Citrus tristiza virus (CTV) is the most-significant viral pathogen of citrus in the world. Rapid decline of trees on sour orange and stem pitting of grapefruit and sweet orange, two diseases induced by CTV, severely jeopardize citrus production worldwide. It is recognized that all future rootstocks should be resistant to this virus, and scion resistance to stem pitting stains is desirable. To facilitate introgression of the CTV resistance gene from Poncirus trifoliata and development of CTV-resistant varieties in citrus, gene mapping projects have been initiated and more than a dozen RAPD markers have been identified with tight linkage to the resistance gene. As part of our efforts to use marker-assisted selection with a large number of crosses, and ultimately to accomplish map-based cloning of the CTV resistance gene, we have been converting the most tightly linked RAPD markers into SCAR (sequence characterized amplified region) markers by cloning, sequencing the marker fragments, and designing locus-specific primers. One codominant and several dominant SCARs have been developed thus far. The updated progress and utilization of these SCARs in marker-assisted selection and possibly in characterization of a BAC library will be presented and discussed.
Citrus Tristeza Closterovirus (CTV) induces mild and/or severe symptoms on Citrus species. It may cause death of trees if the rootstock-scion combination is susceptible. It has been found in other plant/virus combinations that transformation with partial or complete viral genes (e.g., coat protein genes) can confer resistance to the resulting transgenic plants. We previously reported A. tumefaciens mediated transformation and production of two sour orange (C. aurantium L.) plants expressing the coat protein gene of CTV, which was the first report of production of transgenic Citrus using a viral gene. However, in order to properly evaluate resistance, it is necessary to obtain as many transgenic Citrus plants from single transformation events as possible. Therefore, we are currently transforming grapefruit (Citrus paradisi) `Marsh' and `Star Ruby' and sweet orange (C. sinensis) `Valencia' with CTV coat protein genes. These species are susceptible to CTV and more amenable to transformation than sour orange. Epicotyl segments of etiolated seedlings were inoculated with A. tumefaciens strain EHA101 harboring binary plasmid pGA482GG containing the coat protein gene of mild Florida CTV strain T30 (CP-T30) or severe Florida strain T36 (CP-T36). Putatively transformed shoots were regenerated on selection medium containing kanamycin. Regenerated shoots were evaluated with GUS assays; those shoots positively identified by GUS were then evaluated with PCR. We have currently identified 17 `Marsh' grapefruit, 20 `Star Ruby' grapefruit, and seven sweet orange putatively transformed plants.
but with resistance to citrus tristeza virus (CTV)-induced quick decline disease, to which it is especially sensitive ( Bar-Joseph et al., 1989 ). Sour orange has been widely planted throughout the world because of its many positive attributes
Sour orange (Citrus aurantium) has been the dominant citrus rootstock in the Indian River region of Florida since the initial plantings in the 1880s. Use of this rootstock in new plantings has been rare since 1990 because of heightened concern about decline strains of citrus tristeza virus (CTV), to which this rootstock is highly susceptible. Because the proportion of trees remaining on sour orange rootstock and the rate of decline among them are important in predicting the economic consequences for the Indian River citrus industry, two surveys of rootstock usage were conducted for citrus in this growing region. In the first survey, growers were asked about rootstock usage and problems observed across all types of citrus, and responses represented 35% of acreage. In the second survey, growers were restricted to rootstock usage and grower observations on decline for grapefruit (C. paradisi), and responses represented 53% of acreage. Even though 44% of all current Indian River grove area has been planted since 1987, when use of sour orange for new plantings largely ceased, 48% of all citrus and 55% of all grapefruit grove area are currently on sour orange rootstock. The percentage of grapefruit trees on sour orange rootstock that showed significantly health decline in 2000 was 8% based on grower reports. The other root-stocks representing substantial commercial grove area have known problems and limitations that are likely to prevent any of them from gaining the prominence once held by sour orange. Swingle citrumelo (C. paradisi × Poncirus trifoliata) at about 25% of grove area, Cleopatra mandarin (Citrus reticulata) at about 8%, and Smooth Flat Seville (Citrus hybrid) at about 3% all represented similar acreage for grapefruit and across all cultivars, while Carrizo citrange (C. sinensis × P. trifoliata) use was reported for 4% of grapefruit and 13% overall. Evaluation and development of new rootstocks is vitally important for the Indian River area, especially for soils with significant clay and calcium content.
The authors are grateful to S.M. Garnsey for providing CTV-specific polyclonal antibody 1212 and to Phyllis A. Rundell for her help in the lab. Florida Agriculture Experiment Station Journal Series: R-10841.
This project was funded by USDA cooperative agreement 58-6617-4-018. Florida Agricultural Experiment Station Journal Series R-066-91 We thank S.M. Garnsey for providing CTV PCA1212, Phyllis A. Rundell, R.R. Bullock and the staff at the
One hundred single brown citrus aphid (BCA) (Toxoptera citricida Kirkaldy) transmission attempts were made from each of 16 different citrus trees [8 grapefruit (Citrus paradisi Macf.) and 8 sweet orange (C. sinensis (L.) Osbeck)] previously inoculated with decline-inducing (T36-CTV), non-decline-inducing (T30-CTV), a mixture of the two Citrus tristeza virus isolate types, or no CTV. Successful CTV transmission occurred in 1.5% of attempts from grapefruit trees that had been bark-chip-inoculated with T36-CTV, 3% of attempts from orange trees inoculated with T36-CTV, 3% of attempts from grapefruit trees inoculated with both T36- and T30-CTV, 4% of attempts from orange trees inoculated with both T36- and T30-CTV, 1.5% of attempts from grapefruit trees inoculated with T30-CTV, and 3.5% of attempts from orange trees inoculated with T30-CTV. Single BCA were able to recover T30-like-CTV from trees believed to be inoculated only with T36-CTV, and T36-like-CTV from trees believed to be inoculated only with T30-CTV, suggesting that these inoculum sources were also mixtures of T36-CTV and T30-CTV. The T36-CTV was not immunologically detectable in some of the trees from which it was transmitted indicating that single BrCA can recover T36-CTV from a T36-CTV/T30-CTV mixture in which the T36-CTV is an undetectable, minority component.
`Chandler' pummelo [Citrus maxima (Burm.) Merrill] was found to be citrus tristeza virus (CTV)–resistant. The inheritance of this resistance in 84 progeny of two crosses derived from `Chandler' pummelo and trifoliate orange [Poncirus trifoliata (L.) Raf.] was controlled by a single dominant gene designated Ctv2. Progeny analysis of four molecular markers closely linked to the Ctv gene, which confers resistance to CTV in trifoliate orange, demonstrated that Ctv2 was an independently assorting gene from Ctv.
Citrus rootstocks resistant to decline induced by citrus tristeza (CTV) have been developed using resistance found in Poncirus trifoliata (L.) Raf. A breeding line with resistance to CTV infection, US119 [Citrus paradisi Macf. cv. Duncan × P. trifoliata (L.) Raf. × C. sinensis cv. Succory], was developed and crossed with `Ambersweet' orange [C. reticulata Blanco × (C. paradisi × C. reticulata) × C. sinensis] to develop scions resistant to CTV infection. Fifty-one progeny were tested for CTV resistance by grafting each to rough lemon [C. limon (L.) Burm. f.] seedlings infected with CTV. These propagations were assayed repeatedly by ELISA. Seventeen progeny consistently tested negative for CTV (resistant), 24 were consistently positive (susceptible), and 10 gave weak or inconsistent reactions. These results confirm the feasibility of developing CTV-resistant scions by use of US119 to solve the stem pitting disease caused by CTV.
None of 4190 sweet orange [Citrus sinensis (L.) Osb.] nursery trees of `Hamlin', `Midsweet', `Navel', and `Valencia' sampled from five Florida citrus nurseries were infected with a decline-inducing isolate of citrus tristeza virus (CTV) as judged by enzyme-linked immunosorbent assay (ELISA) using isolate-specific monoclonal antibodies. Two of the nurseries had a relatively high level of infection (37% to 100% of composite samples containing tissue from 10 trees) with nondecline-inducing (mild) isolates of CTV, depending on the cultivar. Three of the nurseries had a lower incidence of mild CTV (0% to 22% of 10 tree composite samples). No nursery was CTV-free. ELISA of individual trees used as budwood sources by the nurseries revealed that one tree out of 260 tested contained decline-inducing CTV, and 83 contained mild CTV. These results suggest that the budwood certification program adopted in 1997 has virtually eliminated decline-inducing CTV from commercial budwood supplies.