Plant Biotechnology: Current and Potential Impact For Improving Pest Management In U.S. Agriculture An Analysis of 40 Case Studies June 2002 Viral Resistant Citrus 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.
11. CITRUS Viral Resistant Production Texas citrus production is concentrated in the Lower Rio Grande Valley (LRGV), at the southern tip of the state. There are approximately 30,000 acres of citrus grown in Texas, almost 70% of which is grapefruit, predominately the Rio Red variety [1]. The total value of Texas citrus production in 1999-00 was $48 million. Table 11.1 charts Texas citrus production from 1980 to 2000. As can be seen, there were two periods of significant decline caused by freezes. In both cases, citrus acreage in Texas was replanted. Citrus is a long-lived perennial tree crop. Each tree is a combination of two varieties, grafted together: the roots and lower trunk are one variety, called the rootstock, and the rest of the trunk and canopy are another variety, called the scion. A variety that has good root qualities, such as nematode or soil disease tolerance or efficient nutrient uptake, may not produce edible fruit. Similarly, a variety that produces commercially desirable fruit may be susceptible to soil related production problems. Grafting allows the combination of the desirable root qualities of one variety with the desirable fruit qualities of another, thereby tailoring the tree to growing conditions. Grafting is usually done when trees are seedlings. Cuts are made in the stems of the root stock seedling and buds of the selected scion are inserted into the cuts. Sometimes when an established orchard is planted on a good rootstock, but the scion variety is no longer commercially desirable, growers will topwork the trees in the field, cutting off the scions and regrafting a new scion variety onto the old rootstocks. Approximately 98% of citrus grown in Texas LRGV, including orange and grapefruit varieties, is planted on sour orange rootstock [2]. The soils in the LRGV tend to be very heavy with a high clay content, and sour orange is one of very few rootstocks that do well under those conditions. 2
Relatively tolerant to adverse soil conditions, such as poor internal drainage, high water table, high salt content and high ph, sour orange rootstock has been the universal choice since the Texas industry s beginnings at the turn of the century [9]. Its tolerance to root rot has also been a great benefit to the Texas industry. Sour orange rootstock is also tolerant of citrus tatterleaf virus (CTLV) and citrus exocortis virus (CEV), both of which are economically damaging diseases present in the LRGV. Citrus Tristeza Virus (CTV) CTV is the most devastating disease of citrus worldwide and there is no remedy for an infected tree other than to remove it. Millions of trees on sour orange rootstock have been killed or rendered unproductive by CTV-induced decline [10]. Epidemics of tristeza decline began in the Western Hemisphere in the 1930 s and led to the loss of 30 million trees in Argentina, 10 million trees in Venezuela and 5 million in Brazil [11]. CTV is a complex disease. Citrus tristeza virus is the longest known plant virus, having the shape of a long flexous rod. There are many strains of it, ranging in type and severity of symptoms. The impact on a tree of any CTV strain is also largely dependent on the variety of the tree, both its rootstock and its scion. If the combination of tree variety and CTV strain are right, infected trees may show few symptoms, even if the strain is severe. Similarly, certain combinations of tree variety and CTV strains will lead to the tree showing disease symptoms. Regardless of the symptoms exhibited by a tree infected by a CTV strain, the tree serves as a reservoir of CTV, a potential source of infection for other trees given an efficient means of spreading it. In the United States, the most severe strains of CTV are those that cause quick decline and stem pitting. Grapefruit and sweet orange, regardless of the variety, are susceptible to CTV if they are planted on sour orange rootstock. One of the most economically significant symptoms of the disease is the decline reaction that occurs in infections on sour orange rootstock. The virus infection in the scion causes necrosis of the sour orange trunk and roots. The necrosis has a girdling effect, and trees decline as starch reservoirs in the rootstock are depleted [10]. 3
Symptoms of quick decline include bleaching of leaves and defoliation, resulting in death. This can occur within a matter of weeks. Stem pitting strains of CTV cause stunting, extreme brittleness of twigs, and extended longitudinal pits in the wood of the tree. Reduction in fruit quality and yield result. Infected trees show leaf yellowing, wilting, defoliation and fruit hanging on dead trees [6]. The combination in Texas of sour orange rootstock and grapefruit or orange as scion varieties is very susceptible to the citrus tristeza virus. The Brown Citrus Aphid Aphids are very efficient breeders. Most populations are entirely female, and each female is born pregnant with clones of herself. A single aphid can give birth to live clones at a rate of one or two dozen per day. The brown citrus aphid feeds on citrus and is the most efficient vector of citrus tristeza virus (CTV) [4]. Aphids are piercing-sucking insects. They pierce plant material with their mouthparts and suck out plant juices. If an aphid feeds on a plant infected with a virus, the virus particles are present in the plant juices, and get taken up into the aphid mouthparts when it feeds. When the aphid moves to another plant to feed, it carries the virus particles with it and infects the new plant when it feeds. The brown citrus aphid is not only an efficient vector of CTV, it even seems to selectively transmit the severe strains. Populations of the brown citrus aphid have moved north from South America over the last several decades via wind currents, natural migration and shipments of infested plants. Currently, the northern edge of its range is in Mexico and Florida. It is therefore a matter of time before it is discovered in Texas. Several strains of CTV are present in Texas citrus, including severe strains, but they seem to be contained in symptomless residential trees in the eastern part of the state, where it gets too cold to use sour orange as a rootstock [2, 3]. Without the presence of an efficient vector like brown citrus aphid to spread CTV from residential areas to southern production areas, serious damage to commercial citrus can be prevented by planting certified virus-free seedlings and by removing existing trees found to be infected with CTV. 4
The rapid reproduction of the brown citrus aphid makes controlling them difficult, and eradicating them impossible. Their virus vectoring ability and the fact that it takes only one aphid carrying virus particles to infect a plant, makes controlling CTV transmission through aphid control impossible. Insecticide use may reduce aphid populations and may slow the spread of disease, but it will not eliminate it or completely prevent it. Insecticide use against brown citrus aphids may be most helpful if a small and contained population is discovered. In that case insecticide use may eliminate the population and thereby prevent spread and virus transmission. But once the brown citrus aphid becomes established or widespread, insecticide use is not expected to play a significant role in CTV management [7]. If the brown citrus aphid arrives in Texas, it would move severe strains of tristeza suspected to be present in symptomless hosts in dooryard trees to commercial orchards, potentially destroying the Texas industry. This is because sour orange rootstock, the predominant commercial rootstock in Texas, is susceptible to severe strains. Resistance to CTV Quick decline can be managed by replanting orchards to rootstocks other than sour orange. Declining orchards of trees on sour orange rootstock usually are replanted with CTV-tolerant rootstock, such as rough lemon, Ringspur lime, trifoliate orange and mandarin [10]. Approximately 14% of Florida citrus trees are planted on sour orange rootstock [14]. Florida citrus orchards already have the brown citrus aphid and severe strains of CTV. But, with a wide diversity of soils in Florida, citrus growers in that state have viable options to the sour orange rootstock, and the industry has moved away from replanting trees on sour orange rootstock. In the 1970 s to 1980 s, 20-50% of Florida nursery citrus trees were on sour orange rootstock. Since 1996, no citrus nursery stock in Florida is on sour orange [13]. As a result of moving away from sour orange rootstock, however, there is a new disease problem in Florida citrus (citrus blight). The conditions in southern Texas, including the heavy soils and the presence of CTLV and CEV, mean shifting away from sour orange rootstock to existing CTV-tolerant rootstocks will create other production problems for Texas citrus. For example, trifoliate orange hybrids suffer from 5
severe iron chlorosis when planted in calcerous soil or soil with high clay content [9]. Poor bearing and small fruit size disqualifies mandarin, another rootstock tried unsuccessfully in the Rio Grande Valley [9]. There is a need for new citrus varieties that will tolerate the soils and the diseases of southern Texas, including CTV. There are several breeding programs underway for developing CTV resistant citrus cultivars. Citrus breeding, however, is hampered by the fact that the seeds produced by most commercial varieties are largely asexual they contain genes from only one parent. Therefore traditional breeding programs rely on alternative methods, such as screening for natural or induced mutations that provide desirable traits rather than crossbreeding for them. Innovative laboratory techniques also allow for fusing of cells from different citrus species and then growing them into plants in order to produce crosses. There are at least two new varieties in development that may be CTV tolerant and are adapted to growing conditions in the LRGV. Transgenic Citrus Biotechnology offers another way to produce a citrus variety that has tolerance or even resistance to CTV yet maintains the other desirable horticultural qualities of sour orange rootstock. One method of CTV resistance through biotechnology under investigation uses the natural resistance found in a close relative of citrus, Poncirus trifoliate, and transfers it into the desired commercial citrus variety. This resistance from Poncirus has been bred into commercial citrus through traditional means, but fruit from resulting crosses also contain qualities from Poncirus that make it inedible. Through biotechnology, the CTV-resistance gene can be transferred without the genes for the inedible flavor. Cells of commercial citrus varieties have been fused with CTVresistant but non-sexually compatible relatives by somatic hybridization [18]. Another method through which genetic engineering can confer CTV resistance to citrus is the insertion of CTV coat protein genes into citrus tissue. Rio Red grapefruit has been transformed, via Agrobacterium tumefaciens, with a CTV coat protein gene [8] [17]. Molecular analysis confirmed the presence and expression of the transgenes in the shoot tissue. The transformed shoots were grafted onto sour orange rootstock [8]. 6
The transformation project with the coat protein gene is funded with local commodity funds [12]. The goal of the genetic engineering is to transform a scion with resistance to CTV so that Texas growers can continue to plant on existing sour orange rootstock [12]. Essentially, the coat protein gene insertion works as a form of vaccination in which the plant learns to recognize the viral RNA sequence as foreign and targets the transgene by silencing the coat protein gene. Then when CTV infects the plant, the plant quickly recognizes its genes and is ready to silence them too. In 2000, the USDA granted permission for genetically engineered grapefruit to be planted in a test plot in Texas. Fifty-five trees have been transformed, and 20 have been planted in the field [12]. They were planted in 2000 and will bear fruit in a few years, at which time their horticultural qualities will be assessed. Preliminary laboratory work indicates that CTV resistance is successful, and the studies are being replicated [12]. The most promising engineered tree is the result of grafting a transformed shoot tip onto infected sour orange rootstock. It has remained virus-free for seven months [12]. Pending approval, an additional 70 trees with CTV coat protein genes will be planted. Estimated Impacts It is assumed that a transgenic tristeza resistant cultivar would be planted universally in Texas and thereby prevent the loss of citrus production in the state. 7
TABLE 11.1: Texas Citrus Production Bearing Acres Production Value Year (000) (1,000) Tons (Million $) 1999-2000 29.1 311 47.9 1998-1999 29.1 305 45.4 1997-1998 32.5 256 28.4 1996-1997 29.1 273 29.6 1995-1996 25.5 222 28.6 1994-1995 21.9 230 20.4 1993-1994 18.2 144 20.3 1992-1993 14.5 97 17.0 1991-1992 11.0 4 1.4 1990-1991 8.0 0 0 1989-1990 31.7 131 22.4 1988-1989 28.9 271 39.0 1987-1988 27.1 213 42.2 1986-1987 25.5 114 25.6 1985-1986 21.8 23 5.2 1984-1985 30.5 0 0 1983-1984 66.6 235 20.9 1982-1983 66.2 689 50.6 1981-1982 64.8 808 66.4 1980-1981 66.8 452 47.6 Source: [1] [15] [16] 8
References 1. USDA, Citrus Fruits 2000 Summary, National Agricultural Statistics Service, September 2000. 2. Skaria, M., C.J. Kahlke, N. Solis-Gracia, and R. Prewett, Virus-free Citrus Budwood Production and Tristeza Management Program in Texas Through Industry Partnership, Subtropical Plant Science 49:1-7, 1997. 3. Skaria, Mani, Plant Pathologist, Texas A&M Kingsville Citrus Center. Personal Communication, March, 2001. 4. Costa, A.S. and T.J. Grant, Studies on Transmission of the Tristeza Virus by the Vector Aphis citricidus, Phytopathology 41:105-113, 1951. 5. A Dual Citrus Threat, Agricultural Research, December 1994. 6. Roistacher, C., Citrus Tristeza Closterovirus, available on the internet at www.ecoport.org. 7. Da Graca, J., Deputy Center Director, Texas A&M Kingsville Citrus Center. Personal Communication, March, 2001. 8. Yang, Z.N., I.L Ingelbrecht, E. Louzada, M. Skaria, and T.E. Mirkov, Agrobacteriummediated Transformation of the Commercially Important Grapefruit Cultivar Rio Red (Citrus paradisi Macf.), Plant Cell Reports 19:1203-1211, 2000. 9. What Alternative Rootstocks are There for Valley Citrus?, Citrus Center Newsletter, Texas A&M University, Volume 12, No. 2, June 1994. 10. Whiteside, J.O., et al., Compendium of Citrus Diseases, APS Press, 1988. 11. Texas Citrus Industry Braces for Tristeza, The Grower, April 1995. 12. Mirkov, Erik, Texas A&M University, personal communication, March 2001. 13. Brown, Mark G., Florida Department of Citrus, personal communication, March 2001. 14. Brown, M., The Potential Impact of the Citrus Tristeza Virus on Florida Grapefruit and Orange Production, paper presented at Florida Department of Citrus s Production Workshop, November 1999. 15. USDA, Agricultural Statistics 1980-1999 (separate volumes, National Agricultural Statistics Service. 9
16. USDA, Fruit and Tree Nuts: Situation and Outlook Report, September 1994, Economic Research Service. 17. Transgenic Grapefruit Trees Developed in Texas to Resist Deadly Citrus Tristeza Virus, Western Farm Press, May 6, 2000. 18. McGovern, R.J., et al., Tristeza, SP155, University of Florida, February 1994. 10