This research was supported by the California Tristeza Research Coalition, and the Ira J. Condit Research Fund of the Univ. of California at Riverside. We gratefully thank Dr. Deborah M. Mathews and Professor Allan Dodds in the Dept. of
Sixty-eight percent of the `Pineapple', 52% of the `Navel', 46% of the `Valencia', 38% of the `Hamlin', and 0% of the `Ambersweet' orange [Citrus sinensis (L.) Osh.] trees in five Florida citrus nurseries were infected with severe strains of citrus tristeza virus (CTV), as demonstrated by reaction with a monoclinal antibody specific for severe strains of the virus. Severe strains of CTV infected 4%, 46%, 76%, 30%, and 48% of the trees at each of the five nurseries, respectively, indicating a considerable difference in severe strain prevalence among the nurseries. Thirty-five percent of the trees in the scion blocks (budwood source) of the nurseries also contained severe strains of 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.
185 WORKSHOP 20 (Abstr. 435–437) Strategies to Develop Virus-resistant Fruit Cultivars
Six severe and six mild Florida isolates of citrus tristeza virus (CTV) were used to evaluate the transmission efficiency of the virus from grapefruit seedlings by single brown citrus aphids (Toxoptera citricida Kirkaldy) (BrCA) from colonies initiated by aphids obtained from citrus groves in Fort Pierce, Fla. The transmission rate to 2120 receptor plants [`Mexican' lime (Citrus aurantifolia)] from grapefruit by single BrCA was 1.5%. Single BrCA transmitted four of the six severe isolates and three of the six mild isolates of CTV. The average transmission rate of severe isolates was 1.8%, higher than that (0.9%) of mild isolates. Severe isolate Y-7 had the highest transmission rate among six severe isolates, 3.6%. Mild isolate Y-23 had the highest transmission rate among the mild isolates, 3.0%. The transmission rates of CTV by alatae, apterae, or nymphs of BrCA were 1.5%, 1.5%, and 1.0%, respectively. The results suggested that BrCA is an inefficient vector of CTV when the source plant is grapefruit.
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.
The distribution pattern of citrus tristeza virus (CTV) T-36 isolate in leaves of infected mexican lime [Citrus aurantifolia (Christm.) Swingle] plants was visualized using a whole-leaf-blot immunoassay (WLBIA) procedure in combination with a computer scanning imaging technique and CTV-specific monoclonal antibody 17G11 (CTV MAb 17G11). The distribution pattern of CTV T-36 in leaves varied with the age of the leaves and shoots of infected plants. In the young leaves, especially the about 5-day-old leaves and the completed expanded leaves, CTV T-36 was easily detected in most of the leaf veins, the main veins and the large and small primary veins. In the old leaves, CTV T-36 only was detected in the main veins, sometimes in a few of the large primary veins with weak signals, and seldom in the small primary veins. The distribution density and immunoassay reaction signals of CTV T-36 reacted to CTV MAb 17G11 in leaves from new shoots were much higher than that in leaves from old shoots. ELISA test results using leaves with different ages from different shoots of the same mexican lime plants infected with CTV T-36 supported the visualized-test results obtained by the WLBIA in combination with computer scanning imaging technique. This is the first reported visual analysis of the distribution pattern of CTV in leaves of infected citrus plants. The results indicate that the WLBIA in combination with computer scanning imaging technique is a useful tool for studying the distribution of plant viruses in leaves of virus-infected plants.
Bark chips from six container-grown citrus trees, infected with nondecline-inducing citrus tristeza virus (CTV) isolates and maintained in a vector-free greenhouse for 10 years, 15 commercial grapefruit (Citrus paradisi Macf.) trees, and 16 commercial sweet orange [C. sinensis (L.) Osbeck] trees were used to inoculate three indicator plants each of `Madam Vinous' sweet orange [C. sinensis (L.) Osbeck], sour orange (C. aurantium L.), `Duncan' grapefruit (C. paradisi Macf.), `Mexican' lime [C. aurantifolia (Christm.)], Swingle citrumelo [C. paradisi Macf. × Poncirus trifoliota (L.) Raf.], and sour orange grafted with `Hamlin' sweet orange [C. sinensis (L.) Osbeck]. All plants providing bark chips had repeatedly tested positive by enzyme-linked immunosorbent assay (ELISA) for CTV [reacted with monoclonal antibody (MAb) 17G11], but tested negative for Florida decline-inducing isolates of CTV (did not react with MAb MCA13). After 6 months in vector-free greenhouses, all in oculated trees (except Swingle citrumelo, which is considered CTV resistant) were positive for CTV by 17G11 ELISA. In addition, some indicator plants inoculated from nine (two container, two commercial grapefruit, and five commercial orange trees) of the 37 bark chip source trees also were positive for decline-inducing CTV by MCA13 ELISA. Some of these positive indicators also showed vein-clearing symptoms characteristic of infection with a severe isolate of CTV. No control, noninoculated indicators in the same greenhouse, became infected with either decline-inducing or nondecline-inducing CTV. These results indicate that decline-inducing isolates of CTV can be present as a minor component of a mixture at levels undetectable by ELISA, and that these decline-inducing isolates can become detectable by ELISA and sometimes by symptoms when inoculated into indicator plants.
Four field sources of citrus tristeza virus (CTV) (Y3, Y6, Y7 and Y23) collected from grapefruit trees at groves in Fort Pierce, Florida, and isolate T36 were used to evaluate the transmission and separation of different virus genotypes by single brown citrus aphids (BrCA). Analysis of the field sources of CTV by inoculation to indicator plants, ELISA and RT-PCR showed that Y6 was a decline-inducing isolate and Y23 a nondecline-inducing isolate. Assays of genotype by RT-PCR indicated that Y6 contained the T36 genotype while Y23 contained the T30 genotype. Both Y3 and Y7 were a mixture of decline-inducing and nondecline-inducing CTV isolates and were a mixture of T36 and T30 genotypes. When Y6 and Y23 were the acquisition host for single BrCA, only the T36 or T30 genotypes, respectively, were detected by RT-PCR in `Mexican' Lime receptor plants. Only the T36 genotype was transmitted to receptor plants from infected Y3 and Y7 plants although these acquisition plants contained more than one genotype. No T3 or VT genotypes were detected in any acquisition or receptor plants. CTV genotype mixtures in the various field sources were separated by single BrCA transmission and that the T36 genotype in T36/T30 mixtures was more easily transmitted than the T30 genotype when the acquisition plant was `Duncan' grapefruit and the receptor plant was `Mexican' lime.
The effectiveness of seven different aphid control regimes in delaying movement of decline (DI) and nondecline (NDI) inducing isolates of citrus tristeza virus (CTV) into a CTV-free sweet orange scion on sour orange rootstock block was monitored annually for 5 years beginning in 1999, 2 years after the introduction of the brown citrus aphid (BrCA) into the region. After 5 years, the mean percentages of infection with DI CTV were 19, 19, 17, 29, 23, 19, or 14 for trees treated annually with imidocloprid, every 6 months with imidocloprid, every 3 months with imidocloprid, every 2 months with imidocloprid, annually with Temik, annually with Meta Systox-R, or untreated, respectively. The mean percentages of infection (after 5 years) with only NDI isolates of CTV for the seven treatments were 40, 31, 33, 38, 38, 38, or 33. There was no significant difference (after 5 years) among either the DI or NDI CTV treatment means. The overall 5-year infection percentage for DI CTV (20%) was somewhat lower than that reported before the introduction of the BrCA (27%) (11). Aphid densities (Toxoptera citricidus and Aphis spiraecola) varied considerably from year to year. Good aphid control was achieved with all four imidocloprid treatments, but not with Temik or Meta Systox-R. The level of aphid control did not influence overall CTV infection percentages.