HORTSCIENCE 40(3):694–696. 2005. and/or T30-CTV-infected source tree. They were maintained in an aphid-free greenhouse and served as the acquisition hosts for the Separation of Citrus Tristeza Virus single aphid transmission experiments. GS-1 and GS-2 were replicate ‘Duncan’ grapefruit Isolates in Mixed Infections through seedlings infected with the T36 isolate of CTV. The original source of T36-CTV was a Transfer by Single Brown Citrus Aphids container-grown C. excelsa Wester on rough lemon (C. jambhiri Lush) rootstock tree. The Charles A. Powell and Youjian Lin CTV isolate in this tree (termed T36) causes Indian River Research and Education Center, University of Florida, Fort severe vein clearing, stunting, and stem-pitting Pierce, FL 34945-3138 of ‘Mexican’ lime, mild seedling yellows symp- toms on ‘Eureka’ lemon [C. limon (L.) Burm f.] Additional index words. certification, Toxoptera citricida and sour orange seedlings, and quick decline of sweet orange trees on sour orange rootstock. Abstract. One hundred single brown citrus aphid (BCA) (Toxoptera citricida Kirkaldy) OS-1 and OS-2 were replicate ‘Valencia’ transmission attempts were made from each of 16 different citrus trees [8 grapefruit (Citrus sweet orange seedlings infected with CTV paradisi Macf.) and 8 sweet orange (C. sinensis (L.) Osbeck)] previously inoculated with isolate T36. GM-1 and GM-2 were replicate decline-inducing (T36-CTV), non-decline-inducing (T30-CTV), a mixture of the two Citrus ‘Duncan’ grapefruit seedlings infected with tristeza virus isolate types, or no CTV. Successful CTV transmission occurred in 1.5% of the T30 isolate of CTV. The original source attempts from grapefruit trees that had been bark-chip-inoculated with T36-CTV, 3% of T30 was a container grown C. excelsa on of attempts from orange trees inoculated with T36-CTV, 3% of attempts from grapefruit rough lemon rootstock tree. The CTV isolate trees inoculated with both T36- and T30-CTV, 4% of attempts from orange trees inocu- in this tree causes mild symptoms (vein-clear- lated with both T36- and T30-CTV, 1.5% of attempts from grapefruit trees inoculated ing) on ‘Mexican’ lime, no seedling yellows with T30-CTV, and 3.5% of attempts from orange trees inoculated with T30-CTV. Single on ‘Eureka’ lemon or sour orange seedlings, BCA were able to recover T30-like-CTV from trees believed to be inoculated only with and no symptoms on sweet orange trees on T36-CTV, and T36-like-CTV from trees believed to be inoculated only with T30-CTV, sour orange rootstock. OM-1 and OM-2 were suggesting that these inoculum sources were also mixtures of T36-CTV and T30-CTV. replicate ‘Valencia’ sweet orange seedlings The T36-CTV was not immunologically detectable in some of the trees from which it was infected with CTV isolate T30. transmitted indicating that single BrCA can recover T36-CTV from a T36-CTV/T30-CTV GFS-1 and GFS-2 were ‘Duncan’ grapefruit mixture in which the T36-CTV is an undetectable, minority component. seedlings that were bark chip inoculated from two field grapefruit trees on sour orange root- Citrus tristeza virus (CTV) causes economi- transmission rates have varied considerably stock that had been first inoculated with T30 cally important diseases of citrus world-wide ranging from 0% to 66.6% for single aphid and then challenge inoculated with T36, 12 (Bar-Joseph et al., 1981; Garnsey and Lee, transmission and 0% to 91.6% for multiple months later. OFS-1 and OFS-2 were two field 1988). The virus has several different isolates (3-100) BrCA transmission (Broadbent et ‘Valencia’ sweet orange trees on sour orange that produce significantly different symptoms. al., 1996; Costa and Grant, 1951; Lastra et rootstock that had been first inoculated with Some CTV isolates cause stem-pitting and al., 1992; Nickel et al., 1984; Yokomi et al., T30 and then challenge inoculated with T36, 12 reduced fruit size of grapefruit (Citrus paradisi 1994). Single BrCA transmission of Florida months later. GU-1 and -2 and OU-1 and -2 were Macf.) and/or sweet orange (Citrus sinensis CTV isolates has been low (0% to 5%) (Lin et CTV-free ‘Duncan’ grapefruit and ‘Valencia’ (L.) Osbeck) scions regardless of the rootstock. al., 2002; Powell et al., 1999). However, CTV sweet orange seedlings, respectively. Other isolates induce decline of many citrus and BrCA remain a concern to Florida growers Receptor indicator plants. ‘Mexican’ lime varieties on sour orange (C. aurantium L.) or because of potential hastening of decline of the seedlings were used as the receptor indicator related rootstocks. The severity and chronol- 48% of Florida grapefruit still growing on sour plants. Seeds of ‘Mexican’ lime were sown ogy of decline is influenced by many factors orange rootstock and the potential introduction in trays filled with custom soil mix (Conrad including scion variety, tree age at infection, of exotic stem-pitting CTV isolates into com- Fafard, Inc.), and grown under greenhouse and water availability. Some CTV isolates mercial citrus groves or nurseries. conditions for 6 to 12 months. The seedlings cause no detectable field symptoms on com- CTV isolates frequently occur as mixtures were then transplanted into plastic pots contain- mercial citrus (Garnsey et al., 1987). Only the (Broadbent et al., 1996; Cevik et al., 1997; ing the same soil mix, with one to three plants sour orange decline-inducing and symptomless Moreno et al., 1991; Rosner et al., 1986; per pot. Seedlings with new flush were used isolates are known to be prevalent in com- Powell et al., 2002) in the same tree, and the as receptor indicator plants for single BrCA mercial citrus groves in Florida. isolates sometimes can be separated by graft- transmission of CTV. After inoculation with CTV is vectored in the field by several aphid transmission using bark chips from the trees CTV by single BrCA, the receptor indicator species including Aphis gossypii (Glover), containing more than one CTV isolate (Powell plants were grown in an insect-free greenhouse, Aphis spiraecola (Patch), and Toxoptera cit- et al., 2002). In addition, decline-inducing CTV and tested by in situ immunoassay (ISIA) (Lin ricida (Kirkaldy) (Bar-Joseph et al., 1981), can be recovered by bark chip inoculation from et al., 2000) with CTV monoclonal antibodies commonly called the brown citrus aphid trees in which the decline-inducing isolate is 17G11 and MCA13. (BrCA). BrCA is considered to be the most not detectable using standard immunological Brown citrus aphids. BrCA colonies were efficient vector of CTV (Costa and Grant, tests for CTV diagnosis (Powell et al., 2002). collected, maintained, and characterized as 1951). This aphid was introduced into Florida In this report we show that single BrCA can previously described (Lin et al., 2002). Virus- in 1995 and subsequently spread throughout the acquire decline-inducing CTV from grapefruit free colonies were established by feeding on citrus growing regions of Florida with initial or orange trees in which the virus isolate was uninfected ‘Duncan’ grapefruit or ‘Valencia’ high population infestation. Since these initial not detected immunologically, and transmit it sweet orange seedlings as described (Lin et infestations, the aphid has not flourished, and to ‘Mexican’ lime [C. aurantifolia (Christm.) al., 2002). its reoccurrence has been sporadic. Swingle] indicators. Single aphid transmission. ‘Duncan’ There are several reports of transmission of grapefruit and ‘Valencia’ sweet orange seed- CTV by single and multiple BrCA . Reported Material and Methods ling source plants containing the T30, T36, or T30 plus T36 isolates of CTV or uninfected Received for publication 24 Mar. 2003. Accepted Virus isolates and source plants. Sixteen seedlings were pruned several weeks before for publication 20 Oct. 2003. Florida Agricultural container-grown seedlings were inoculated aphid acquisition feeding to stimulate flushes Experiment Station journal series R-09322. with bark chips from an uninfected, T36-, of new growth. When the leaves on the new 694 HORTSCIENCE VOL. 40(3) JUNE 2005 flushes had completely expanded, groups of the initial assay. On rare occasions when the non-MCA13 reactive (nondecline-inducing) 200 to 400 virus-free aphids were transferred repeat assay failed to confirm the initial assay, CTV from each of the seedlings (GFS-and onto the new flushes for a 24-h acquisition ac- a third assay was performed. OFS) inoculated with both T30-CTV and cess feeding. One hundred single aphids were T36-CTV resulting in ‘Mexican’ lime receptor then transferred from each source plant, using Results plants that assayed positive for T30-CTV, but a small paintbrush, to a new flush on one hun- not T36-CTV. dred healthy ‘Mexican’ lime receptor seedlings The source trees for aphid transmission Four source trees, two orange and two (one aphid per receptor seedling). Only alatae could be placed in three virus infection catego- grapefruit, assayed negative for T36-CTV, but aphids were used. The aphids were allowed ries based on ISIA. These included infection positive for T30-CTV by ISIA (OM-1, OM- 24 h on the receptor seedlings for inoculation with decline-inducing CTV (reacted with MAb 2, GM-1, GM-2). Single-aphid transmission access feeding. After the inoculation access MCA13), infection with non-decline-induc- from these source plants resulted in several feeding, pesticides (Marathon) or 1 soap : 1 ing CTV (reacted with MAb 17G11, but not ‘Mexican’ lime seedlings that were infected oil : 30 water mixed were sprayed to kill the MCA13), and uninfected (reacted with neither with T30-CTV (reacted with MAb 17G11, but aphids. The receptor ‘Mexican’ lime seedlings MAb) (Table 1). ISIA results are consistent not MCA13), as expected. were kept under insect-free green house condi- with the virus originally introduced into the However, four of the ‘Mexican’ lime indica- tions (23 to 30 °C) for 4 to 6 months.