Plant Biotechnology: Current and Potential Impact For Improving Pest Management In U.S. Agriculture An Analysis of 40 Case Studies June 2002 Viral Resistant Squash Leonard P. Gianessi Cressida S. Silvers Sujatha Sankula Janet E. Carpenter National Center for Food and Agricultural Policy 1616 P Street, NW Washington, DC 20036 Phone: (202) 328-5048 Fax: (202) 328-5133 E-mail: ncfap@ncfap.org Website: www.ncfap.org 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.
2. SQUASH Viral Resistant Production Squash varieties that were developed to be eaten at a very immature stage, such as zucchini, are called summer squashes. Other summer squash varieties include yellow crookneck and yellow straightneck cultivars. Most other squash varieties are called winter squashes because they are stored and eaten during the winter. Harvest of summer squash usually begins about 50 to 60 days after sowing or transplanting. Fruit develops rapidly, and fields are harvested every two or three days. Daily harvesting is often practiced. Summer squash are harvested very young and tender for the fresh market, usually when they are six to eight inches long. Annual production statistics for summer squash are not collected for all producing states. Annual statistics on summer squash production are available for Georgia and Florida, where approximately 27% of the nation s squash acreage is located [1]. In recent years, Florida s squash acreage has totaled about 13,000 acres with production of about 150 million pounds and a total value of $54 million per year [36]. Georgia s summer squash production is estimated at 15,772 acres with production of 182 million pounds valued at $54 million per year [35]. US squash acreage totals 67,000 acres [1]. Virus Problems Worldwide, production of summer squash is severely limited by a complex of mosaic viral diseases, each of which attacks a wide range of crop and non-crop plants. Four mosaic viruses are of particular concern to U.S. summer squash production: cucumber mosaic virus, watermelon mosaic virus, zucchini yellow mosaic virus and papaya ringspot virus type W. These viruses are found throughout U.S. summer squash growing regions, they all affect a wide range of host plants, and all are spread by aphids. Within a given growing region, though, the viruses or combinations of viruses that strike may vary from year to year, as may the infection incidence for each virus. 2
Cucumber mosaic virus (CMV) is one of the most devastating viruses affecting summer squash. Its symptoms are first manifested on younger leaves, causing them to curl downward and wrinkle, develop a mosaic pattern, and decrease in size. Plants are stunted because leaves and stems are smaller than normal. Fruits are also discolored with mosaic patterns, reduced in size and misshapen with a warty texture. Watermelon mosaic virus 2 (WMV) symptoms show up in leaves as yellowing between the veins and reduced leaf area around the veins, giving them the look of tendrils rather than leaves. New leaves are mottled, blistered and distorted, and the plant becomes stunted. Fruit can be bumpy and distorted in shape and color. Zucchini yellow mosaic virus (ZYMV) makes leaves yellow and distorted, either with a blistered surface or tendril-like appearance. Plant growth is stunted and fruit is bumpy and deformed. Symptoms of papaya ringspot virus type W (PRSV-W) can be very similar to those of ZYMV, with leaves showing green mosaic patterns, fruits and leaves misshapen, and plant growth stunted [2,3]. Level of yield loss depends on what point in crop development infection occurred. If a field is infected when plants are young, 100% loss of yield is likely. In such extreme, though not uncommon, cases, the grower will abandon the field. Similarly, in areas where virus infestation is unmanageable, growers will no longer plant the crop [4]. If a squash field is infected later in the season when plants are older and perhaps already flowering or fruiting, it may be harvested until symptoms become apparent in the fruit, at which point harvests would be abandoned. More general or areawide estimates of losses to mosaic viruses, when given, are often in the form of a range, commonly 20% to 80% yield loss. The Department of Plant Pathology at North Carolina State University for several years in the 1980s published statewide estimates of crop losses due to disease. Their most recent estimates, for 1987 and 1988, were that 25% of the state s annual squash crop was lost to the mosaic virus complex [5]. The University of Georgia s Department of Entomology estimated the yield loss of summer squash to viruses transmitted by aphids in 1997 at $2.6 million [35], which translates 3
approximately to a 5% overall yield loss. This loss occurred even though 10 applications of insecticides were made to each squash acre at a cost of $10 per application [35]. In 1998, losses to viral diseases in Georgia increased. The statewide yield loss to squash viruses in 1998 is estimated at 15% [34]. Several factors interact to determine how severe damage is, but the most influential seems to be how early infection takes place. One study in Georgia calculated average yield loss in summer squash from WMV to be 43%, 28% and 9%, respectively, when plants were infected early, mid and late in the season. Marketable yield loss ranged from almost 100% for early and mid season infections, to 70% for late [6]. With such a wide range and no way to foresee where in that range the damage to a particular field may fall in a given year, one of the most trying aspects of mosaic virus infections for squash growers is the uncertainty when they plant of whether or not there will be a crop to harvest [7]. Generally growers can expect to lose about 20% of their summer squash crop to viruses. They may therefore plant and cultivate excess acreage as insurance. Increases in per acre costs of production since the 1980s have made such insurance measures uneconomical. Without the ability to afford acreage buffers against crop losses to viruses, fewer acres of squash are planted and there is a greater economic risk attached to each acre that is planted. Virus Biology and Transmission A virus is a microscopic particle consisting of two parts: a piece of genetic material, either DNA or RNA, surrounded by a protein coat. A virus is not even an entire cell. In order to replicate, or survive, it uses the resources of its host s cells. Unlike other plant pests, viruses do not physically damage the plant or destroy its tissue. Rather, by using up space and resources within plant cells, they disrupt normal metabolism and compromise the functioning of the plant s various systems [8, 9]. A virus must be introduced into a plant cell. It cannot enter unaided. Something must create a wound -- even a microscopic wound will do -- in the plant to allow the virus entry. Once inside 4
a plant cell, the virus sheds its protective protein coat. The exposed genetic material begins to replicate using the genetic replication mechanisms of its host plant cell. When enough virus particles have been produced, the virus begins to spread into other neighboring cells through the normal intercellular transport channels of the plant. Virus particles continue slowly making their way through the plant in this manner, until they reach the conductive tissue that distributes water and nutrients throughout the plant. Once inside the conductive tissue, virus particles are rapidly spread to other parts of the host plant [8, 9]. The four mosaic viruses discussed here are transmitted from infected plants to healthy plants primarily by aphids. Aphids are small, sucking insects that feed on plant sap. Their protracted mouthparts form a piercing tube, which they use to probe leaves and stems and to siphon out the plant s liquid nutrients. When an aphid feeds on a plant infected with a virus, virus particles become lodged in the mouthparts of the aphid. When the aphid relocates to a non-infected plant, it carries the virus particles with it and injects them into the new plant when it probes to feed. The viruses CMV, WMV, PRSV-W, and ZYMV are classified as nonpersistent, which means that an aphid need only feed on an infected host for a few seconds in order to pick up virus particles, and a few seconds on a new host to pass on the virus. [10] Two additional aspects of the aphid-virus interaction contribute to the ubiquitous nature of these diseases. Firstly, aphids reproduce at a very high rate. This is because during most of the season populations consist of females reproducing asexually, with no males present or needed. Each female is said to be born pregnant, full of miniature, live clones of herself. Throughout the course of her lifetime, a female aphid may give birth to innumerable of these live clones, each of which is in turn born pregnant with her own supply of live clones. This method of reproduction allows aphid populations to amplify at a rapid rate, easily reaching over a million within a short time. [10, 11] Not only are the aphids which vector the mosaic viruses present in high numbers, but sources of the viruses are also abundant. All four diseases affect a wide range of cucurbits as well as other types of plants, including other crops and weeds. A squash field is therefore surrounded by potential sources of mosaic viruses in the form of neighboring crops and weed populations. The 5
combination of potentially limitless host sources and insect vectors of mosaic viruses makes prevention of infection very difficult, if not impossible, in squash production. Virus Management Because there is no pesticide that can kill the individual virus particles themselves, the only way to manage viruses in crops is to manage their transmission. It only takes one aphid feeding for a few seconds to spread infection, so simply reducing aphid population levels will not effectively prevent nonpersistent virus transmission. Only complete elimination of every single aphid -- an impossible task -- will accomplish that. Even if insecticide treatments do reach and kill all aphids, the aphids will still have enough time to feed on the treated plant before the insecticide kills them. That is enough time to transmit a virus. Therefore insecticide applications are not effective in controlling virus infection. [10, 12, 13] Foliar applications of stylet oil (highly refined petroleum oil), on the other hand, provide a barrier between the aphid and the plant, interfering with virus transmission [7, 12]. Viral particles that may be released from the aphid mouthparts will travel through the oil layer, and ideally be stopped there, before being introduced into the plant tissue. Frequent applications must be made in order to ensure that new foliage, which is attractive to aphids, is constantly coated. It is also suggested that every two or three stylet oil applications be combined with an insecticide application. The insecticide prevents the development of aphid colonies that would increase the potential for secondary in-field spread of the viruses [12]. Proper oil application may delay the introduction of aphid-borne mosaic viruses into squash fields and also delay the secondary spread of the virus within a field. This allows plants more time to produce marketable fruit before virus symptoms are manifested. But virus control with oil applications is temporary and incomplete, and plants will eventually succumb to virus infection. [10, 12] It is estimated that each oil application contains eight pounds of oil per acre. Half of the oil applications are estimated to be mixed with endosulfan at an average of 0.75 pound of active ingredient per acre [31]. Each of the applications is estimated to cost $10 per acre [35]. In the 6
late 1990 s it was estimated that 10 applications were made per acre for control of aphids and virus transmission [35]. The most effective management technique for consistent and prolonged virus control in summer squash is the use of masking and resistant varieties [3, 10]. Virus resistance traits from other squash and close relatives have been horticulturally bred into some commercially grown summer squash varieties. A few of these resistant varieties perform well even when used in late season plantings when virus pressure is highest. Certain varieties of yellow squash have been bred for a different kind of virus resistance. Called precocious yellows, these varieties are susceptible to mosaic virus infections and their foliage shows viral symptoms, but symptoms in the fruit are delayed or avoided. The yellow fruit remains yellow, masking the green mosaic coloring that viral infections usually produce. Fruit is therefore harvestable for a longer period of time, and marketed despite having come from virus infected plants. One study in Pennsylvania found most precocious yellow squash varieties to have between 15 and 25% of fruit showing mosaic symptoms as compared to 98% of the fruit from a standard variety [19]. In another study in Kentucky, precocious yellow varieties produced, on average, 50% more marketable fruit than the standard varieties of yellow straightneck squash [20]. The fruit of precocious yellow varieties, however, have yellow stems and are less appealing to consumers than green-stemmed fruit. Transgenic Squash Recently, a third type of resistance, called pathogen derived resistance, has been developed in summer squash. By inserting certain nonvirulent genes from a virus into a squash plant, scientists are able to endow the plant with resistance to that virus. The genes that produce the coat protein of the virus were introduced into squash via agrobacterium mediated transformation. Specifically in summer squash, coat protein-mediated resistance is used. This does not cause virus symptoms in the transgenic squash because the viral genes that are introduced are not the disease causing genes, but rather are genes that encode for the virus coat protein. Virus coat protein genes are inserted into the plant s gene sequence so that the plant itself produces the viral 7
coat protein. Any virus particles that invade the plant are somehow prevented from causing an infection. The specific mechanism for coat protein-mediated virus resistance is not known, but it has been speculated upon. Usually, after a virus particle enters a plant cell, it must shed its coat protein in order to replicate and cause disease. If it is unable to replicate, the virus has no impact on the host cell or the plant itself. It is thought that somehow by enabling an invaded plant cell to inundate invading virus particles with plant-made viral coat proteins, the invading particles will not be able to remain coatless, and therefore will not be able to replicate and cause infection. Experimental evidence also suggests that other mechanisms may be involved, including interference with virus spread within the plant. While some similar viruses may be thwarted by the same viral coat protein, it seems clear that most virus resistance is achieved through multiple mechanisms and that different viruses may require differing combinations of coat protein and host environment in order to be resisted. [17, 18] An advantage transgenic virus resistant varieties have over traditionally bred varieties is the reduction in the field of sources of virus for new and prolonged infections. While traditionally bred virus resistant squash may reduce the number of fruit damaged by virus, the plants themselves are still infected. The infected plants serve as reservoirs of the virus from which aphids can spread infection to other plants and other areas. Transgenic virus resistant plants remain virus free, so the incidence of virus is reduced in the field. This helps prevent further spread of the virus in time and space because the reservoir of inoculum is reduced. [19, 21, 22, 23, 24, 25] The first line of summer squash with transgenic resistance to viruses was deregulated by EPA in 1994 and made available commercially in1995 [26]. It carried coat protein mediated resistance to two viruses, ZYMV and WMV 2. In 1996, EPA deregulated another line that carried resistance to these two viruses as well as to CMV [27]. Varieties were made commercially available in 1998 and are planted on an estimated 5,000 acres [33]. 8
Transgenic summer squash plants with resistance to three viruses (CMV, ZYMV and WMV 2) produce as many or more marketable fruit than nontransgenic squash. The increase in yield resulting from virus protection depends on which viruses the plants are challenged with and how great the virus pressure is. In many regions, such as North Carolina, virus pressure is low in the spring and greater in the fall, which often limits squash planting to the spring. In a normal year, planting transgenic squash may not provide a significant yield benefit in the spring, but in the fall, transgenic varieties with resistance to three viruses produce more marketable fruit than standard varieties do [16]. Similar results were found in Alabama [29] and in Florida, where the number of symptomatic fruits produced per plant were 0 for transgenic squash and 5.4 for the standard variety [25]. In Pennsylvania, between 0 and 3% of the fruit harvested from transgenic virus resistant squash had virus symptoms, compared to between 15 and 51% in precocious yellow squash and 98% symptomatic fruit from the standard variety [20]. A Cornell University study found that transgenic squash with resistance to three viruses produced a 50-fold increase in marketable yield over nontransgenic varieties [30]. There are two major limitations to the commercially available, transgenic squash with virus resistance. One is that they do not carry protection against the fourth virus that affects squash production, PRSV. Therefore even though transgenic lines have resistance to CMV, ZYMV and WMV 2, they can still be, and in fact are, limited by the presence of PRSV [20, 29, 30]. Research is underway to produce commercially viable lines with resistance to all four viruses using both transgenic and traditional resistance breeding. There are a great variety of summer squash cultivars grown in order to appeal to the variety of consumer preferences. A wide variety of cultivars is needed to meet differing requirements of various growing regions, with their different soil types and different rain and temperature patterns. In California alone, there are dozens of summer squash varieties grown [28]. To date, the background varieties that have been transformed for virus protection are varieties grown in southeastern states, while most of the varieties used in other regions have yet to be given transgenic virus protection. 9
Given the many summer squash varieties, the many viruses, the possible levels of disease pressure and combinations thereof, it seems ambitious to expect a transgenic cultivar for every situation. But research shows that planting varieties with resistance to even one or two viruses can be profitable, with yields of up to 40 times the yield without resistance [23, 24, 30, 31]. It is important to note, also, that even when plants are not challenged with virus infection, the transgenic lines produce fruit that is equal to nontransgenic fruit in both quantity and quality [25, 29]. Estimated Impacts It is estimated that transgenic squash with virus protection has been planted on 5,000 acres mostly in Georgia and Florida. The transgenic squash was planted on approximately 17% of the two states combined acreage. It is assumed that the yield increase on the transgenic acreage in comparison with an insecticide/oil-treated acre is 10% on average, equaling approximately 1,200 pounds of squash per acre, with a value of $380 per acre. In the aggregate the 10% yield increase on 5,000 acres translates to an increase of 6 million pounds of production with a value of $2 million. These impacts are assigned to Georgia (55%) and Florida (45%) base on acreage. It has been estimated that this yield increase will occur primarily in fall plantings, where disease incidence is highest [7]. Planting transgenic squash increases the length of the marketing season and increases the number of harvests per acre [7]. No impacts on insecticide use are projected since the 10 insecticide sprays that control aphids also control whiteflies and will be made even to the transgenic acreage. Traditional seed for yellow squash costs about $60 per acre. Transgenic seed costs approximately $135 per acre [37]. Assuming that growers are paying a premium of $75 per acre for planting the transgenic squash implies a total increased cost of $375,000 on the 5,000 acres. The net benefit of the transgenic squash planting is estimated at $1.6 million per year. Transgenic summer squash varieties with resistance to three of the four major productionlimiting viruses offer several benefits to growers. They can produce greater marketable yields of high quality fruit, particularly in production areas where high virus incidence limits the growing 10
season both in terms of number of plantings made and the number of harvests per planting. Transgenic virus resistant squash also reduces disease incidence in the field by preventing plants from serving as reservoirs for infection spread by aphids. 11
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