1. Field of the Invention
The field of the invention is plant breeding, and, in particular, the development of squash plants having high resistance to geminiviruses, such as Squash leaf curl virus (SLCV). The invention also pertains to squash plants having high resistance to geminiviruses as well as resistance to potyviruses, such as Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus 2 (WMV-2), and Papaya ringspot virus (PRSV).
2. Description of Related Art
Squash is the common name for a collection of plants that produce edible seeds, fruits and flowers. Squashes are divided into two general groups, summer and winter. Fruits from the summer group are harvested and consumed at an immature state. Winter squash (which includes market classes sometimes called pumpkin) fruits are harvested at maturity, after the fruits have ripened and developed a hard rind.
Winter squashes are the mature fruits of three Cucurbit species: Cucurbita maxima, Cucurbita moschata and Cucurbita pepo. Fruit from winter squash varieties are grown to physiological maturity and typically stored for consumption during the winter months or used for ornamental purposes. Examples of common winter squashes are acorn, butternut, hubbard, and spaghetti squash, as well as the Halloween type pumpkins.
Most summer squash varieties are Cucurbita pepo, and their fruits are typically harvested and consumed at an immature stage. The flowers of summer squash can also be harvested for consumption. There are many types of summer squash, including yellow crookneck, yellow straightneck, scallop, Lebanese, and green and gray zucchini. Green zucchini is the type of C. pepo squash preferred by consumers in Europe and many parts of the North America, as well as in other regions. Unlike winter squashes, summer squash fruit have a short shelf life, and are typically consumed within days of harvest. Because of the extended ability to ship produce over long distances there are some markets where the terms “summer” and “winter” squash no longer reflect a restriction on availability and all types can be found in these markets year round.
All squash are indigenous to North and South America, and the Cucurbita pepo varieties are most likely domesticated from the wild Cucurbita texana and Cucurbita fraterna species (Sauer (1993) Historical geography of crop plants—a select roster. CRC Press, Boca Raton, Fla.). Squash, along with beans and maize, were the primary agricultural crops of Native North Americans for at least 300 years before the arrival of the Europeans. These crops were grown in an intercropping system. Because of their importance in the culture, the crops were coined ‘The Three Sisters’ in the Iroquois tribe's creation myth (Erney, Diana. 1996. Long live the Three Sisters. Organic Gardening. November. p. 37-40).
After introduction to Europe by explorers in the 1500's, squash spread as an agricultural crop around the world. Summer squash varieties show considerable diversity, and are classified commercially into market types by their biological species, fruit shape, and fruit coloring. The green zucchini, white scallop and yellow crookneck types are some examples of diversity found in Cucurbita pepo. 
Many pathogens infect squash, though studies have shown that viral diseases cause the most severe damage. For many years, four viral pathogens have presented the main challenge to squash production in almost every growing region in the world. Of these four most common viral pathogens, three belong to the Potyvirus genus. These are ZYMV, WMV-2 and PRSV. Potyvirus virions contain one or two linear, positive-sense single stranded RNA(s). Viruses in this genus have a genome between 8,500 and 10,000 nucleotides.
The International Committee on Taxonomy of Viruses (ICTV) operates under the auspices of the Virology Division of the International Union of Microbiological Societies. In the universal scheme developed by the ICTV, virion characteristics are considered and weighted as criteria for making divisions into families, in some cases subfamilies, and genera. The ICTV database is similar in structure to the Dewey Decimal Classification System. The ICTV virus code for PRSV is 00.057.0.01.045. For ZYMV, the ICTV code is 00.057.0.01.077. The ICTV virus code for WMV-2 is 00.057.0.01.073.
The fourth traditional viral pathogen of squash is Cucumber mosaic virus (CMV), a cucumovirus. Cucumoviruses belong to the family Bromoviridae (ICTV Virus Code: 00.010.0.04.001).
There are serological and molecular diagnostic tests to diagnose which of these traditional viral pathogens have infected squash plants, as it is difficult to visually distinguish viral symptoms caused by any one of these viruses.
The visual diagnosis of virus infection can be even more difficult in growing areas where infection by more than one virus is commonplace. Common foliar symptoms caused by these traditional pathogens are plant stunting due to shortening of the internodes and an overall reduced growth rate. Foliar symptoms can include mottling, puckering, curling and distortion of the leaves; mosaic patterns are also common and range in color from green to yellow. Generally, the younger the plant at the time of infection, the more pronounced the symptoms.
In addition to these foliar symptoms, fruit defects have been described as being warty, bumpy and deformed in shape. These defects typically make the fruits unmarketable (Bernhardt et al. Cucumber Disease: A Practical Guide for Seedsmen, Growers and Agricultural Advisors, Petoseed Company. 1988; Zitter et al. Compendium of Cucurbit Diseases, American Phytopathological Society, 1996). Many plant viruses can be mechanically transmitted, but by far the most common mode of transmission of these four viruses is by aphids in a non-persistent manner. Aphids feed on plant sap, and acquire the nutrients therein through a specialized feeding tube designed both for probing leaves and for siphoning off the sap. If an aphid feeds on an infected plant, the virus particles stay in their mouthparts. When the insect then feeds on an uninfected plant, the virus particles are transmitted to the new host as the aphid probes again for its food. It takes only a few seconds for virus transmission to occur.
Both commercial or volunteer crops, and weeds, can harbor squash infecting viruses and serve as sources for additional infection. Some viruses that infect squash, such as CMV, have a very broad host range. CMV is an aphid-transmitted virus with worldwide distributions that can infect up to 800 plant species. Other viruses, such as SLCV (ICTV virus code is 00.029.0.03.035), infect a much more limited but diverse (beans and squash) group of plants. SLCV is transmitted persistently by the sweet potato whitefly, Bemisia tabaci, not by aphids. Removing weeds or volunteers, or growing the commercial crop away from these other potential hosts or when the insect vectors are not plentiful can help reduce the incidence of viral diseases.
Once the virus has been identified, a control strategy that includes insect suppression or eradication, sanitation, crop rotation, and planting schedules can be developed. The diversity of insect vectors makes controlling viruses by controlling the vector difficult, especially if conditions are conducive for vector proliferation. In some cases, the extent of virus infection can be limited by controlling the vector. Weather and crop timing can be of great influence on insect vector populations and the subsequent amount of virus that develops. In arid and semi-arid growing regions, aphids proliferate in the spring and early summer when temperatures are generally below 90-95° F., but their numbers dramatically fall when temperatures exceed 95° F. In contrast, whiteflies develop and increase in number quickly at temperatures in excess of 90-95° F. Therefore, viruses that are associated with each of these vectors also will be most common during the respective weather conditions. Crops that are planted late in the growing season are often at highest risk because insect vector populations and virus levels may have built up on earlier crops.
Insecticide or mineral oil sprays can be used for controlling insect vectors. Good results have been reported using a combination of Platinum™, a soil-applied insecticide that provides control of aphids and whiteflies, and Fulfill™, a foliar applied insecticide that suppresses whitefly populations and stops aphid feeding within 1-2 hours of initial ingestion, to reduce the transmission of viruses. Platinum™ contains the active ingredient thiamethoxam. Its effectiveness is a function of its systemic properties; because of its high water solubility, moderate soil absorption and low the partition coefficient of thiamethoxam, it moves quickly through the plant to provide protection
Fulfill™ is a selective, foliar aphicide with a unique mode of action that is not harmful to some beneficial insects. Fulfill™ also has some limited activity against whiteflies in vegetable crops. The active ingredient in Fulfill™ is pymetrozine.
Additional to chemical control methods, growers can use several other horticultural practices to reduce virus-induced damage. One of these is known as mid-bedding, where seed is planted in V-shaped trenches between plant beds in the early spring, and the trenches are covered with plastic. The main benefit is increased soil temperatures to hasten plant development for early production, but a side benefit is a possible reduction in early aphid populations and associated viruses on the young plants. Reflective mulches, which are intended to disorient aphids, have been used with varying degrees of success for limiting aphid-transmitted viruses. For the viruses that are transmitted mechanically as well as by insects, cultivation and other equipment should be cleaned and disinfected prior to being moved from infected to non-infected fields.
While chemical and other controls can sometimes be somewhat helpful in curtailing virus-induced diseases, alternative and better control strategies are clearly needed. Chemical control methods are a particular concern. Their efficacy can be a problem, first of all, as insects can escape the spray.
Of greater concern is the fact that whiteflies develop resistance to a pesticide when used frequently. In tomato, pesticide resistant whiteflies of B. tabaci have developed that can vector over 20 different tomato-infecting begomoviruses. If resistance to the pesticide develops, the mechanism to limit the virus spread is lost to all viruses transmitted by that vector (Morales, F. J., P. K Anderson: The emergence and dissemination of whitefly-transmitted geminiviruses in Latin America, Archives of Virology, Volume 146, Issue 3, March 2001, Pages 415-441; Polston, J. E., P. K. Anderson (1997): The emergence of whitefly transmitted geminiviruses in tomato in the western hemisphere, Plant Disease 81:1358-1369; Zeidan, M., et al., 1999: Molecular analysis of whitefly-transmitted tomato geminiviruses from Southeast and East Asia, Trop. Agric. Res. Ext. 1, 107-115.). Further, there are increasing pressures from regulatory agencies to withdraw approvals for pesticide use, and from environmental and consumer advocates, who have concerns about the environmental consequences of pesticide use and about the effect these pesticides may have on human health Therefore, there is a long-standing need in commerce for alternate strategies to control these viral diseases.
In recent years, squash varieties having some level of resistance to certain plant viruses have been developed. Examples include the varieties Noche and Contender (Rogers), which are dark green zucchini varieties with reported resistance to ZYMV and WMV-2. Resistant varieties can be incorporated into a virus control strategy but are generally only resistant to specifically designated viruses, and not broadly resistant to the complete assortment of squash infecting viruses.
Seminis has created and obtained regulatory approval in the United States to sell multiple virus resistance transgenic squash. One transformation event is referred to as ZW-20. A second event, called CZW-3, confers resistance to ZYMV, WMV-2 and CMV. Both transformation events express the viral coat protein genes that were inserted into the plant DNA using Agrobacterium mediated transformation. Varieties containing the ZW-20 event have resistance to ZYMV and WMV-2; those containing the CZW-3 event are also resistant to CMV.
Except for the virus resistance phenotype, the transgenic lines look and behave the same way as their non-transgenic counterparts. Seminis markets a number of varieties that contain these transgenic events, including the varieties ‘Declaration II’ (green zucchini), ‘Independence II’ (green zucchini), ‘Patriot II’ (yellow straightneck), ‘Destiny III’ (yellow crookneck), ‘Liberator III’ (yellow straightneck), ‘Justice III’ (green zucchini), ‘XPT 1832 III’ (yellow straightneck), and ‘Judgement III’ (green zucchini). In addition, one transgenic variety called ‘Conqueror III’ contains non-transgenic intermediate resistance to PRSV. The addition of ‘II’ or ‘III’ to the variety name refers to the transformation event, respectively ZW-20 and CZW-3.
Varieties which are well matched to their target growing regions, such as Conqueror III targeted to the South-eastern USA, will have resistance to all the viral diseases likely to be encountered in that region. Similarly Justice III and Judgement III (green zucchini varieties) and XPT 1832 (yellow precocious straightneck) have high resistance to the three viruses likely to be encountered in the Northeastern and Midwestern USA (CMV, WMV-2, ZYMV).
Unlike some of the well-established common viral diseases described above, SLCV is the causative agent of a relatively new squash disease called squash leaf curl. SLCV is a geminivirus belonging to the genus Begomovirus. Geminiviruses are plant viruses that belong to the family Geminiviridae, first described by Goodman in 1977 (Goodman, 1977a, 1977b). Geminiviruses are characterized by the unique twin shape of a fused icosahedral viral particle. The geminate virions contain a circular single-stranded DNA (ssDNA) genome. The family Geminiviridae is comprised of three genera, all of which share similarities in genome organization, insect transmission, and host range.
The genus Begomovirus consists of viruses with monopartite and bipartite genomes. Begomoviruses are transmitted by whiteflies in a persistent, circulative, non-propagative manner, and infect dicotyledonous plants. There is agreement that plant pathogenic Begomoviruses have a complex association with the whitefly, their insect vector, though aspects concerning viral genetic activity (genome replication and gene transcription) within the insect remain controversial.
During the past 25 years geminiviruses have become a particular problem on squash in dry regions where the whitefly vectors are present in large numbers. There are now three begomoviruses identified in squash and it is expected that this number will grow. The current list includes Squash mild leaf curl virus (SMLCV) from California, SLCV from the United States (Arizona, California, and Texas), Mexico (Sinaloa and Sonora), Guatemala, Nicaragua and Panama, and Cucurbit leaf curl (crumple) virus (CuLCV) from the USA (Arizona, California, and Texas) and Northern Mexico (Coahuila). Most of these begomoviruses are capable of forming viable recombinants with related viruses in the squash leaf curl virus group. This ability to recombine is thought to expand diversity within the virus group, and can explain why these geminivirus-induced diseases have spread so quickly. In addition to the pathogens' rapid evolution, the development of irrigation in arid and semi-arid growing regions has led to a longer growing season, and to increases in host plant densities. These in turn, have resulted in increased whitefly populations and the proliferation of begomoviruses.
SLCV is a typical example of the viruses in this group. SLCV has geminate particles, 22×38 nm in size (Cohen, S.; Duffius, J. E.; Liu, H. Y. (1989) Acquisition, interference, and retention of cucurbit leaf curl viruses in whiteflies. Phytopathology 79, 109-113), and the virus is associated with maturing phloem sieve tube elements (Hoefert, L. L. (1987), Association of squash leaf curl virus with nuclei of squash vascular cells. Phytopathology 77, 1596-1600). SLCV was first identified in the late 1970's and was originally transmitted by Bemisia tabaci biotype A whitefly, but became a more severe problem in the late 1980's when it became transmissible by biotype B. In addition to vectoring SLCV, extensive feeding of the Bemisia tabaci, biotype B whitefly on squash causes a leaf silvering disease. The combination of SLCV and silvering disease can be devastating. SLCV causes severe losses of squash, melons and related cucurbits in Arizona and California (USA) (Dufflis, J. E.; Flock, R. A. (1982) Whitefly-transmitted disease complex of the desert southwest. California Agriculture 36, 4-6; Nameth, S. T.; Laemmlen, F. F.; Dodds, J. A. (1985) Viruses cause heavy melon losses in desert valleys. California Agriculture 39, 28-29). Typical symptoms of SLCV include a bright yellow mottling or mosaic of the leaves, which are accompanied by pronounced upward leaf curling and enations on the underside of leaves. Infected squash plants are stunted and normally do not produce additional foliage after infection, and fruit set will be greatly reduced. Fruit on infected plants may become misshapen and discolored. Plants may be killed when the disease is severe, especially if infection occurs early during the plant's development.
Silvering disease is described in Yokomi, R. K, K. A. Hoelmer, and L. S. Osborne. 1990. Relationships between the sweet potato whitefly and the squash silverleaf disorder. Phytopath. 80:895-900. (Also Schuster, K. J., J. B. Kring, and J. F. Price. 1991. Association of the sweet potato whitefly with a silverleaf disorder of squash. HortSci. 26:155-156; Costa, H, S., D. E. Ullman, M. W. Johnson, and B. E. Tabashnik. 1993. Squash silverleaf symptoms induced by immature, but not adult, Bemisia tabaci. Phytopath. 83:763-766; Jimenez, D. R., J. P. Shapiro, and R. K Yokomi. 1993. Biotype-specific expression of dsRNA in the sweet potato whitefly. Entomol. Exp. Appl. 70: 143-152). Silvering disease is seen on cucurbits infested by Bemisia tabaci B-type, but is not necessarily associated with SLCV. It has been suggested that silvering disease could be due to infection by another virus (Bharathan, N.; Narayanan, K. R.; McMillan, R. T. 1992. Characteristics of sweet potato whitefly-mediated silverleaf syndrome and associated double-stranded RNA in squash. Phytopathology 82, 136-141.), but it is now generally thought to be induced physiologically by the feeding of the B-type (hence its proposed name B. argentifolii) whitefly (Perring, T. M., Cooper, A. D., Rodriguez, R. J., Farrar, C. A., and Bellows, T. S. 1993. Identification of a whitefly species by genomic and behavioral studies. Science 259:74-77 and Brown, J. K, Frohlich, D. R, and Rosell, R. C. 1995. The sweet potato or silverleaf whiteflies: Biotypes of Bemisia tabaci or a species complex? Ann. Rev. Entomol. 40: 511-534).
SLCV was first reported in California damaging crops of Cucurbita pepo. The SLCV group evolves rapidly (Brown J. K, A. M. Idris, C. Alteri and D. C. Stenger (2002) Phytopath. 92:734-742). This has created some concern over the potential emergence of a new cucurbit-infecting begomovirus species capable of forming viable recombinants with related viruses in the squash leaf curl virus cluster.
Control of SLCV mainly aims at eliminating or excluding the vector Bemisia tabaci. Endosulfan and other insecticides have been used in an attempt to reduce whitefly density. Protecting rows of seedlings with spun-bonded polyester as a floating cover was found more effective than any other mechanism to date (Natwick, E. T.; Durazo, A. (1985) Polyester covers protect vegetables from whiteflies and virus diseases. California Agriculture 39, 21-22.). This is an extreme and expensive measure to take for a field crop, as the cloth must be lifted to ensure pollination. In many growing areas, the additional material cost (cloth) and labor is prohibitively expensive, and can be ineffective in windy environments. Transmission of SLCV by mechanical means has not been documented.
While some level of resistance in Cucurbita spp. to SLCV was reported in 1984 (McCreight, J. D. (1984) Tolerance of Cucurbita spp. to squash leaf curl. Report, Cucurbit Genetics Cooperative, USA No. 7, 71-72), to date no resistance or high resistance to SLCV has been reported in any commercial squash varieties (C. pepo).