HORTSCIENCE 40(3):694–696. 2005. and/or T30-CTV-infected source tree. They were maintained in an -free greenhouse and served as the acquisition hosts for the Separation of 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 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, citricida and sour orange seedlings, and quick decline of sweet orange trees on sour orange rootstock. Abstract. One hundred single brown citrus aphid (BCA) ( 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- (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 -free greenhouse, (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. Since source trees by bark chip inoculation. The ISIA tors (one from each source plant), that were BrCA alatae can travel several meters from cannot distinguish between a tree infected with reactive with MAb 17G11, were also reactive one plant to another, the receptor plants were only decline-inducing CTV and a tree infected with MAb MCA13. This suggests that GM- caged individually. In addition, donor plants, with both decline-inducing and non-decline- 1, GM-2, OM-1, and OM-2 also contained a receptor plants on which aphids were feeding, inducing CTV. mixed infection of T36-CTV and T30-CTV, and receptor plants after treatment At least one successful aphid transmission and the T36-CTV was not detectable in the were housed separately. Control, uninoculated, (out of 100 attempts) occurred from each source plant by ISIA. In spite of this, a single uninfected plants were housed with the donor source tree except the uninfected controls, BrCA was able to acquire the T36-CTV and and receptor plants to signal contamination. from which no transmissions occurred (Table transmit it to an indicator plant. Virus analysis. Both acquisition and recep- 1). Transmission from GS-1 (grapefruit inocu- tor plants were assayed for CTV infection by lated with T36-CTV) resulted in one tree that Discussion ISIA using monoclonal antibodies (MAb) reacted with both MAbs 17G11 and MCA13 17G11 and MCA13. MAb 17G11 reacts with and one tree that reacted only with 17G11. Previous results have shown that decline- most Florida isolates of CTV including both This second tree was only infected with inducing isolates of CTV can occur as mixtures T30 and T36. MAb MCA13 reacts with most T30-CTV (as detectable by ISIA), indicating in trees also infected with non-decline-inducing Florida decline-inducing isolates of CTV that this T36-CTV source tree was infected isolates of the virus. In some cases, the de- (including T36), but not with nondecline-in- with both MCA13 reactive (T36-CTV) and cline-inducing isolate is not immunologically ducing isolates of CTV (including T30). The MCA13-nonreactive (T30-CTV) virus, and detectable, but can be recovered by bark chip ISIA procedure has been previously described the aphid vector could selectively transmit inoculation to indicator plants (Powell et al., (Lin et al., 2000). the T30-CTV from the mixture. Aphids did 2002). We have now shown that BrCA can Each receptor plant was sampled three not separate any non-MCA13 reactive CTV also acquire decline-inducing CTV from citrus times, and each sample was assayed inde- from the remaining three source trees (GS-2, plants in which it was not immunologically pendently. A plant was considered positive if OS-1, or OS-2) believed to be inoculated only detectable. An explanation for this result is any of the three samples was positive. Each with T36-CTV. not clear. Perhaps the decline-inducing CTV receptor plant was assayed again 6 weeks after Aphids were able to selectively transmit is limited to a low number of cells in which it occurs at a transmissible concentration. Aphids Table 1. Transmission of decline-inducing and nondecline-inducing isolates Citrus tristeza virus (CTV) may be able to acquire virus from these cells, from grapefruit and sweet orange seedlings with single brown citrus aphids. but they may not have been included in an in situ Immunoassy of ISIA sample. acquisition Transmissionx The transmission efficiency of CTV by treey No. positive/No. inoculated BrCA from either grapefruit or sweet orange Source plantz 17G11 MCA13 17G11 MCA13 was low compared to some other reports GS-1 + + 2/100 1/100 (Broadbent et al., 1996; Costa and Grant, 1951; GS-2 + + 1/100 1/100 Lastra et al., 1992; Nickel et al., 1984; Yokomi OS-1 + + 3/100 3/100 et al., 1994). The reasons for this are unclear, OS-2 + + 3/100 3/100 but the same low transmission efficiency has GFS-1 + + 3/100 2/100 been observed by other Florida virologists (R. GFS-2 + + 3/100 1/100 Brlansky and R. Lee, personal communica- OFS-1 + + 5/100 4/100 OFS-2 + + 3/100 1/100 tions). The conclusion that aphids can transmit GM-1 + – 2/100 1/100 decline-inducing CTV from trees in which the GM-2 + – 1/100 1/100 virus is immunologically undetectable has OM-1 + – 3/100 1/100 significance for the citrus industry and CTV OM-2 + – 4/100 1/100 control. Currently most certification programs GU-1 – – 0/100 0/100 rely heavily on immunological detection to GU-2 – – 0/100 0/100 insure budwood sources are free of disease- OU-1 – – 0/100 0/100 causing CTV. In many cases nondecline-induc- OU-2 – – 0/100 0/100 ing isolates of CTV are ignored or deliberately zGS = grapefruit with decline-inducing CTV; OS = orange with decline-inducing CTV; GFS = grapefruit, propagated for cross-protection purposes. bud-inoculated with both decline-inducing and nondecline-inducing CTV; OFS = orange, bud-inoculated The observation that decline-inducing CTV with decline-inducing and nondecline-inducing CTV; GM = grapefruit with nondecline-inducing CTV; OM can be recovered from trees, that would have = orange with non-decline-inducing CTV; GU = CTV negative grapefruit; OU = CTV negative orange. See Materials and Methods for details of source plants. passed these immunological tests, by aphids y17G11 + source plant was infected with CTV. 17G11 = source plant was not infected with CTV as detect- or grafting (Powell et al., 2002), suggests that able by ISIA. MCA13 + source plant was infected with a decline-inducing isolate of CTV. MCA13 = source procedures for certifying budwood sources plant was not infected with a decline-inducing isolate of CTV as detectable by ISIA. as free of disease-causing CTV need to be xNumber of successful aphid transmissions versus the number of attempts as detected by ISIA using MAbs, reevaluated. 17G11 and MCA13.

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