Evidence for Trifoliate Resistance Breaking Isolates of Citrus Tristeza Virusin New Zealand

Fifteen isolates of citrus tristeza virus (CTV) have been identified in New Zealand citrus which are infectious after transmission from trifoliate orange. These isolates were identified during routine virus screening for other citrus viruses following filtering through trifoliate orange. Bark patches from Citrus excelsa infected with transmissible CTV isolates were budded onto visually and isozyme tested, Ctv -R, true-to-type trifoliate orange seedlings. Clean buds of Mexican lime and Madam Vinous were then budded both above and below the inoculation site. Symptoms of vein clearing and stem pitting were produced on the Mexican lime and Madam Vinous indicators. Stem pitting and leaf curling symptoms were produced on some trifoliate orange rootstock shoots. Double stranded (ds) RNA extractions were then obtained from bark material of the components of the composite plant. Reverse transcription polymerase chain reaction (RT-PCR) was performed on the extracted dsRNA, using primers specific for the coat protein sequence. Ten PCR products were obtained then sequenced and compared with each other and with known coat protein sequences of the Florida T36 and T3 and the South African B7 strains. In studies for citrus viruses other than tristeza, latent citrus tristeza virus (CTV) can cause severe symptoms in the indicator plants, which can mask the symptoms of the virus of interest. It was, therefore, necessary to eliminate CTV from the field source material prior to screening for other viruses. CTV elimination was achieved using a filtering step through trifoliate orange (12). The success of this technique is based upon resistance or immunity that true-to-type trifoliate orange has to CTV (3, 12, 13). CTV is widespread in New Zealand (NZ) citrus orchards but the status of other viruses is unknown. When screening for the presence of viruses other than CTV, test buds from commercial orchard plants grown in the Kerikeri district and known to be viroid free, were budded on to either trifoliate orange Rubidoux or Flying Dragon seedlings in order to filter out CTV. Buds from basal shoots on these trifoliate orange plants were then used to inoculate the selected indicator plants. After checking the cause of unexpected symptom expression on these indicator plants, the presence of CTV was confirmed by double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) and double-stranded (ds) RNA analysis. This obviously raised the possibility that there are isolates of CTV in New Zealand that were able to replicate and be transmissible through trifoliate orange. This paper reports on the studies to investigate the movement of CTV through trifoliate orange and the partial characterization of these trifoliate orange transmissible isolates. MATERIALS AND METHODS Biological Characterization. Initial indexing was carried out using the following indicators: Madam Vinous sweet orange, Mexican lime, Duncan grapefruit, Eureka lemon. Three plants of each indicator were graft-inoculated with three bark patches. Madam Vinous sweet orange buds from plants that had previously been inoculated with CTV isolates, were budded onto Brazilian sour orange indicator seedlings to test for quick decline. Symptom expression on the indicator plants were assessed 4 to 5 mo and 9 mo after plant inoculation. 70 Fourteenth IOCV Conference, 2000—Citrus Tristeza Virus Transmission through trifoliate orange. Seed of trifoliate orange Rubidoux was obtained from commercial sources. The trifoliate orange seedlings were initially culled on visual symptoms, then isozyme tested (8), and samples of trifoliate orange sent to Citrus Research and Education Centre, Florida, for checking for presence of the CTV resistance gene using SCAR markers (1). Buds obtained from basal rootstock shoots of trifoliate orange plants that had been previously budded with commercial citrus cultivars were budded onto either Citrus excelsa or sour orange seedlings. Inoculum buds from the C. excelsa seedlings, which had tested CTV positive by DAS-ELISA (Sanofi, CTV polyclonal antibodies), were Tbudded onto tested true-to-type trifoliate orange Rubidoux seedlings 12 cm above the base of the stem. Each inoculum source was replicated on three plants. After 3 wk, wrapping tapes were removed and inoculations checked for survival; 3 wk later virus-free indicator buds of Mexican lime and Madam Vinous were budded 5 cm above and 5 cm below the inoculation sites, respectively. The indicator bud tapes were removed 3 wk later, and buds checked for survival; at this time the trifoliate orange stem was cut back above the Mexican lime bud. The buds of the two indicators and a bud of the trifoliate orange rootstock were allowed to sprout and grow (Fig. 1). After 5 mo growth, the shoots were examined for symptoms. Bark was collected separately from Madam Vinous and Mexican lime indicator shoots and from the trifoliate orange shoot for dsRNA analysis. No ELISA were carried out as dsRNA analysis was performed on the component parts of the composite plant. The peeled shoots were examined for signs of stem pitting. The trial was carried out in an insect-proof glasshouse with mean maximum temperature of 28 ° C and mean minimum temperature of 18 ° C. Extraction of CTV specific dsRNA. DsRNA analysis was carried out by phenol/chloroform/isoamyl alcohol extraction of nucleic acids from 3 to 5 g of fresh bark tissue, followed by purification by CF11 cellulose chromatography, concentrated by ethanol precipitation, and analyzed by polyacrylamide gel electrophoresis following the method of Gillings et al. (4). The dsRNA was visualized by staining with silver nitrate. cDNA cloning. Reverse transcription polymerase chain reaction (RT-PCR) was carried out on the dsRNA. 4.5 μ l of sample dsRNA was heat-treated under three drops of paraffin oil for 5 min at 94 ° C then chilled on ice. The cDNA synthesis and amplification of the CTV coat protein gene followed the procedure of Gillings et al . (4). Thermal cycling was performed in a Stratagene Robocycler 40 temperature cycler. The PCR products were separated from the overlay of paraffin oil by sequential phenol/chloroform extractions, the cDNA was precipitated by the addition of 1 / 10 vol. of 3M sodium acetate and 2.5 vol. of absolute ethanol, and overnight incubation at -20 ° C. The PCR product was collected by Fig. 1. Indexing methodology for testing CTV replication and movement in trifoliate orange Rubidoux seedlings. Inoculum source was from Citrus excelsa plants previously inoculated by trifoliate orange buds. Fourteenth IOCV Conference, 2000—Citrus Tristeza Virus 71 centrifugation and the pellet washed with 70% ethanol, dried under vacuum and resuspended in 20 μ l sterile water. An aliquot (10%) of the PCR product was checked on 1.2% agarose/TBE gels and visualized by ethidium bromide staining. Amplified cDNA products were separated on 1.5% agarose gels and eluted using a Bresaclean gel extraction kit according to the manufacturer’s protocol (Bresatec Pty Ltd. Australia). Sequence analysis. The purified coat protein gene (CPG) PCR products were sequenced directly in a commercial laboratory using the forward CTV CPG primer and the Applied Biosystems International protocol. Sequence analysis was performed using GCG PileUp and Clustal V sequence analysis programs (2, 6) on these sequences and compared to those of T3, T36 and B7. A dendrogram showing the relationship of the deduced amino acids was constructed using Tajima and Nei correction, neighbour joining and mid-point rooting (11).