Evaluation of Changes Which Occurred in a Mild Protective Citrus Tristeza Virus Isolate in Pera Sweet Orange Trees by Using RFLP and SSCP Analyses of the Coat Protein Gene

The cross protection program to control citrus tristeza virus (CTV) in Pera sweet orange through the use of preimmunization with a mild isolate of CTV has been working satisfactorily for 30 yr with approximately 80 million preimmunized trees in the State of Sao Paulo (SP.), Brazil. Recently, however, 3to 4-yr-old plants (daughter plants) grown from budwood selected from 20-yr-old vigorous preimmunized Pera sweet orange trees are showing severe CTV symptoms. The protective CTV isolate present in the mother trees was compared with the isolates occurring in the daughter plants that were showing severe symptoms by restriction fragment length polymorphism (RFLP) and single-strand conformation polymorphism (SSCP) analyses of the CTV coat protein gene (CPG). RFLP analyses detected no polymorphism between the CTV isolates of the mother trees and the original isolate maintained in stock mother plants in Cordeirópolis County, Sao Paulo, Brazil. However, the CTV in the daughter plants were quite different from the CTV isolates present in the mother plants. SSCP analyses revealed complex and different patterns in almost all the CTV isolates, including both groups of mother plants. The ramification of these results are discussed in relation to symptoms in the daughter plants and with cross protection. The control of severe citrus tristeza virus (CTV) isolates in Pera sweet orange scions budded on rootstocks has been satisfactorily achieved by preimmunization with a mild isolate of CTV. Presently, 80 million preimmunized trees are growing in the State of Sao Paulo and other parts of Brazil as nursery plants, young or bearing orchards. However, more recently, 3to 4-yrold plants propagated from 20-yr-old vigorous preimmunized Pera sweet orange trees have been showing severe CTV symptoms. This could be attributed to a segregation of the protective isolate or the arising of severe isolates able to supplant the former after many years of exposure. Techniques such as RFLP (restriction fragment length polymorphism) and SSCP (single-strand conformation polymorphism) of coat protein gene (CPG) have been used in order to characterize CTV isolates (2, 5). RFLP consists in the digestion of DNA from PCR amplifications using restriction enzymes. Fragments of different sizes are generated and separated by electrophoresis. SSCP analyses is based on the fact that denatured doublestranded DNA from PCR amplifications migrates as two single stranded DNA (ssDNA) bands in non-denaturing polyacrylamide gel electrophoresis. The migration of the two strands depends on the sequence of nucleotides and, therefore, on their conformation under the electrophoretic conditions used. It has been demonstrated that small changes in the sequence may alter conformation of the ssDNA and therefore their electrophoretic profile (4). The purpose of this work was to compare the RFLP and SSCP patterns of the CPG of the CTV mild protective isolate from the 20-yr-old Pera IAC trees used as mother trees with those from the isolates found in daughter plants showing severe symptoms. MATERIALS AND METHODS The evaluations were carried out using 10 20-yr-old Pera IAC mother trees (preimmunized) without sympFourteenth IOCV Conference, 2000—Citrus Tristeza Virus 137 toms (Fig. 1A), and a tree between 3to 4-yr-old showing severe decline with severe stem pitting and which was representative of several other trees (Fig. 1B) propagated from budwood from the mother trees. The trees were growing in the Matão County, SP. The control mild protective CTV isolate was that in a Pera IAC tree maintained in the greenhouse at the Centro de Citricultura Sylvio Moreira (CCSM), Cordeirópolis County, São Paulo, Brazil. Young leaf and young bark samples collected from the plants were lyophilized and stored at -20 ° C until used for CTV dsRNA extraction. Ds RNA was isolated according to modifications of the procedure described by Valverde et al. (8). The dsRNA was used as a template for the first strand cDNA using random primers. The dsRNA was denatured at 75 ° C for 10 min and chilled on ice. The cDNA reaction was carried out at 37 ° C for 60 min, using M-MLV reverse transcriptase (GIBCO) (6). About 1 / 10 of the cDNA reaction was used for the PCR to amplify the CPG using CTV specific primers (RFL33: 5’TCAACGTGTGTTGAATTT3’, and RFL34: 5’ATGGACGACGAAACAAAG3’). The DNA amplification was performed in 35 cycles of denaturation for 2 min at 94 ° C, annealing for 2 min at 55 ° C, and synthesis for 2 min at 72 ° C, followed by a single chain extension period of 10 min at 72 ° C. The visualization of the amplified product was carried out by electrophoresis in 1% agarose gels containing ethidium bromide and observed under UV light. The amplified CPG was digested with Hinf I, previously described for discriminating CTV isolates (2), according to the manufacturer's instructions (Biolabs). Digests were analyzed by electrophoresis in 8% polyacrylamide gels (16 × 20cm × 1mm BIO-RAD Protean II) using TBE 0.5 X (6) as electrophoresis buffer, and constant voltage of 200 V for 2 h at 25 ° C. The gels were silver stained using the procedure of Beidler et al. (1). The RFLP patterns were evaluated in function of size and number of fragments generated after digestion. SSCP analysis was performed directly on the PCR products of the CPG of CTV isolates. Usually, 1 to 3 μ l of the PCR product was mixed with equal volume of the denaturing solution (95% formamide, 20 mM EDTA, 0.05% xylene-cyanole and 0.05% bromophenol blue), heated for 10 min. at 95 ° C, and chilled on ice. In preliminary experiments, several conditions (gel size, voltage, polyacrylamide concentration, run time, temperature, and presence or absence of glycerol) were determined. The denatured DNA was elecFig. 1. A). Twenty-yr-old Pera TAC (preimmunized) sweet orange plant used as mother trees. B) Threeto 4-yr-old trees raised from tree shown in (A); tree on the left is showing severe CTV symptoms and the tree on the right is a good plant. 138 Fourteenth IOCV Conference, 2000—Citrus Tristeza Virus trophoresed using the conditions determined: non-denaturing 8% polyacrylamide gels (16 × 20 cm × 1mm BIO-RAD Protean II xi) using TBE 0.5 X (6) as electrophoresis buffer, and constant voltage of 200 V for 7h, at 25 ° C. The gels were silver stained using the procedure of Beidler et al. (1). The SSCP patterns were evaluated in function of position and number of electrophoretic bands.