1.11.72
ANALYSIS OF APPLE STEM GROOVING CAPILLOVIRUS QUASISPECIES BY SINGLE-STRAND CONFORMATION POLYMORPHISM

H MAGOME, N YOSHIKAWA and T TAKAHASH

Plant Pathology Laboratory, Faculty of Agriculture, Iwate University, Ueda3-18-8, Moriokao 20, Japan

Background and objectives
Apple stem grooving capillovirus (ASGV) is the type species of the genus capillovirus group and is distributed worldwide in Rosaceae fruit trees such as apple, pear, Japanese pear, apricot and cherry [1]. The virus also infects citrus and lily plants [1]. ASGV has very flexuous filamentous particles, 600-700 nm long, that contain apolyadenylated, plus-sense ss RNA of 6496 nt, excluding the poly(A) tail and a single 27-kDa coat protein (CP). The genome of ASGV contains two overlapping open reading frames (ORFs). From the results of sequence analyses of the 3'-terminal region of the genomes, we recently reported that the ASGV isolates from apple, Japanese pear, and European pear comprise at least two to four 'sequence variants' (sv) that differ considerably from each other [1]. In this study, we analyzed the ASGV quasispecies in fruit trees by immunocapture (IC)-RT-PCR and single-strand conformation polymorphism (SSCP).

Materials and methods
Leaves collected from apple (AF38), Japanese pear (N4) and European pear (Bartlett and La France) trees were subjected to RT-PCR to amplify the 'variable region' of ASGV genome [1]. The nested PCR with asymmetric primers and SSCP analysis of the nested PCR products were conducted as described by Enomotoet al. [2].

Results and conclusions
In preliminary experiments, SSCP analysis was performed on the PCR product of the cDNA clone of 14 isolates and sequence variants of ASGV described earlier [1]. All clones were detected as a single band and most of clones were differentiated under conditions used. The result indicated that SSCP analysis can be used to analyze ASGV quasispecies within an individual tree. SSCP analysis of mixed clones showed that minor subpopulations with dilutions of up to 0.5:1.0 ng were visible as discrete bands. Therefore, SSCP analysis can detect minor sequence variants representing at least 5% of the whole population. IC- RT-PCR and SSCP analysis of leaf samples from four trees showed that two distinct bands were detected in N4 and Bartlett trees, three bands in La France, and four bands in F38. The band pattern was also different between leaf samples with in an individual tree. These results indicated that the ASGv genome in a single virus-infected tree is composed of a mixture of sequence variants.

References
1. Magome H, YoshikawaN, TakahashiT, ItoT, MiyakawaT, 1997. Phytopathology 87, 389-96.
2. Enomoto N, Kurosaki M, Tanakay, Marumo F, Sato C, 1994. Journal of General Virology 75, 1361-69.