Emergence and Spread of Severe Strains of Citrus Tristeza Virus in the Dominican Republic

Fourteenth IOCV Conference, 2000—Citrus Tristeza Virus

S. M. Garnsey, T. R. Gottwald, M. E. Hilf, L. Matos, and J. Borbón

ABSTRACT. Following the establishment of the brown citrus aphid, Toxoptera citricida (Kirkaldy), in the Dominican Republic, rapid diffusion of mild isolates of citrus tristeza virus (CTV) into all major commercial citrus areas was observed between 1992 and 1995. Decline and stem pitting isolates were not detected initially, but since 1996, isolates that react to the monoclonal antibody MCA-13 have been detected in several areas. An isolate causing decline in trees on sour orange rootstocks was discovered near Hato Mayor. An extensive survey of the area surrounding the original focus of infection of this decline isolate was conducted using a hierarchical sampling method. Increases in the area and the number of trees infected with decline isolates were documented. An isolate that severely affects Persian limes and causes stem pitting in grapefruit was also discovered in the Monte Plata area north of Santo Domingo. Biocharacterization tests and marker profiles using an immunocapture PCR protocol with selective primers indicated that this isolate is distinct from the decline-inducing isolate at Hato Mayor. While the mild isolate of CTV widely prevalent in the Dominican Republic appears similar to mild isolates from Florida, the MCA-13-positive isolates discovered do not have the marker profile associated with the typical Florida decline isolate T36. Hierarchical sampling methods and genotype-specific probes are being used to determine rates and patterns of movement of CTV and the probable origin of the MCA-13-positive isolates in new locations.

Early events in new epidemics of tablished in 1992, surveys for CTV citrus tristeza virus (CTV), includ- were made in the major commercial ing rates and patterns of spread of production areas (1, 5). These plant- the virus, are poorly understood. ings had been established 4 to 8 yr While CTV epidemics have been de- earlier using primarily budwood scribed in different locations (18), sources imported from California the presence of CTV was often not that were virus-free. Some trees recognized until a number of trees were also propagated with budwood were showing symptoms. By this from Florida of unknown CTV sta- time, important early details, such tus. Most of the trees tested in 1992 as the probable focus of initial infec- were not infected. Those trees that tion, and spread associated with were infected apparently carried a movement of infected nursery trees, mild isolate (1). Repeated surveys were difﬁcult to determine. Methods were made in several different com- to rapidly identify infections in mercial citrus growing areas to doc- symptomless trees and to differenti- ument the rate and pattern of ate isolates were also lacking. A spread of CTV (5, 8). Rapid diffusion good opportunity to study the early of CTV into commercial sweet or- phases of CTV spread in the pres- ange plantings was observed in all ence of the brown citrus aphid areas between 1992 and 1995 while (BrCA), Toxoptera citricida, has oc- movement into grapefruit was much curred in the Dominican Republic slower (5). None of these isolates (DR). Shortly after the BrCA was es- tested reacted to the strain-selective monoclonal antibody MCA-13 (5), and no visible evidence for decline Mention of a trademark, warranty, propri- or stem pitting was found in these etary product, or vendor does not constitute a survey areas through 1995. Nearby guarantee by the U.S. Department of Agricul- Persian and Mexican lime trees ture and does not imply its approval to the exclusion of other products or vendors that were free of obvious vein clearing or may also be suitable. stem pitting symptoms.

58 Fourteenth IOCV Conference, 2000—Citrus Tristeza Virus

A change in isolate severity was Analysis of the pattern of spread noted in 1996 with the appearance of of CTV in the Dominican Republic, decline symptoms in Valencia sweet Costa Rica, Florida, and Spain in orange trees on sour orange within a the presence of two different aphid limited portion of a large commercial vectors demonstrated that the pat- planting. This site is several km tern of spread of CTV is inﬂuenced from one of our original survey loca- by the biology of the vectors (9). Ag- tions near Hato Mayor in the east- gregation of new infections near ex- ern Dominican Republic (Fig. 1) (5). isting infected trees is common Isolates of CTV from these declining when BCA is the vector, while inci- trees reacted to MCA-13, but normal dence appears more random when appearing trees were either free of the melon or cotton aphid (Aphis CTV or were infected with isolates gossypii) is the primary vector. that did not react to MCA-13. In ad- While the dynamics of short range dition, Persian lime trees with con- spread of CTV has been described spicuous vein clearing and stem (8, 10), information is still lacking pitting symptoms were discovered in on longer range spread of CTV by the Monte Plata area north of Santo aphids between areas within large Domingo. This area, which is some- plantings, and between plantings in what isolated from the major citrus separate locations. It is frequently areas we had previously surveyed, assumed that rapid spread of CTV is contains a number of small citrus inevitable in the presence of the plantings. Persian lime trees with BCA, but some observations have conspicuous leaf symptoms reacted indicated slow movement over rela- positively with MCA-13, and MCA- tively small distances between dis- 13-positive isolates were also found crete plantings (4). in nearby symptomless mandarin The original survey work in the and sweet orange trees. These obser- Dominican Republic was based on vations indicated dispersion of two individual testing of all trees in se- distinct new isolates of CTV more selected plots by ELISA (5). While this vere than those detected previously. provided detailed information on The apparent foci of these isolates CTV infections within these plots, were favorably located to study fur- surveys over larger areas were not ther local and long range movement. possible with the limited technical

Fig. 1. Relative locations (±91 M) of test plots used to monitor spread of citrus tristeza virus in the Dominican Republic as determined by global positioning satellite (GPS) data and Universal transverse mercator (UTM) conversions. LC = Citricos La Cumbre, VAG = Villa Alta Gracia (DR1), MP = Monte Plata, B = Bayaguana, SG = Sabana Grande, HM = Hato Mayor, and DR3 and 4 = Citricos Don Juan, North of Hato Mayor.

Fourteenth IOCV Conference, 2000—Citrus Tristeza Virus 59 resources available. The recent de- mentation. Universal transverse velopment of the hierarchical sam- mercator conversions were used to pling (HS) method (14) provides a determine approximate (±91 M) dis- means to monitor changes in CTV tances between plots and locations. infection within larger areas by col- Extensive testing was done in an ar- lecting relatively moderate numbers ea near Hato Mayor called Sabana of systematically arranged samples. Grande that had not been surveyed It has also been adapted for survey previously. This area is approxi- use where non random incidence of mately 14 km northwest of Hato CTV infections associated with vec- Mayor, and 7 to 9 km west of two toring by BrCA occurs (15). The HS plots established as DR3 and DR4 method is especially useful in situa- (Fig. 1) in 1992. There are approxi- tions where CTV has been newly es- mately 720 ha of citrus planted on tablished and incidence of infection rolling terrain in this location. Indi- is low (6, 11, 14). vidual blocks are irregular in size MCA-13 was used to distinguish and frequently separated for short putatively severe isolates of CTV distances by streams and/or forested from mild isolates of CTV in our ini- areas (Fig. 2). A few declining trees tial studies. However, MCA-13 does on sour orange were noted in one not discriminate isolates that induce block of trees on the southern edge decline from those that cause stem of this large area in 1996. The sam- pitting. A serological method to spe- ples collected from declining trees ciﬁcally identify isolates that cause tested positively against MCA-13 stem pitting in sweet orange (OSP) and the whole block of trees was im- has been recently described (16). mediately removed by the owner. Other approaches to discriminate Sampling of individual trees nearby CTV isolates based on molecular indicated that incidence of MCA-13- properties have also been described. positive isolates in the general area These are based on differences in was low. the conserved region of the CTV ge- The Monte Plata area lies be- nome (2, 7) as well as differences in tween survey areas previously es- the more divergent 5’ region of the tablished at Villa Altagracia and CTV genome (12, 13). Bayaguana (Fig. 1) and has not been This paper reports new informa- previously surveyed. Samples were tion on the movement of distinct de- taken at six locations in the area. cline and stem-pitting isolates of Unless otherwise noted, all sam- CTV in the Dominican Republic ob- ples were collected from commercial tained by use of hierarchical sam- plantings. Most plots were in plant- pling methods and use of sequence- ings of Valencia sweet orange, but based markers to determine genetic some samples were taken from relationships between isolates found grapefruit, mandarin, and Persian in different hosts and locations. lime trees. Plot design and analysis. Two METHODS AND MATERIALS types of plots were used. The ﬁrst was intensively sampled plots (re- Plot locations. Test plots were ferred to in the remainder of the pa- located in five general locations ar- per as IS plots) in which all trees in ranged from east to west near Hato a 20 by 20 tree area were sampled. Mayor, Bayaguana, Monte Plata, This is the same protocol described Villa Altagracia, and La Cumbre. for an earlier survey (5). The second Most sites have been previously type was plots sampled by the hier- described (5), and the relative loca- archical method (hereafter called tions are shown in Fig. 1. Plot loca- HS plots) in which 25 composite tions were mapped by global samples were taken in a 20 × 20 tree positioning satellite (GPS) instru- area. Each composite sample con-

60 Fourteenth IOCV Conference, 2000—Citrus Tristeza Virus

Fig. 2. Location of survey sites in the Sabana Grande area. Circles indicate location of plots sampled by hierarchical methods (HS plots). Solid circles indicate plots with trees that tested positively with MCA-13. The rectangular box indicates location of a 400 tree plot in which each tree was sampled individually (IS plot), and the star indicates the site where MCA-13-positive isolates were ﬁrst discovered in the Sabana Grande area. sisted of tissue taken from four adja- ture PCR analysis were collected cent trees in a two by two and processed in the same manner, conﬁguration. The sampling sites except that 1 to 5 g quantities of tis- were arranged systematically with- sue were collected from each tree. in the 400 tree area (11, 15). Calcu- Samples for biological assay were lation of estimated infection levels collected as short pieces of budwood, on an individual tree basis in HS surface sterilized, and sent to the plots from composite sample data quarantine facility at Beltsville, was as previously described. When MD, under permit (3). all composites tested positive indi- ELISA. The dried tissue samples vidual tree infection levels above were rehydrated in extraction buffer 60% were assumed (15). (PBST), pulverized with a Kleco tis- Sample collection and pro- sue pulverizer (Kinetic Laboratory cessing. Samples for ELISA from Equipt. Co. Visalia, CA 93292) and IS plots were collected as previously the extracts were tested in a double described (5), and consisted of peti- antibody indirect (DAS-I) ELISA, as oles from young leaves or pedicel previously described (5). A mixture bark from young fruit. For HS plots, of two broadly reactive monoclonal two petioles were collected from antibodies (3E10 and 11B1) was each of the four trees that comprised used as the intermediate antibody the sample and were pooled. Tissue for general detection of all isolates samples were placed in paper enve- (5). Extracts that tested positively lopes and dried over silica gel (5). were also tested using the selective Samples collected for immunocap- Mab MCA-13 (17) as the intermedi-

Fourteenth IOCV Conference, 2000—Citrus Tristeza Virus 61 ate antibody. Select samples were of MCA-13-positive isolates of 5.4%. also tested with a combination of The distribution of HS plots con- trapping and intermediate antibod- taining MCA-13-positive isolates is ies selective for CTV isolates that shown in Fig. 2. In June of 1998, we cause sweet orange stem pitting (16). sampled six HS plots in the same The percentage of individual trees area. The calculated incidence of infected in the HS plots was calcu- MCA-13-positive trees ranged from lated from the number of composite 3.1 to 20.5%. samples that tested positive for CTV An IS plot with 400 trees was es- as previously described (11, 15). tablished about 0.5 km north of the Immunocapture PCR. Sam- original decline site (Fig. 2). In ples to be evaluated for genetic June, 1997, 344 of the 400 trees grouping by PCR were extracted as tested positively for CTV infection indicated for ELISA and incubated and six tested positively against in the presence of paramagnetic MCA-13. Five of these six trees were beads (Dynal, Inc., Lake Success, removed, but by June, 1998, the NY 11042) that had been pre-coated number of infected trees was 394 with CTV-specific polyclonal anti- and the number of MCA-13-positive bodies (12, 13). Complementary DNA trees had increased to 20. The data (cDNA) was synthesized using ran- from this plot indicated that the in- dom hexamers and RNA associated fection levels estimated from the HS with virions trapped by the antibody plots in the surrounding area are re- coated beads. Analysis by PCR was alistic. An HS plot established sev- done using primer pairs designed to eral km farther north tested free of amplify a general CTV molecular MCA-13-positive isolates both years. marker (the capsid protein gene) A few declining trees were ob- and by primers designed to amplify served in 1997 in blocks that were sequence-specific markers for differ- adjacent to the original 1996 site of ent CTV molecular groups (12, 13). decline. By June 1998, large num- Isolates were differentiated by their bers of trees were in various stages respective marker profiles. of decline. Trees in more advanced stages of decline typically showed RESULTS inverse pitting (honey combing) (19) in the inner face of the bark im- Diffusion of MCA-13-positive mediately below the budunion, and isolates in Sabana Grande. From sometimes a yellowish stain was al- June through December 1997, 43 so present (Fig. 3c). No quick decline HS plots were sampled within 1-3 symptoms were observed. While km of the site where decline was most trees in this location are on first noted in 1996 (Fig. 2). In 36 of sour orange, some blocks contain the 42 plots sampled by HS, 100% of trees on Alemow rootstock. Stunted the pooled samples tested positively or unthrifty trees were also ob- against the Mab mix. Individual tree served on Alemow, and these typi- infection levels exceeding 60% were cally have extensive stem pitting in assumed for these plots (11) and cal- the trunk below the bud union. culated infection levels in the re- MCA-13-positive isolates in maining plots ranged from 12 to prior test locations. No MCA-13- 56%. In 25 of the HS plots, all pooled positive isolates have yet been re- samples tested negatively against covered in the original DR3 and MCA-13. In 16 plots the maximum DR4 IS plots that are 5 to 8 km east incidence of pooled samples that of the Sabana Grande location (Fig. tested MCA-13-positive was 8% 1). However, MCA-13-positive iso- (equivalent to individual tree inci- lates have recently been found in a dence of 2% or less). One plot had a nearby HS plot and infection levels calculated individual tree incidence were comparable to those in the Sa-

62 Fourteenth IOCV Conference, 2000—Citrus Tristeza Virus

Fig. 3. Symptoms of CTV infection in different locations in the Dominican Republic A) Vein clearing in Persian lime leaf from tree in Monte Plata area. B) Stem pitting in twig from Persian lime tree at Monte Plata. C) Decline symptoms in Valencia orange on sour orange rootstock at Sabana Grande (inset shows budunion symptoms). D) Stem pitting in limb of naturally-infected red grapefruit tree in Monte Plata area.

Fourteenth IOCV Conference, 2000—Citrus Tristeza Virus 63 bana Grande location. The DR4 plot north of Villa Altagracia, and sepa- has been sampled for MCA-13-posi- rated by some hills, remained free of tive isolates by the HS method since MCA-13-positive isolates through 1996 when the plot was essentially 1998. fully infected by mild isolates. The Stem pitting isolates in Monte DR3 plot, which is in a grapefruit Plata. Surveys for CTV among scat- block, has been sampled as an IS tered smaller commercial citrus plot continuously since 1992. The plantings in the Monte Plata region overall CTV infection level has were made for the ﬁrst time in 1997. climbed from 0.5% in 1995 to 13% in This is north of Santo Domingo and 1998, but no MCA-13-positive trees between the Bayaguana and Villa have been detected to date. Altagracia locations (Fig. 1). Pro- No MCA-13-positive samples were nounced vein clearing and stem pit- collected in the original DR2 plot at ting symptoms were seen in Persian Bayaguana through 1997. Sampling lime trees in six different plantings in 1996 and 1997 to detect MCA-13- (Fig. 3a and b) and tree vigor was re- positive isolates was by the HS duced, even though most infections method after incidence of mild iso- were apparently relatively recent. lates exceeded 90%. An additional Some unaffected trees could still be HS plot (HS3) was established found, and growers reported that about 1 km south of DR2 in 1997, young trees obtained from nurseries and it also tested negatively for in other locations grew normally MCA-13-positive isolates. In June, when ﬁrst planted and then devel- 1998, one composite sample in DR2 oped symptoms. All Persian lime and three in HS3 tested positively trees with pronounced vein clearing by MCA-13 (individual tree infec- symptoms indexed positively by tion levels of 1 and 3% respectively). MCA-13. Most samples collected Neither plot is on sour orange, but from nearby symptomless sweet or- trees on sour orange in nearby ange and mandarin trees also tested blocks have remained free of decline positively with MCA-13. Additional symptoms. surveys were made in the Monte The DR1 plot at Villa Altagracia, Plata area in 1998. Stem pitting was approximately 45 km west of Bay- found in both grafted and seedling aguana (Fig. 1), remained free of grapefruit trees near affected Per- MCA-13-positive isolates until 1997. sian lime trees (Fig. 3). Valencia Two MCA-13-positive trees were trees grafted on sour orange were discovered in the IS plot survey found with mild stem pitting in the made in June, 1997, and seven were scion and a slight honeycombing re- found in December, 1997. These in- action at the budunion, but no dis- fected trees were removed in the tinct decline symptoms were seen. spring of 1998, but seven new infec- All eight MCA-13-positive samples tions with MCA-13-positive isolates from the Monte Plata area that were found in June of 1998. Three of were tested using the serological the four HS plots established nearby test for sweet orange stem pitting in March of 1998 also contained isolates gave a positive reaction. MCA-13-positive samples and the Four samples infected with MCA- calculated MCA-13-positive virus 13-negative isolates and two infect- incidence was from 1 to 4%. An HS ed with MCA-13-positive decline iso- plot approximately 3 km west of lates did not react. DR2 was negative in 1997 for MCA- Bio-indexing results: Limited 13-positive isolates, but had a calcu- testing has been done on selected lated single tree infection level of Dominican Republic isolates that 10% in 1998. HS plots at the La had been established in the collec- Cumbre location, which is 10 km tion of exotic CTV isolates at Belts-

64 Fourteenth IOCV Conference, 2000—Citrus Tristeza Virus

ville. Two MCA-13-negative isolates sour orange and only one isolate collected from IS test plots between produced definite decline effects in 1993 and 1995 produced a mild re- grafted sweet/sour indicators. Prop- action in limes and no stem pitting agations of several other isolates in orange or grapefruit seedlings. from this area produced stem pit- Two isolates from declining trees at ting in the Madam Vinous seedlings Sabana Grande did not produce a used to maintain the isolates. strong effect on grafted combina- Immunocapture PCR results: tions of sweet orange on sour orange Tests were run with tissue collected or seedling yellows (SY) in sour or- from infected plants established in ange seedlings, but produced a mild the CTV collection at Beltsville and stem pitting in grapefruit. Neither from field-collected tissues. Sam- isolate caused stem pitting in Mad- ples from test plots infected with am Vinous sweet orange seedlings. isolates which do not react to MCA- Isolates from five Persian limes and 13 yielded reaction products similar one Valencia sweet orange at Monte to those expected for the T30 mild Plata all induced a strong reaction isolate from Florida (14). No amplifi- in Mexican lime, strong stem pitting cation products were obtained with in grapefruit, and moderate to strong T36 or T3 specific primers (see Table stem pitting in Madam Vinous seed- 1, Samples 15 to 17). Isolates from lings. None produced strong SY in declining trees on sour orange at Sa-

TABLE 1 SUMMARY OF MARKER PROFILES OF SELECTED ISOLATES OF CITRUS TRISTEZA VIRUS FROM THE DOMINICAN REPUBLIC USING SELECTIVE PRIMERS AND IMMUNO- CAPTURE PCR

Primersx

VT VT T30 T30 T36 T36 T3 MCA Sample Hostz Locationy Pol k17 Pol k17 Pol k17 K17 13

1 P. lime MP-1 +++ 0 + +++ 0 0 +++ + 2 Grapefruit MP-1 +++ 0 0 +++ 0 0 +++ + 3 Valencia MP-1 +++ + +++ +++ 0 0 ++ + 4 Mandarin 1 MP-1 +++ 0 +++ ++ 0 0 ++ + 5 Mandarin 2 MP-1 +++ 0 0 ++ 0 0 ++ + 6 P. lime MP-2 +++ + +++ +++ 0 0 +++ + 7 P. lime MP-3 +++ NT +++ NT 0 0 +++ + 8 P. lime MP-4 +++ NT 0 NT 0 0 +++ + 9 Valencia SG-1 ++ NT +++ NT 0 0 0 + 10 Valencia SG-2 +++ NT +++ NT 0 0 0 + 11 Valencia SG-3 0 0 +++ +++ 0 0 0 + 12 Valencia Sg-4 + + +++ +++ 0 0 0 + 13 Valencia HM-DA +++ 0 0 +++ 0 0 ++ + 14 Navel HM-DA +++ 0 +++ +++ 0 0 0 + 15 Valencia DR1 0 NT +++ NT 0 0 0 0 16 Valencia DR2 + NT +++ NT 0 0 0 0 17 Valencia DR4 0 NT +++ NT 0 0 0 0

zP. lime is Persian lime, mandarin 1 and mandarin 2 are two different unidentified mandarins. yMP = Monte Plata area, SG = Sabana Grande, HM-DA = nursery at Hato Mayor, DR1 = test plot at Villa Alta Gracia, DR2 = test plot at Bayaguana, and DR4 = test plot at Hato Mayor. See Fig. 1 for relative locations. xPrimers were designed from polymerase (pol) and k17 regions of the genomes of isolates T3, T30, T36, and VT as described by Hilf et al. (12, 13). Relative amounts of amplification product, as determined by electrophoresis of PCR reaction products, are indicated by +, ++, and +++. 0 indicates no product formed, and NT indicates no test was made.

Fourteenth IOCV Conference, 2000—Citrus Tristeza Virus 65 bana Grande yielded amplification parent foci of these isolates were not products with VT and or T30 prim- included in the earlier surveys, in- ers, but did not yield amplification gress of these isolates into commer- products with either T3 or T36 prim- cial plantings has apparently been a ers (Table 1, Samples 9 to 12). recent event. The levels of infection In contrast, isolates from symp- at Sabana Grande are still relative- tomatic Persian limes, and also from ly low and there was no prior histo- mandarin, grapefruit, and sweet or- ry of decline of trees on sour orange ange trees in the Monte Plata area in this area. The increase of decline yielded a reaction product to the T3 isolates (based on MCA-13 reactivi- k17 and VT pol primers (Table 1, ty) near the original focus discov- Samples 1 to 8). Some of these sam- ered in 1996, and the failure to find ples also yielded an amplification MCA-13-positive isolates several km product with T30 primers indicating to the north, suggests that spread a probable dual infection. No ampli- has occurred from a localized source. fication product was obtained for The original source of inoculum is any of the Monte Plata samples unknown and occurrence of primary with the T36 primers. Similar reac- infections from an undetermined lo- tion products were obtained from cation outside the immediate area isolates from Persian lime, grape- may also be involved. Although the fruit, two types of mandarin, and MCA-13-positive isolates found in sweet orange collected from a single Monte Plata were already wide- location in the Monte Plata area spread when our first survey was (Table 1, Samples 1 to 5). Samples made in that area in 1997, field ob- from Persian lime at four different servations suggest that much of this locations in the Monte Plata area movement has occurred recently, es- yielded similar VT and T3 primer pecially in Persian lime. amplification profiles (Table 1, Sam- Appearance of MCA-13-reactive ples 1 and 6 to 8). Samples which isolates at the Bayaguana and Villa gave an amplification product to the Altagracia locations during the last T3 k17 primer also reacted positive- year is further indication of in- ly with an antibody combination creased movement of MCA-13-posi- that reacts selectively with CTV iso- tive isolates. It is unlikely that these lates that induce stem pitting in isolates would have remained unde- sweet orange (data not presented). tected during the repeated earlier Two different T3 and VT primer surveys and no symptoms in limes product profiles were seen among in these areas were seen earlier. samples from an old Department of Differences in symptom expres- Agriculture nursery block near Hato sion, reaction in the OSP serological Mayor (Table 1, Samples 13, 14). All test, and marker profiles obtained samples yielded a product to VT pol, by IC-PCR analysis suggest that the but only two samples yielded a prod- MCA-13-positive isolates found in uct to T3 k17. the Monte Plata area are different from the isolates discovered at Sa- DISCUSSION bana Grande. The difference in re- sponse to the T3 k17 primer be- The results obtained indicate tween isolates from these two loca- that following the previously report- tions is especially clear. The exact ed widespread dissemination of be- origin of isolates with the T3 k17 nign isolates of CTV in commercial marker in the Monte Plata area re- plantings (5), at least two new and mains undetermined, but the only distinctly different isolates of CTV other location where we have found are now also spreading into com- a T3 k17 marker is in the old nurs- mercial plantings. Although the ap- ery site at Hato Mayor where some

66 Fourteenth IOCV Conference, 2000—Citrus Tristeza Virus imported mandarin budwood was immediate area, and also between reportedly propagated approximate- spatially well separated plantings ly 15 yr ago. These trees were re- provides a unique opportunity to portedly destroyed because of con- learn more about patterns and rates cerns about CTV. of spread of CTV. In contrast to the Marker profiles suggest that the situation in most areas where CTV MCA-13-positive isolates tested has moved into uninfected trees, we from five different cultivars at Mon- have the opportunity to observe the te Plata may have a similar origin. pattern of super infection by a chal- This indicates that one of these cul- lenge isolate in trees with pre-exist- tivars is the primary source of infec- ing infections of a mild isolate. tion or that all have been infected by The results presented clearly in- a similar isolate from an unknown dicate the potential usefulness of source. It is unlikely that either the CTV group-specific primers for epi- Persian lime or grapefruit plants demiological applications. We were were the primary source, since test- able to determine a probable differ- ing and examination of Persian lime ence between MCA-13-positive iso- trees in numerous locations had in- lates in several locations, the rela- dicated that most trees were free of tionship of isolates from different CTV or carried only a mild isolate. hosts at a single location and indica- Grower observations that nursery tions for mixed infections. The simi- trees shipped from other areas grow larity of primer reaction between the vigorously at first and then develop Dominican Republic mild isolates symptoms, is a further indication and the Florida mild isolate T30 fur- that the Persian lime trees were not ther supports the previous sugges- an original source. CTV infection in tion that the original source of inocu- grapefruit by any isolate of CTV is lum may have originated in Florida. relatively uncommon at other loca- Failure to find MCA-13-positive iso- tions in the Dominican Republic, lates that were amplified by the T36 and no stem pitting symptoms have primers suggests that Florida was been seen elsewhere. probably not the origin of either of The detection of MCA-13-positive the two MCA-13-positive isolates isolates in Hato Mayor, Bayaguana, discovered. Several isolates of CTV and Villa Altagracia indicates that from Japan have yielded biocharac- the initial stages of natural spread terization, serological, and marker of these isolates is now beginning to profiles similar to those observed occur. It is unlikely that these iso- with the Monte Plata isolates. lates were present earlier but went The spread of more severe iso- undetected. Tests are in progress to lates of CTV is a matter of concern determine if the MCA-13-positive for the citrus industry of the Domini- isolates discovered at Bayaguana can Republic and increased losses of and Villa Altagracia are related to trees on sour orange are anticipated. those discovered at the Sabana While many of the newer plantings Grande and Monte Plata locations. are on decline-tolerant rootstocks, While introduction of these isolates the isolates discovered in the Monte via infected nursery stock into these Plata area can cause significant in- areas is possible, trees for the com- jury to Persian lime and grapefruit mercial plantings where the plots plantings regardless of rootstock. are located are grown on site from Some adverse effects on fruit size existing budwood source trees. may also occur in sweet orange. The The presence of two distinct increased incidence of trees infected sources of challenge infection and with decline isolates at Hato Mayor the ability to monitor movement of over a short time frame, plus models these isolates into plots within an generated from previous studies on

Fourteenth IOCV Conference, 2000—Citrus Tristeza Virus 67 movement of CTV by BrCA indicate Zeng provided expert technical as- that rapid spread of these isolates is sistance for ELISA and Bio-indexing likely. Initial ﬁeld observations sug- tests. ELISA tests for sweet orange gest that the presence of a pre-exist- stem pitting were conducted by Olga ing infection by benign isolates has Nikolaeva, CREC, University of not provided useful levels of cross Florida, Lake Alfred. Consorcio Cit- protection. Occurrence of super in- ricos Don Juan, Consorcio Citricos fection by new isolates over existing Dominicanos, Plantaciones Oscar de mild isolates is also indicated by IC- la Renta, and R. A. Citricos de la PCR analysis. Cumbre generously provided access to experimental plot sites and tech- ACKNOWLEDGMENTS nical personnel to assist in collection of samples. Junta Agro- Ascites of CTV-speciﬁc mono- empresarial Dominicana provided clonals 3E10 and 11B1 were gener- facilities and technical support for ously made available by M. C. Tsai the project. Portions of the study and H. J. Su, National Taiwan Uni- were also supported by a National versity, Taipei. C. Behe, C. Bierman, Citrus Research Council Grant C. Halliday, T. Riley, and Chung Yi funded by USDA, ARS.

LITERATURE CITED

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