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Virus Research 71 (2000) 97 – 106 www.elsevier.com/locate/virusres

Progress on strain differentiation of Citrus tristeza 6irus and its application to the epidemiology of citrus tristeza disease C.L. Niblett a,*, H. Genc a, B. Cevik a, S. Halbert b, L. Brown b,1, G. Nolasco c, B. Bonacalza c, K.L. Manjunath a, V.J. Febres a, H.R. Pappu d, R.F. Lee e b

a Plant Pathology Department, Uni6ersity of Florida, Gaines6ille, FL 32611 -0680, USA Di6ision of Plant Industry, Florida Department of Agriculture and Consumer Ser6ices, FL, USA c Uni6ersidade do Algar6e, Faro, Portugal d Uni6ersity of Georgia, Coastal Plain Experiment Station, Tifton, GA 31793, USA e Citrus Research and Education Center, Lake Alfred, FL 33823, USA

Abstract Citrus tristeza 6irus (CTV) occurs in most citrus producing regions of the world, and it is the most serious viral pathogen of citrus. With the recent establishment of the brown citrus aphid, Toxoptera citricida, its most efficient vector, on Madeira Island (Portugal) and in Florida (USA) and the countries of the Caribbean Basin, the impact of CTV is likely to increase in these regions. Since there are many strains of CTV and CTV infections frequently occur as mixtures of several strains, it is necessary to be able to distinguish the strains for regulatory purposes, disease management and epidemiology. We describe the evolution of techniques developed to detect CTV and to differentiate the individual strains, and present the results of tests using these latest methods on CTV isolates from mainland Portugal, Madeira Island and Florida. Mild and decline-inducing strains of CTV were detected in mainland Portugal and mild, decline-inducing and severe stem pitting strains on Madeira Island. In Florida we demonstrated the presence of infections that reacted with probes made against stem pitting strains not previously detected there. It is concluded that CTV presents a significant threat to citrus production in mainland Portugal, on Madeira Island and in the neighbouring countries of the Mediterranean Basin, as well as in Florida, elsewhere in the USA and throughout the Caribbean Basin, especially following the widespread establishment of T. citricida throughout the region. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Citrus; Citrus tristeza 6irus; Virus strains; Aphid vector; Toxoptera citricida

1. Introduction * Corresponding author. Tel.: +1-352-3923814; fax: +1352-3923633. E-mail address: [email protected] (C.L. Niblett). 1 Present address: Center for Plant Health Science and Technology, USDA/APHIS/PPQ, Raleigh, NC 27607, USA.

Citrus tristeza, caused by Citrus tristeza 6irus (CTV; Family: Clostero6iridae; Genus: Clostero6irus), is the most economically important virus disease of citrus in the world (Bar-Joseph et al., 1989; Rocha-Penã et al., 1995). The virus has

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C.L. Niblett et al. / Virus Research 71 (2000) 97–106

been well-characterized (Bar-Joseph et al., 1979b; Bar-Joseph and Lee, 1990), being a flexuous virion c. 12× 2000 nm in dimensions (Fig. 1) and containing a monopartite positive-sense RNA genome of c. 20 kb, the largest known for any plant virus. The genomes of several CTV strains have been sequenced (Karasev et al., 1995; Mawassi et al., 1996; Vives et al., 1999), the genome organization determined (Fig. 2) and a full-length clone has been developed which can infect protoplasts of Nicotiana benthamiana (Satyanarayana et al., 1999). CTV is transmitted in nature by several species of aphids (Raccah et al., 1976; Bar-Joseph and Lee, 1990), of which Toxoptera citricida, the brown citrus aphid (Fig. 3), is the most efficient (Yokomi et al., 1994). CTV generally has been disseminated widely between and within countries and continents by man, in illegally imported infected bud-wood or plants. This has resulted in devastating epidemics, causing the debilitation and death of many millions of citrus trees (Rocha-Penã et al., 1995; Cambra, et al. 2000). Recent information on the transmission and spread of CTV by T. citricida is presented in Gottwald et al. (1998, 1999). Strains of CTV have been identified primarily on the basis of their biological activities (the symptoms they cause) on a defined group of Citrus spp. indicator plants (Garnsey et al., 1987). The major

groups of strains are: mild strains, which cause barely detectable clearing or flecking of the leaf veins of Mexican Lime (Fig. 4); decline-inducing strains, which cause the decline and death of citrus scions grafted on sour orange rootstocks (Figs. 5 and 6); stem pitting strains, which cause mild to severe pitting of the stems and branches of grapefruit and orange (Fig. 7), resulting in decreased tree vigour and significant reductions in fruit size and number (Fig. 8). Moreover, some strains cause yellowing of seedlings, but their significance in the field is uncertain (Garnsey et al., 1987; Rocha-Penã et al., 1995). There is also biological (Moreno et al., 1993) and biochemical (Genc, 1998; AlbiachMarti et al., 2000) evidence that individual citrus trees may be infected with several different strains of CTV. Currently, the only method available to differentiate CTV strains is to inoculate the appropriate indicator plants and observe the symptoms. The major disadvantages of this biological indexing are the time required to complete the test (usually 12–15 months) and the associated expenses (greenhouse space, technical personnel, plant and pest management, etc.). However, it is necessary

Fig. 1. An electron micrograph of the virion of Citrus tristeza 6irus (CTV).


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Fig. 2. Organization of the CTV genome. The capsid protein gene is designated CP. See Karasev et al. (1995) for details.

to be able to differentiate strains of CTV quickly to facilitate data-based quarantine and eradication policies, to prevent movement of severe strains into new areas and for meaningful epidemiology. With the recent knowledge of the nucleotide sequence of the CTV genome, additional progress has been made on differentiation of CTV strains. This progress is reviewed here.

2. Evolution of detection techniques and results CTV was purified and a polyclonal antibody having good specificity was prepared and used in SDS-immunodiffusion tests to detect CTV (Garnsey et al., 1979). The same investigators then used ELISA (Bar-Joseph et al., 1979a) to improve the efficiency and throughput of samples. Several investigators (Lee, 1984; Dodds et al., 1987; Moreno et al., 1990) used electrophoresis patterns of the double-stranded replicative RNA forms of CTV to detect and characterize strains of CTV. However, some of their conclusions have been made uncertain by recent research on the generation of defective RNAs of CTV (Ayllon et al., 1999). The development of monoclonal antibodies (MCAs) (Vela et al., 1986; Permar et al., 1990) was a major breakthrough in CTV diagnosis. MCA 13 is especially useful because it reacts primarily with most severe strains. This positive reaction has been associated with tree decline in Florida, the Orient and Latin America, but not in Spain or California. Hence, for the first time it was possible to test many samples and to conclude that those reacting with MCA13 may contain a severe strain of CTV. However, it was not possible using MCA 13 to conclude whether a reactive sample contained a stem pitting or a

decline strain or a mixture of these severe strains. Likewise, with MCA 13 it was not possible to conclude if the non-reactive samples were uninfected or infected with a mild strain or a nonMCA 13-reactive severe strain. Thus parallel tests were needed with polyclonal or monoclonal antibodies. Using the capsid protein (CP) gene and polymerase chain reaction (PCR), Gillings et al. (1993) developed a restriction fragment length polymorphism (RFLP) assay to differentiate CTV strains. They found that Hin f1 restriction digests of PCR products of the CP gene produced seven different characteristic patterns which were associated with specific biological activities. This permitted a categorization of CTV strains without having to clone and sequence the CP gene. Again using PCR, Mawassi et al. (1993) and Pappu et al. (1993b) determined the nucleotide sequences of the CP gene of diverse strains of CTV. They found that the sequences were conserved c. 90% and that there was a relationship

Fig. 3. Photographs of the brown citrus aphid, Toxoptera citricida, the most efficient vector of CTV.

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Fig. 6. A close-up of a citrus tree expressing decline symptoms. Fig. 4. Symptoms of vein clearing caused by a mild strain of CTV in Mexican lime.

between the sequences and the symptoms caused by the CTV strains (Fig. 9). Pappu et al. (1993a) demonstrated that the critical amino acid in the epitope for MCA-13 was a phenylalanine (F) residue at amino acid position 124 of the CP of MCA-13-reactive severe strains, and that it was replaced by tyrosine (Y) at this position in the MCA-13 non-reactive mild strains (Fig. 10). Sitedirected mutagenesis confirmed that the F to Y change and the differential reactivity of MCA-13 was determined by a single base change at position 371, with the resulting codons TTT and TAT

Fig. 5. Citrus trees on sour orange rootstock in south Florida with some trees expressing decline symptoms due to decline-inducing strains of CTV.

for MCA-13-reactive and non-reactive strains, respectively. The discovery of the single base change controlling the MCA-13 epitope enabled the development of bi-directional PCR for differentiating CTV strains. Two oligonucleotide primers were designed to initiate DNA synthesis at base 371 (Fig. 11) and amplify the sequence to opposite ends of the gene. To detect mild strains, the 3% base of the oligonucleotide was T and it amplified the upstream sequences to yield a DNA fragment of c. 400bp (Fig. 12). To detect severe strains, the

Fig. 7. Pitting symptoms on branches of Washington navel orange infected with a severe stem pitting strain of CTV in Colombia.


Fig. 8. Reduction in fruit size of grapefruit caused by a severe stem pitting strain of CTV in Colombia.

3% base of the oligonucleotide was A and it amplified downstream sequences to yield a DNA fragment of c. 300bp. Both the 300 and 400 bp fragments are produced if a plant is infected with both mild and severe strains. Thus for the first time it was possible to detect both severe (MCA 13-reactive) and mild strains in the same plant (Cevik et al., 1996). Cloning and sequencing individual CP genes to differentiate CTV strains provided much informa-

Fig. 9. A dendrogram showing the relationships of the nucleotide sequences of the CP genes of decline-inducing (T3, 36 and 66), stem pitting (B18,12 and 53) and mild strains (T4, 30, 55 and 26) of CTV.

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Fig. 10. Characterization of the epitope of the monoclonal antibody MCA13. Polyclonal antibodies react as shown with both mild (B35) and severe (B227) strains of CTV, whereas MCA13 reacts only with the severe B27. The enlargement of the capsid protein (CP) region from amino acid 114 – 133 shows tyrosine (Y) present at position 124 for B35 and phenylalanine (F) present at position 124 for B227. The A or T in position 371 of the CP gene determines whether the amino acid will be Y or F, respectively, and MCA13 reacts only when F is present.

tion, but it was laborious and expensive. Rubio et al. (1996) utilized single-strand conformation polymorphism (SSCP) of the CP gene to distinguish strains of CTV and Febres (1995) further demonstrated its usefulness with the p18, p20 and p27 genes of CTV. SSCP was developed to detect single base mutations in genes. Its principle and application are simple and it is relatively inexpensive. PCR products are denatured and then electrophoresed on non-denaturing gels. When the denatured, single DNA strands leave the denaturing medium and enter the non-denaturing gel, they form intra-molecular hydrogen bonds (rather than annealing to their complementary strands) and separate based on their relative conformations. The (usually) two bands are visualized by silver staining (Fig. 13). Thus the characteristics and relationships among CTV strains can be ob-

Fig. 11. Cartoon demonstrating the theory of bi-directional PCR. Oligonucleotide CN219 amplifies sequences upstream of position 371 for mild strains of CTV and yields a 400bp product, whereas oligonucleotide CN218 amplifies sequences downstream of position 371 for severe strains and yields a 300bp product.

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Fig. 12. Photograph showing the results of bi-directional PCR. Lane 1 contains DNA standards, and lanes 2, 3, 4 and 5 contain the PCR-amplified products from citrus inoculated with a mild strain (400bp), a severe strain (300bp), no virus (no product), and a mixture of a mild and a severe strain (300 and 400bp products).

tained by simple, rapid and inexpensive gel analyses. Infections by multiple strains produce a complex gel pattern (Fig. 13). More expensive analyses can then be applied to those samples of specific interest. Knowledge about the MCA-13 epitope also provided the basis for a strategy to develop probes for specific groups of CTV strains. To confirm that the desired mutations had been introduced at the epitope, it was necessary to distin-

Fig. 13. A typical pattern of the p20 gene of CTV when analyzed by single-strand conformation polymorphism. Samples A, C, G, H and I are from trees infected with a single strain of CTV, and G and H contain the same strain. Samples B, D, E, and F are from trees infected with two or more strains, and D and F contain the same strains.

guish between bacterial colonies which carried the wild type and the mutant CP gene. This was accomplished by radio-labelling the mutagenic oligonucleotide used to induce the single T/A mutation and hybridizing it to the DNA from the individual transformed colonies. The single nucleotide difference between the wild type and mutant genes was detected reliably by nucleic acid hybridization. Following these results, Cevik et al. (1996) analyzed in detail the CP gene sequences for many biologically and geographically diverse strains of CTV. They grouped the strains by known biological activity and found minor but consistent differences in the nucleotide sequences for several groups of CTV strains. Oligonucleotides (15–20 bases) containing these one or two nucleotide differences were synthesized and labelled with biotin. The CP gene was then amplified from CTV-infected samples of known or unknown biological activity. The DNA was blotted on nylon membranes, hybridized to the various probes and the probes detected by chemiluminescence. Probe 0 contains a sequence conserved in the CP gene of all known strains of CTV and hence is a universal probe and hybridizes with all known strains of CTV. Probe I hybridizes with decline-inducing strains; Probes II, III, IV and V hybridize with different groups of stem pitting strains; Probe VI hybridizes with mild strains typical of those found in Florida; Probe VII hybridizes with mild strains from the Orient; and Probe VIII hybridizes with all mild strains. Fig. 14 demonstrates the specificity of the probes in that each reacts only with the two strains from the group to which it was made. The background reactions with probes VII and VIII can be eliminated by increasing the wash stringency. The only exception to this specificity is the reaction of sample B1 with Probes II and V. This was clarified by sequencing several clones of the B1 CP gene and finding two sequences. One contained the Probe II-reactive sequence and one contained the Probe V-reactive sequence; Probe V predominated. These findings provided confidence that the probes could be used to determine whether individual citrus plants were infected with more than one strain of CTV. For this purpose CTV-infected


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Fig. 14. A dot blot demonstrating the specificity of the CTV probes. Following reverse transcription and PCR, the 700bp CP gene products were separated on a gel and blotted to a nylon membrane. The membrane was then probed successively with Probes 0 to VIII.

samples from mainland Portugal, Madeira Island and Florida were tested. G. Nolasco (Nolasco et al., 1997 and unpublished information) had performed a survey for CTV in 1995 shortly after the establishment of T. citricida on Madeira Island and, he detected severe (MCA13-reactive) strains in isolated areas. In 1997 he found that the MCA13-reactive strains were widely distributed on Madeira Island. Similar surveys for CTV in mainland Portugal indicated that the MCA13-reactive strains remained in isolated areas in the absence of T. citricida. Some of the MCA13-positive samples from Madeira Island and mainland Portugal were tested and the results are shown in the left half of each of the dot blots in Fig. 15. The CP gene was amplified from most of the samples, as shown by the reaction with Probe 0. The samples hybridized with Probes I, III, IV, V and VI which indicates the presence of decline-inducing strains, three different types of stem pitting strains and mild strains of CTV. Close scrutiny reveals that sample A2 reacted with Probes I, III and V; samples B2, C2 and D2 reacted with Probes I and III, and samples E3, F3 and G3 reacted with Probes III and V, indicating that some samples were infected with several strains of CTV.

A similar situation occurred in Florida where T. citricida became established in southern areas in 1995 (Halbert and Brown, 1996). An extensive survey for CTV had been performed there just before it arrived (Brown and Davidson, 1997) and repeat spot surveys were performed in 1997 and 1998. CTV was then found to be much more widely distributed, and it was detected in many previously uninfected plants. Some of the plants also contained strains which hybridized with Probes III, IV and V, indicative of stem pitting strains not previously detected in Florida (Fig. 15, left half of the dot blots and other data not shown). Samples of CTV-infected plants from Florida and several locations throughout the world were analyzed (Genc, 1998). Often up to 25% of the samples were infected with more than one strain of CTV. This caused concern that with low level probe reactions or higher background reactions, such as the Probe 0 reaction with sample H4 in Fig. 15, biologically important strains might not be detected if they occurred in low concentration. To investigate this, the specific probes were hybridized directly with the CP gene PCR products of infected plants as usual (referred to as direct PCR hybridization, or DPH). With some samples, strong reactions were observed with several differ-

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ential probes (Table 1, sample B57). With other samples there were only strong reactions with a single differential probe (sample B23), or one strong reaction and several weak or barely detectable reactions (not shown). The PCR products were then cloned from both types of samples. The DNA isolated from individual bacterial colonies was then used as PCR templates and the PCR products were tested with the specific probes (referred to as colony PCR hybridization or CPH). We also tested direct hybridization of the probes to disrupted colonies, but CPH was superior. The CPH approach revealed that samples such as B57 which were seemingly mixtures of strains were indeed mixtures (Table 1); other samples which seemingly contained a single strain (B52, T36, DP9 and DP15) contained only that strain; but some samples which seemingly contained a single strain (B23, DP11 and DP16) actually contained one or more additional strains not detectable by DPH. This would not present a serious threat if the undetected strain was a mild one, as in samples B23 and DP16, but it would be extremely

serious if the contaminating strain induces a decline, as in samples B23 and DP11, or stem pitting (data not shown).

3. Summary and conclusions Considerable progress has been made in recent years in the development of techniques for the detection and differentiation of strains of CTV. It is now possible to detect CTV reliably, and to identify and differentiate various strains of CTV, even if several strains infect a single tree. Techniques are still needed which are sufficiently sensitive to detect low concentrations of diverse strains and also enable large scale surveys and testing for budwood certification programmes. Current techniques have demonstrated the complexity of citrus tristeza disease and suggest that it will soon become much more destructive than formerly in several parts of the world. Hopefully, as tristeza becomes more important, more resources will be devoted to developing new techniques and these

Fig. 15. A dot blot of CP genes amplified from CTV-infected samples from mainland Portugal and Madeira Island (left halves of blots) and from Florida (right halves of blots) and then probed with the CTV strain-specific probes.


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Table 1 Population diversity of Citrus tristeza 6irus (CTV) isolates detected by hybridization of PCR products directly from RT-PCR of infected tissue (DPH) and from clones derived from the RT-PCR products (CPH) with the CTV strain-specific probes Isolatesa

B23 B52 B57 T36 DP9 DP11 DP15 DP 16

DPHb

0, 0, 0, 0, 0, 0, 0, 0,

III VI V, VI I III III III III

CPHc Probe 0

Probe I

Probe II

Probe III

Probe IV

Probe V

21C 24 14 14 19 19 31 32

3 0 0 14 0 1 0 0

0 0 0 0 0 0 0 0

10 0 0 0 19 18 31 30

0 0 0 0 0 0 0 0

0 0 13 0 0 0 0 0

Probe VI 8 24 1 0 0 0 0 2

a Samples having the B code are from the Collection of Exotic Citrus Pathogens maintained under quarantine in Beltsville, Maryland; T36 is a well characterized Florida decline-inducing strain and those with DP codes are field-collected samples from Florida. b Direct hybridization of RT-PCR products from infected tissue with strain-specific probes (DPH). Probes that hybridized with the corresponding isolates are shown. c Hybridization of the strain-specific probes with clones derived from RT-PCR products from infected tissue (CPH). The number of clones that hybridized with each probe is shown (note that the probe 0 is universal and hybridizes with all strains).

could then be applied to other important virus diseases.

Acknowledgements This research was supported in Florida by grants from the Florida Citrus Production Research Advisory Committee, a T-STAR-CBAG Special Grant in Tropical Agriculture no. 9434135-0652 and USDA Specific Cooperative Agreement 58-6617-0-102. It was supported in Portugal by the grant Praxis 2159-95, ‘Developing the bases for protection against citrus tristeza virus and its vector, Toxoptera citricida’. This paper is Florida Agricultural Experiment Station Journal Series No. R-07345.

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