed Thompson Seedless, Merlot and Chardonnay plants through the insertion of the cecropin gene. The transgenic plants are currently being tested for resistance to Pierce’s Disease.[26] Research is ongoing to learn more about the genetics of Pierce’s disease in order to identify other strategies for its control within plants. But most projects on Pierce’s disease control are in their first year and so are still gathering basic information. In the meantime, work is being done on grape genetics and biology so that when a resistance strategy is developed, it can be implemented quickly. Research to improve the efficiency of grape transformation via Agrobacterium is being conducted, as is research to improve the efficiency of producing vines from cell culture [20]. Because Pierce’s disease is concentrated in the xylem tissue of plants, any resistance strategy that is developed must be focused in the xylem. Work is therefore also being done on targeting gene expression The coordinated efforts for areawide GWSS suppression in infested regions have been successful in reducing populations. Preliminary research has helped to identify potential control strategies, but it may be several years before the details of an effective PD/GWSS Most likely, areawide suppression of GWSS populations will continue in order to reduce their influx into vineyards. In vineyards, this may include releases of parasitoids and applications of systemic insecticides compatible with the parasitoid releases, such as imidacloprid, either throughout the vineyard or along borders. The recommended rate for imidacloprid application on grapes is 0.25 lbs. a.i. per acre, at a cost of $79 per acre [21]. But the GWSS would have to feed on vines in order to be killed by the systemic insecticide, so PD infection may still occur. In order to prevent GWSS from feeding on vines, researchers are looking to kaolin, either as a border treatment or throughout the vineyard. Researchers suggest at least three applications of kaolin at 25 lbs. per acre, at a cost of $17.50 per acre per application [11, 21]. It is estimated that transgenic grape cultivars that resist Pierce’s disease would essentially be planted on all of California’s grape acreage (780,000 acres) and would eliminate the potential spraying of 59 million pounds of insecticides (75.25lbs/A) costing $105 million/year ($131.50/A). Current insecticide use in California grapes is approximately 252,000 lbs. AI/yr. [25]. The 59 million pounds projected for GWSS control would be a Table 21.1. Projected costs of insecticide treatments for GWSS in grapes.
Insecticide $/Application #
Applications
Total Cost/Acre
Table 21.2. Projected insecticide amounts used for GWSS suppression in grapes.
Insecticide Lbs
AI/Acre #
Applications
Total Lbs AI/Acre
1. USDA, Noncitrus Fruits and Nuts 1999 Summary, National Agricultural Statistics 2. Wine Institute, “Pierce’s Disease Update”, available on the internet at www.wineinstitute.org/communications/pierces_disease/pierces_disease_update.htm. 3. University of California, Grape Pest Management, Division of Agriculture and Natural Resources Publication 3343, Second Edition, 1992. 4. USDA, Crop Profile for Grapes (Wine) in California, available on the internet at 5. Craig Weaver, Calaway Vineyards, Riverside County, CA. Personal communication, 6. Purcell, A.H., “Xylella fastidiosa Web Site”, available at www.cnr.berkeley.edu/xylella/index.html. 7. California Department of Food and Agriculture, “Glassy Winged Sharpshooter”, available on the internet at http://plant.cdfa.ca.gov/gwss. 8. Blua, M.J., P.A. Phillips, and R.A. Redak, “A New Sharpshooter Threatens Both Crops and Ornamentals”, California Agriculture 53(2): 22-25. 9. UC IPM, “Pest Management Guidelines”, Division of Agriculture and Natural 10. Redak, R., “Impact of the Glassy-Winged Sharpshooter on Pierce’s Disease Spread in California and New Approaches to Disease Management”, American Vineyard Foundation, Viticulture Project Summary, available on the internet at www.avf.org. 11. “Kaolin Clay Film Repels Insect Pests in Fruits, Vegetables”, Ag Alert, April 18, 12. CDFA, “Report to the Legislature”, Pierce’s Disease Control Program, January, 2001. 13. University of California, “A New Pest Transmitting Pierce’s Disease Spreads in California; UC Scientists Study Control of the Insect and Diseases It Carries”, DANR News, available on the internet at http://danr.ucop.edu/news/July- 14. “Report of the Pierce’s Disease Research and Emergency Response Task Force”, available on the internet at http://danr.ucop.edu/files/reportpiercesdisease.pdf. 15. “Farmers Speak Out on Sharpshooter Control Program”, Ag Alert, April 18, 2001. 16. Meredith, C.P., Professor, Department of Viticulture and Enology, UC Davis. 17. California Rare Fruit Growers, Inc., “Muscadine Grape”, Fruit Factsheet, available on the internet at www.crfg.org/pubs/ff/muscadinegrape.html. 18. Walker, M.A., Associate Professor, Department of Viticulture and Enology, UC Davis. Personal communication, April 2001. 19. Walker, M.A., “Genetics of Resistance to Pierce’s Disease”, American Vineyard Foundation, Viticulture Project Summary, available on the internet at www.avf.org. 20. Meredith, C.P., “Genetic Transformation: A Means to Add Disease Resistance to Existing Grape Varieties”, American Vineyard Foundation, Viticulture Project Summary, available on the internet at www.avf.org. 21. “Glassy-winged Sharpshooter Pilot Project Compliance Agreement”, Kern County Department of Agriculture, California Department of Food and Agriculture, and 22. Pearson, Roger C. and Austin C. Goheen, eds., Compendium of Grape Disease, APS 23. Varela, Lucia G., et al, Pierces Disease, University of California, Agriculture and Natural Resources, publication 21600, 2001. 24. “On the Grapevine”, New Scientist, May 18, 2001. 25. USDA, Agricultural Chemical Usage, 1999 Fruit and Nut Summary, National Agricultural Statistics Service, July 2000. 26. Gray, Dennis, University of Florida, Personal Communication, October 2001. 27. Gray, D. J., et al, “Transgenic Grapevines”, In: Khachatourians, McHughen, Scorza, Nip and Hui (Eds). Transgenic Plants, Marcel Dekker, 2001 (in press). 28. Scorza, R. and D. J. Gray, “Disease Resistance in Vitis”, US Patent No. 6,232,528

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From: Mike Anderson, The Wilderness Society Re: Date: April 8, 2005 Following is a brief summary and analysis of the national forest planning directives that were published in the Federal Register on March 23, 2005 (70 Fed. Reg. 14637).1 The directives supplement the National Forest Management Act planning regulations that the Bush Administration issued on December 22, 2004 and published in the F

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Protein phosphatase assay This protocol describes the standard strategy for measuring Ser/Thr protein phosphatase (PPase) activity in our laboratory using an artificial substrate (ex. Fzy-S50), a recombinant protein kinase (ex. Cdk) and [γ-32P]-ATP. This protocol can be modified/utilized to measure various PPase activity of your interest by changing substrate and kinase. 1, Purification o

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